WO2025085782A1 - Systems and methods for rna-guided dna integration - Google Patents

Abstract

The present disclosure provides Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems and components thereof which are fully or partially derived from orthogonal CAST systems. More particularly, the present disclosure provides engineered CAST systems comprising one or more Cas proteins selected from: Cas5, Cas6, Cas7, Cas8, Cas12, and combinations thereof; and one or more transposon-associated proteins selected from TnsA, TnsB, TnsC, TnsD, TniQ, and combinations thereof, wherein at least one of TnsB, TnsC, or TniQ is a chimeric protein comprising amino acid sequences derived from respective TnsB, TnsC, or TniQ proteins, or homolog thereof, of at least two CAST systems.

Description

Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 SYSTEMS AND METHODS FOR RNA-GUIDED DNA INTEGRATION FIELD The present disclosure relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems and components thereof which are fully or partially derived from orthogonal CAST systems. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 63/591,987, filed October 20, 2023, and 63/687,572, filed August 27, 2024, the contents of which are herein incorporated by reference in their entirety. SEQUENCE LISTING STATEMENT The content of the electronic sequence listing titled COLUM_42515_601_SequenceListing.xml (Size: 1,742,207 bytes; and Date of Creation: October 18, 2024) is herein incorporated by reference in its entirety. S^TATEMENT R^EGARDING F^EDERALLY S^PONSORED R^{ESEARCH OR} D^EVELOPMENT This invention was made with government support under HG011650 awarded by the National Institutes of Health. The government has certain rights in the invention. B^ACKGROUND In bacteria and archaea, CRISPR/Cas systems provide immunity by incorporating fragments of invading phage, virus, and plasmid DNA into CRISPR loci and using corresponding CRISPR RNAs (“crRNAs”) to guide the degradation of homologous sequences. Transcription of a CRISPR locus produces a “pre-crRNA,” which is processed to yield crRNAs containing spacer-repeat fragments that guide effector nuclease complexes to cleave dsDNA sequences complementary to the spacer. Several different types of CRISPR systems are known, (e.g., type I, type II, or type III), and classified based on the Cas protein type and the use of a proto-spacer-adjacent motif (PAM) for selection of proto-spacers in invading DNA. Although RNA-guided targeting typically leads to endonucleolytic cleavage of the bound substrate, recent studies have uncovered a range of noncanonical pathways in which CRISPR protein-RNA effector complexes have been naturally repurposed for alternative functions. For example, some Type I (Cascade) and Type II (Cas9) systems leverage truncated Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 guide RNAs to achieve potent transcriptional repression without cleavage and other Type I (Cascade) and Type V (Cas12) systems lie inside unusual bacterial Tn7-like transposons and lack nuclease components altogether. SUMMARY Provided herein are proteins, systems, kits, and methods for use in nucleic acid modification (e.g., RNA-guided nucleic acid editing). In some embodiments, the systems comprise an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system or one or more nucleic acids encoding the engineered CAST system. In some embodiments, the engineered CAST system comprises: one or more Cas proteins selected from: Cas5, Cas6, Cas7, Cas8, Cas12, and combinations thereof; and one or more transposon-associated proteins selected from TnsA, TnsB, TnsC, TnsD, TniQ, and combinations thereof. In some embodiments, at least one of TnsB, TnsC, or TniQ is a chimeric protein comprising amino acid sequences derived from TnsB, TnsC, or TniQ proteins, or homologs thereof, of at least two CAST systems. In some embodiments, the system comprises a chimeric TnsB. In some embodiments, the chimeric TnsB has a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a first CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a second at least one second CAST system. In some embodiments, the C- terminal domain comprises the C-terminal hook and at least a portion of the C-terminal linker region of TnsB. In some embodiments, the system further comprises a TnsA protein derived from the second CAST system. In some embodiments, the system further comprises a TnsC protein having an amino acid sequence fully or partially derived a TnsC protein or homolog thereof from the first CAST system. In some embodiments, the TnsC protein is a chimeric protein comprising a C-terminal region having an amino acid sequence derived from the first CAST system. In some embodiments, the system further comprises a TniQ protein derived from the first CAST system. In some embodiments, the one or more Cas proteins are derived from the first CAST system. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the system comprises a chimeric TnsC. In some embodiments, the chimeric TnsC has a C-terminal domain comprising an amino acid sequence derived from a TnsC protein, or homolog thereof, of a second CAST system and N-terminal domain derived from a TnsC protein, or homolog thereof, of a first CAST system. In some embodiments, the system further comprises a TnsA and a TnsB protein derived from the second CAST system. In some embodiments, the system further comprises a chimeric TnsB having a C- terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of the second CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, from the first CAST system or a third CAST system. In some embodiments, the system further comprises a TnsA protein derived from same CAST system as the N-terminal domain of the TnsB protein. In some embodiments, the system further comprises a TniQ is derived from the first CAST system. In some embodiments, the one or more Cas proteins are derived from the first CAST system. In some embodiments, the system comprises a chimeric TniQ. In some embodiments, the chimeric TniQ has an N-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, of a second CAST system and C-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, derived from the first CAST system. In some embodiments, the chimeric TniQ is fused to a second TniQ derived from the first CAST system. In some embodiments, the system further comprises a TnsC protein fully or partially derived from the second CAST system. In some embodiments, the system further comprises a TnsA and TnsB protein derived from the second CAST system. In some embodiments, the TnsC protein is a chimeric protein comprising a C-terminal region having an amino acid sequence derived from a TnsC protein, or homolog thereof, of a third CAST system. In some embodiments, the system further comprises a TnsA and TnsB protein derived from the third CAST system. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the system further comprises a chimeric TnsB protein having a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or a homolog thereof, of the third CAST system. In some embodiments, the system further comprises a TnsA protein derived from same CAST system as the N-terminal domain of the chimeric TnsB protein. In some embodiments, TnsB is a chimeric protein comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 33-35 and 257-288. In some embodiments, TnsC is a chimeric protein comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 48-53. In some embodiments, TniQ is a chimeric protein comprising comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 36-47. In some embodiments, the one or more Cas proteins are derived from the first CAST system. In some embodiments, the TnsB or chimeric TnsB is provided as a fusion protein with TnsA. In some embodiments, one or more of the at least one Cas protein and the at least one transposon-associated protein comprises a nuclear localization signal (NLS). In some embodiments, the system further at least one guide RNA (gRNA) complementary to at least a portion of a target nucleic acid sequence, or at least one nucleic acid encoding thereof. In some embodiments, the system further a target nucleic acid. In some embodiments, the system further a donor nucleic acid. In some embodiments, the donor nucleic acid comprises a cargo nucleic acid sequence flanked by at least one transposon end sequence. In some embodiments, the system further the at least one transposon end sequence is configured to be bound by TnsB. In some embodiments, the system is a cell-free system. Also provided are cells (e.g., prokaryotic or eukaryotic cells) and compositions comprising the disclosed system or any or all components thereof. Further provided are methods for nucleic acid modification. In some embodiments, the methods comprise contacting a target nucleic acid sequence with a system disclosed herein or one or more components thereof. In some embodiments, the modification comprises nucleic acid integration. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the target nucleic acid sequence is in a cell. In some embodiments, contacting a target nucleic acid sequence comprises introducing the system into the cell. In some embodiments, the cell is in a subject. In some embodiments, the introducing the system into the cell comprises administering the system to a subject. In some embodiments, the subject has a disease or disorder which is treated by the method. Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description. B^RIEF D^{ESCRIPTION OF THE} D^RAWINGS FIGS. 1A-1F show cryoEM structure of the TniQ-Cascade (QCascade) complex from PseCAST (which is encoded by transposon Tn7016, and is sometimes synonymously referred to as Tn7016). FIG.1A shows the phylogenetic tree of type I-F CRISPR-associated transposons (CASTs), adapted from Klompe, S. E. et al. Mol. Cell 82, 616-628.e5 (2022). Systems with previously solved QCascade structures are marked with red arrows, while PseCAST is marked with a green arrow. Phylogenetic clades are colored. FIG. 1B shows an exemplary design to investigate both DNA binding and overall integration activities for CAST systems in human cells. DNA binding is extrapolated from two different transcriptional activation assays, one in which VP64 is fused to Cas7 (left), and one in which VP64 is fused to TnsC (right). Overall integration efficiencies are measured via amplicon sequencing. FIG.1C shows a comparison of VchCAST (which is encoded by transposon Tn6677, and is sometimes synonymously referred to as Tn6677) and PseCAST across different assays in human cells. Although PseCAST exhibits consistently weak transcriptional activation compared to VchCAST, its absolute integration activity is approximately two orders of magnitude greater. DNA integration data is adapted from Lampe, G. D. et al. Nat. Biotechnol.42, 87–98 (2023). FIG. 1D shows the operonic architecture of PseCAST components from the PseCAST transposon, with genes encoding the QCascade complex labeled accordingly. FIG. 1E shows dominant reference-free 2D cryoEM class averages (left) and cryoEM densities with colored map regions corresponding to Cas8, Cas7 monomers 1- 6, Cas6, TniQ monomers 1-2, crRNA, and target DNA indicated (right). FIG.1F is a refined UWLMT NWZ \PM 7I[1 p&PMTQKIT LWUIQV IVL Q\[ XW[Q\QWVQVO ZMTI\Q^M \W \PM EVQB LQUMZ QV\MZNIKM' FIGS. 2A-2E show the role of crRNA in the PAM-distal region of PseQCascade. FIG. 2A is an overall view of the cryoEM reconstruction of the PseCAST QCascade complex. FIG. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 2B is a magnified view of the dashed region in FIG.2A, highlighting the cryoEM density (colored and semi-transparent) for interactions between the indicated crRNA nucleotides and protein subunits. FIG.1C is a magnified view of the dashed regions in FIG. 1B, highlighting interactions between the crRNA and Cas6 (left), TniQ.1 (middle), and both TniQ.2 and Cas7.6 (right). Key interacting residues are labeled. FIG. 1D shows normalized RNA-guided DNA integration efficiency at AAVS1 in HEK293T cells, as measured by amplicon sequencing. The indicated alanine mutations were designed to perturb specific RNA-protein interactions highlighted in FIG. 1C, and were compared to WT. NT, non-targeting crRNA. Data are shown as mean ± s.d. for n=3 biologically independent samples. FIG.1E shows a comparison of the crRNA conformation within the PAM-distal region, adjacent to the site of RNA hairpin stabilization by Cas6, for VchCAST (PDB: 6PIJ) and PseCAST (this study). The region around nucleotide G41 exhibits a distinct configuration for PseCAST, likely affecting the behavior of the adjacent TniQ dimer. FIGS. 3A-3F show TniQ recruitment to the Cas6-Cas7.6 interface of Cascade requires hydrophobic and electrostatic interactions. FIG. 3A shows an overall view of the PseCAST QCascade complex, oriented to highlight the TniQ dimer (dark/light orange). FIG.3B shows a magnified view of the region indicated in FIG. 3A, showing how TniQ.1 (dark orange) interacts with a hydrophobic cavity on Cas6. The two visual renderings are colored either by Cas6 surface (top) or hydrophobicity (bottom). FIG.3C is a comparison of the hydrophobic interactions between TniQ.1 and Cas6 in PseCAST (left) and VchCAST (right, PDB: 6PIJ), with residues labeled. FIG.3D shows normalized RNA-guided DNA integration efficiency at AAVS1 in HEK293T cells, as measured by amplicon sequencing. The indicated arginine point mutations were designed to perturb TniQ.1-Cas6 hydrophobic interactions. NT, non-targeting crRNA. e, FIG. 3E shows magnified views of hydrogen bonding (top) and electrostatic (bottom) interactions between Cas7.6 (blue) and TniQ.2 helix (yellow). FIG.3F shows normalized RNA- guided DNA integration efficiency at AAVS1 in HEK293T cells, as measured by amplicon sequencing. Alanine mutations perturbing Cas7.6-TniQ interactions are generally tolerated. Data in FIGS.3D, 3F are shown as mean ± s.d. for n=3 biologically independent samples. FIGS. 4A-4H show structural and functional consequences of PAM and target DNA recognition by PseQCascade. FIG. 4A shows an overall view of the PseCAST QCascade complex, oriented to highlight the target DNA recognition (top) and magnified views of the Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 PAM binding pocket, with Cas8 and DNA shown in blue and red, respectively (bottom). Residues A243 and A244 lack any base-specific, hydrogen-bonding interactions with the DNA. FIG. 4B shows magnified view of the experimental cryoEM density map around Cas7.1 and Cas7.2, showing interactions with the crRNA (gray) and DNA target strand (TS, red). NTS, DNA non-target strand. FIG.4C shows normalized genomic integration efficiencies at AAVS1 for the indicated Cas8 mutants (top), plotted above the WebLogo for PAM preferences in the -1 and -2 positions (bottom) derived from integration into pTarget. For additional PAM specificity data, see FIG.16E. Integration efficiency data are shown as mean ± s.d. for n=3 biologically independent samples. FTRPAAV (SEQ ID NO: 251); YPNSASI (SEQ ID NO: 254); RPAAV (SEQ ID NO: 255); KPQNI (SEQ ID NO: 256). FIG.4D is an overlay of the refined atomic model and cryoEM density (semi-transparent) for the seed region of QCascade bound to the DNA target strand. FIG. 4E is a schematic representation showing angles for the first five RNA- DNA base pairs (BP 1–5) within the R-loop. FIG.4F shows a view of the RNA-DNA heteroduplex at right, highlighting the unfavorable base-pairing surrounding flipped out nucleobases within the first 18 base pairs of the R-loop. FIG.4G shows a magnified view of the RNA-DNA heteroduplex segments aligned at the flipped out base pair, revealing consistent unfavorable angles at the adjacent base pairs. FIG. 4H shows normalized RNA-guided DNA integration efficiency at AAVS1 in HEK293T cells for the indicated Cas7 mutations, as measured by amplicon sequencing. Data are shown as mean ± s.d. for n=3 biologically independent samples. FIGS. 5A-5E show AlphaFold-guided engineering of TnsABC to generate chimeric CAST systems. FIG. 5A is a schematic showing the approach to generate a chimeric CAST system by combining optimal DNA targeting and DNA integration machineries from distinct CAST systems. FIG. 5B is an AlphaFold-generated structure prediction of the TnsABC co- complex from PseCAST. The C-terminal “hook” region of TnsB that putatively interacts with TnsC is marked. FIG. 5C is a visualization of select TnsB graft points within the predicted PseTnsABC structure. Residues where Pse-Vch chimerism was introduced are colored in blue, and the three top performing graft points (V585, S589, Q594; PseTnsB numbering) from panel FIG. 5E are labeled. FIG. 5D shows an exemplary workflow to test chimeric TnsAB constructs for RNA-guided DNA integration activity. E. coli BL21(DE3) cells containing a pEffector encoding VchQCascade and VchTnsC were transformed with a plasmid encoding a mini- Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 transposon (mini-Tn) and TnsAB, with TnsAB derived from either VchCAST, PseCAST, or a chimeric combination thereof. Integration efficiency was measured by qPCR (bottom). FIG.5E shows DNA integration efficiencies for each tested TnsAB chimera. The amino acid listed represents the position at which the reading frame was grafted from PseTnsB (red) to VchTnsB (blue). “Custom” denotes a variant in which multiple different VchTnsB sequences were substituted (see Table 1 for details). Data are shown as mean for n=2 biologically independent samples. FIGS. 6A-6B show structure-guided engineering of chimeric TnsAB proteins. FIG.6A is an Alphafold model of the PseCAST TnsABC co-complex. The C-terminal interacting regions of TnsB with TnsC are marked with red asterisks. FIG.6B shows sequence conservation of the C-terminus of PseCAST TnsB (generated via consurf.tau.ac.il/). Residues that represent graft points are marked with red arrows. Sequence shown in SEQ ID NO: 237. FIGS. 7A-7B show chimeric TnsAB proteins to generate VchCAST-PseCAST (Tn6677-Tn7016) chimeric CASTs. FIG. 7A shows structural visualization of graft points chosen to generate TnsAB chimeras. Residues that represent graft points are colored blue with their side chains shown. FIG. 7B is a zoom-in of TnsB-TnsC interacting region and visualization of graft points along PseCAST TnsB, which generated TnsAB chimeras that were functional for DNA integration. Functional chimeric graft points are marked in blue with their side chains shown. The most optimal functional graft points are marked with blue asterisks, and occurred at residues V585, S589, or Q594. FIGS. 8A-8B show structure-guided engineering of chimeric TnsC proteins. FIG.8A shows the structure of VchCAST TnsC (Hoffman, Kim, Beh et al., Nature 609, 384-393 (2022)). The final residue that is resolved on all subunits in the TnsC heptamer (H314) is colored red for each monomer. FIG. 8B shows the sequence conservation of the C-termini of VchCAST TnsC (generated via consurf.tau.ac.il/). Residues that represent graft points are marked with red arrows. Sequence shown is SEQ ID NO: 238. FIGS. 9A-9C show structure-guided engineering of chimeric TniQ proteins. FIG.9A shows a modeled structure of PseCAST TniQ-Cascade with TnsABC; TniQ-TnsC interactions are modeled (marked with a dashed square) from several known structures (Hoffman, Kim, Beh et al., Nature 609, 384-393 (2022), Wang et al., Cell 186, 4204-4215 (2023)) (left) and a zoom- in of the TniQ-TnsC interface, with TniQ1 colored from N-terminus to C-terminus (blue to red, Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 respectively), (right). The N-terminal region of TniQ1 interacts with TnsC. FIG.9B is a schematic showing chimeric TniQ dimer designs to mutate only one subdomain of the expressed dimer. FIG.9C shows sequence conservation of N-termini of VchCAST and PseCAST TniQ (generated via consurf.tau.ac.il/). Residues that represent graft points are marked with red arrows. Sequence shown are SEQ ID NOs: 239 and 240 for VchCAST and PseCAST, respectively. FIGS. 10A-10B show chimeric TniQ proteins to generate VchCAST-PseCAST (Tn6677-Tn7016) chimeric CASTs. FIG. 10A is a schematic showing chimeric TniQ dimers would enable targeted transposition with divergent CAST modules. Through short, N-terminal peptide modifications in only one of the two TniQ monomers, there can be minimal disruption to the overall TniQ-Cascade interactions, while still successfully recruiting the TnsABC complex to perform integration. FIG. 10B shows integration efficiencies for various TniQ chimeras with VchCAST Cascade and PseCAST TnsABC in E. coli. Integration efficiencies are measured using junction quantitative PCR, normalized against a reference locus in the E. coli genome. The bar graph on the right reports the genomic integration activity for chimeric TniQ fusion proteins, containing chimeras of the TniQ polypeptide at the indicated amino acid junction point (residue listed to the left of the construct cartoon). TniQ1 or TniQ2 indicates if the first, or the second, TniQ monomer (respectively) in the fused polypeptide was modified. These chimeras contain the VchCAST (Tn6677) TniQ polypeptide sequence (light blue), except for an N-terminal modification of varying lengths in either of the two monomers, in which the corresponding sequence from PseCAST (Tn7016, dark salmon) was used. Integration efficiencies were measured across two replicate experiments, and negative controls are shown at the bottom, using TniQ homodimers derived from either VchCAST or PseCAST, both of which would fail to coordinate transposition between divergent modules. FIGS. 11A-11D show purification of QCascade from PseCAST (Tn7016). FIG. 11A shows two schematized expression plasmids (left) encode E. coli codon-optimized PseCAST QCascade genes and a crRNA cassette, with a strong ribosome binding site (half-circle) upstream of each protein-coding gene. After transformation of BL21(DE3) cells and IPTG induction, PseQCascade was purified via Ni-NTA affinity chromatography and size exclusion chromatography (SEC). Codon-optimized expression plasmids were used after the native operon failed to generate detectable QCascade complexes after SEC. FIG. 11B shows a SEC Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 chromatogram of PseQCascade showing 2 distinct peaks. FIG.11C shows SDS-PAGE gel of representative Ni-NTA elution fractions that were pooled and used for SEC. QCascade subunits are labeled. FIG.11D shows an SDS-PAGE gel of both peaks from SEC. Elutions from peak 1, marked with a red dashed box, were pooled and used for cryoEM. FIGS. 12A-12D show cryoEM imaging, data processing, and model refinement. FIG. 12A shows preliminary sample characterization for cryoEM grid optimization. Left, Talos L120C microscope analysis showing exemplary negative staining micrograph (left) and cryogenic micrograph (right). Corresponding reference-free 2D class averages from particles obtained from 10 images are shown below each image, with a calibrated pixel size of 2.5 Å. Right, two grids from the Talos L120C screening were recovered and loaded into a Titan Krios G3i microscope, and a large dataset was collected at a pixel size of 0.644 Å. Two images at different foci are shown with their corresponding CTF images (inset). Reference-free 2D class averages are shown on the right, with multiple different views revealing details compatible with protein secondary structure. FIG. 12B shows the image processing workflow implemented in Relion4 for high-resolution structure determination. Briefly, from left to right: an ab-initio 3D model was reconstructed after selection of 2D class averages; using this as a reference, a consensus refinement was generated; inspection of this preliminary map revealed heterogeneity, especially in the region of the TniQ dimer. However, after unbinning and multiple rounds of 3D refinements, the map still exhibited residual heterogeneity in the region adjacent to the Cas6 protein, suggesting mobility of the TniQ dimer with respect to Cascade. To improve the maps and to analyze TniQ dynamics, two masks were designed, yielding improved the densities and B- factors for the first body, but the second body exhibited a significant improvement in terms of resolution and general density quality. FIG.12C shows Fourier Shell Correlation (FSC) curves for the half-maps and model-maps. FIG.12D shows local resolution depictions of the final map before and after the multibody approach. FIGS. 13A-13B show visualization of TniQ dimer dynamics with cryoDRGN. FIG. 13A shows cryoDRGN analysis, using the same set of particles (~128,000) identified using Relion4 classifications, revealed dynamics of the TniQ dimer and uncovered multiple conformational states. cryoDRGN was trained on the dataset with multiple values of the zdim (2, 4 and 8), and it was found that the derived latent space for different runs was similar. Shown is a principal component analysis of the latent space derived from the run at zdim = 2. FIG.13B Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 shows segmentation of this latent space via k-mean clustering reveled multiple TniQ dimer KWVNWZUI\QWV[3 IV hWXMVi XW[Q\QWV% QV _PQKP \PM EVQB LQUMZ Q[ LQ[\IV\ NZWU \PM 7I[1 p&PMTQKIT domain (cluster 3, green); an intermediate position, where the distal end of the TniQ dimer UIZOQVITTa KWV\IK\[ \PM 7I[1 p&PMTQKIT LWUIQV #KT][\MZ /% OZMa$4 IVL I KWUXIK\ KWVNWZUI\QWV% QV _PQKP \PM EVQB LQUMZ KTW[MTa IXXZWIKPM[ \PM 7I[1 p&PMTQKIT LWUIQV #KT][\MZ 1% XQVS$' <V ITT KZaW8C;@&OMVMZI\ML UIX[% \PM 7I[1 p&PMTQKIT LWUIQV ZMUIQV[ QV I [QUQTIZ XW[Q\QWV IVL conformation. with only the TniQ dimer exhibiting pronounced fluctuations :<;D' *-5&*-6 [PW_ 7I[1 p&PMTQKIT LWUIQV LMTM\QWV IJWTQ[PM[ C@5&O]QLML 8@5 integration. FIG. 14A shows a comparison of select regions of the DNA-bound QCascade complex from VchCAST (left, PDB: 6PIJ) and PseCAST (right), including the crRNA (grey), \IZOM\ 8@5 #ZML$% IVL 7I[1 #JT]M$' EPM 7I[1 p&PMTQKIT LWUIQV NZWU PseCAST (residues 274– 423) is shown in light blue, and was replaced with a flexible, 10-amino acid GS linker in subsequent integration assays. FIG.14B shows normalized efficiency of RNA-guided DNA integration at AAVS1, tested in HEK293T cells and measured by amplicon sequencing. Experiments used WT Cas8 and either a non-targeting (NT) or targeting (T) crRNA, or a targeting crRNA and Cas8 mutant, in which residues N274–K243 were replaced with a 10-amino acid GS linker. Data are shown as mean ± s.d. for n=3 independent biological samples. FIGS. 15A-15B show Cas7 loops chosen to selectively perturb Cas7-TniQ.2 interactions. FIG.15A shows a view of overall PseCAST QCascade complex, with specific regions for panel FIG. 15B highlighted. FIG.15B shows magnified view of the different Cas7 loop interactions. Loop C participates in interactions at the interface between Cas7 monomers (left) and was therefore left intact. Amino acid sidechains in loops A and B (pink) that interact more closely with TniQ.2 were selected for mutagenesis, as detailed in FIGS. 3E-3F. FIG.16A-16E show experimental design and results for PAM library screening with Cas8. FIG.16A shows visualization of PAM binding pockets for diverse type I-F Cascade complexes (from left to right): PseCAST (this study), AsaCAST (PDB: 7U5D), VchCAST (PDB: 6PIJ), and PaeCascade (PDB: 6NE0). The top inset shows core PAM-interacting residues; the bottom inset shows the wedge residue and additional interacting residues. FIG.16B shows amino acid sequence conservation within PAM-interacting regions of PseCas8, with the WebLogo derived from a multiple sequence alignment (MSA) of 66 homologs; the PaeCas8 WT sequence is shown below (SEQ ID NO: 241, right). FIG.16C shows MSA of the same regions from FIG. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 16B, shown for diverse type I-F Cas8 homologs from both CAST and canonical type I-F1 CRISPR-Cas systems. Conserved residues are colored in blue. SEQ ID NOs: 241-247. FIG.16D shows a mammalian PAM library assay workflow. A target plasmid (pTarget) was generated that contains an AAVS1 target flanked by a 4-bp randomized PAM library. Individual Cas8 mutants were screened in each transfection via a plasmid-based integration assay, in which junction PCR and next-generation sequencing revealed PAM sequences enriched within integration products. FIG. 16E shows detailed PAM library data for all active Cas8 variants, showing the identity of the mutation(s) (top), WebLogo of the top 10% of enriched library members (middle), and PAM wheel of all library members (bottom). The PAM wheel is displayed with the inner and outer rings representing the -1 and -2 PAM positions, respectively. FTRPAAV (SEQ ID NO: 251); YPNSASI (SEQ ID NO: 254); RPAAV (SEQ ID NO: 255); KPQNI (SEQ ID NO: 256). FIGS. 17A-17C show the investigation of integration activity via engineering DNA- binding ability of Cas8. FIG.17A shows a comparison of N-terminal regions of PaeCas8 (PDB: 6NE0) and PseCas8. While PaeCas8 (left) shows a vise domain clamped around the dsDNA backbone, PseCas8 shows an unstructured region at the N-terminus that does not exhibit clear dsDNA backbone interactions. FIG.17B shows grafting N-terminal sequences from PaeCas8 to PseCas8 to restore the vise domain and improve overall integration efficiencies. The amino acid listed represents the position at which the reading frame was grafted from PaeCas8 (green) to PseCas8 (blue). FIG. 17C shows fusing diverse DNA-binding proteins and domains onto Cas8. 13 unique archaeal 7kDa DNA-binding proteins, two helix–hairpin–helix DNA-binding motifs (“HhH”), and a binding domain from a Pyrococcus abyssi DNA ligase were individually fused to PseCas8; overall integration activity was then determined for each fusion variant. Both a non- targeting (NT) and targeting (T) crRNA were tested with a WT Cas8 variant. Data in FIGS.17B and 17C are shown as mean for n=2 biologically independent samples. FIGS. 18A-18C show a detailed view of Cas7 interactions with the RNA-DNA heteroduplex. FIG. 18A shows a view of overall PseQCascade complex, with the five similar Cas7-crRNA interactions highlighted. FIG.18B is a visualization of Cas7 residues that interact with the crRNA at each flipped out nucleobase; residues with bulky and hydrophobic sidechains are highlighted and labeled. FIG.18C shows PseCas7 sequence conservation at residues in panel FIG. 18B, from a multiple sequence alignment of 98 homologs; the WT sequence is shown Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 below the x-axis (SEQ ID NOs: 248-250). Specific residues selected for functional investigation are marked with red arrows. FIGS. 19A-19C show DNA binding and integration activity of diverse CAST systems in E. coli. FIG.19A shows a schematic of E. coli transcriptional repression and DNA integration assays to investigate CAST-encoded QCascade activity in bacteria. Using an engineered E. coli strain that constitutively expresses mRFP and sfGFP, transformation of a QCascade expression plasmid driven by a medium-strength J23101 promoter leads to target DNA binding (red triangle) and mRFP repression. Alternatively, when cells are co-transformed with QCascade, TnsABC, and pDonor, RNA-guided DNA integration occurs at the mRFP target site. FIG.19B is a bar graph showing the fold change in mRFP fluorescence for each CAST-encoded QCascade [a[\MU% ZMTI\Q^M \W I KWV\ZWT M`XMZQUMV\ TIKSQVO B7I[KILM #fB7I[KILM$4 VchCAST (aka Tn6677) and PseCAST (aka Tn7016) are highlighted in bold text. CAST systems are colored by phylogenetic clade, as shown in FIGS.1A. FIG.19C is a bar graph comparing DNA integration activity for VchCAST and PseCAST at the same mRFP target site used for repression assays, as measured by qPCR. As observed in human cells, PseCAST yields higher levels of DNA integration activity despite exhibiting apparent weaker QCascade-based DNA targeting and repression. Data in FIG.19B-19C are shown as mean for n=2 independent biological samples. FIGS. 20A-20C show the rational design of chimeric CAST systems. FIG. 20A shows the holo transpososome structure from type V-K ShCAST system (PDB 8EA3), with a magnified view (right) showing how the C-terminal hook of TnsB docks into the TnsC ATPase. FIG. 20B shows the QCascade-TnsC structure from type I-B PmcCAST system (PDB 8FF4), with a magnified view (right) showing the N-terminus of a monomeric TniQ interacting with the TnsC ATPase. FIG.20C shows the predicted QCascade-TnsC structure from type I-F CAST, based on previous modelling but with PDB ID: 7U5D, for which the PAM-distal DNA is better resolved. The magnified view (right) highlights the putative TniQ-TnsC interface, with the N-terminus of just one TniQ monomer within the dimeric arrangement interacting with the TnsC ATPase. FIGS. 21A-21D show additional chimeric TnsB designs are functional for RNA- guided DNA integration. FIG. 21A shows the investigation of reciprocal chimeric designs to coordinate transposition between PseQCascade-TnsC and VchTnsAB. WT TnsAB sequences for both VchCAST and PseCAST and three unique chimeras inspired by the most active variants in FIG. 5E (variants V585, S589, and Q594) were tested. Chimeric TnsAB variants enabled Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 coordinated DNA integration activity when combining PseQCascade and PseTnsC with VchTnsAB and Vch mini-transposons. FIG.21B shows the exploration of chimeric CASTs across multiple type I-F systems. Chimeric TnsAB variants enable coordinated transposition when combining VchQCascade-TnsC with TnsAB constructs sourced from diverse Type I-F CASTs; Tn numbers were defined previously in International Patent Publication WO 2022261122, see FIG.9. Tn7005 is derived from Vibrio cholerae strain M1517, Tn7010 is derived from Photobacterium ganghwense strain JCM 12487, Tn7011 is derived from Pseudoalteromonas sp. strain P1-25, and Tn7015 is derived from Shewanella sp. strain UCD- KL21. FIG.21C shows the design of chimeric CASTs across evolutionarily distinct CAST families. Chimeric ShCAST TnsB constructs (inspired by functional chimeric PseTnsABs) can coordinate low levels of transposition between type I-F and type V-K CAST systems. For chimera 1, only one of two biological replicates exhibited detectable integration. FIG.21D shows insertion site orientation preference of VchCAST, PseCAST TnsAB chimeras, and ShCAST TnsB chimeras. VchCAST TnsAB and PseCAST TnsAB chimeras adopt the common T-RL preference; ShCAST TnsB chimeras invert the insertion site orientation preference, adopting the previously observed T-LR preference for ShCAST systems. Data shown as mean for n=2 independent biological samples. Chimeras 1, 2, and 3 for all homologs are listed in Table 1. DETAILED DESCRIPTION In bacteria and archaea, CRISPR/Cas systems provide immunity by incorporating fragments of invading phage, virus, and plasmid DNA into CRISPR loci and using corresponding CRISPR RNAs (“crRNAs”) to guide the degradation of homologous sequences. Transcription of a CRISPR locus produces a “pre-crRNA,” which is processed to yield crRNAs containing spacer-repeat fragments that guide effector nuclease complexes to cleave dsDNA sequences complementary to the spacer. Several different types of CRISPR systems are known, (e.g., type I, type II, or type III), and classified largely based on the Cas protein type and the use of a proto-spacer-adjacent motif (PAM) for selection of proto-spacers in invading DNA. Although RNA-guided targeting typically leads to endonucleolytic cleavage of the bound substrate, recent studies have uncovered a range of noncanonical pathways in which CRISPR protein-RNA effector complexes have been naturally repurposed for alternative functions. For example, some Type I (Cascade) and Type II (Cas9) systems leverage truncated Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 guide RNAs to achieve potent transcriptional repression without cleavage, and other Type I (Cascade) and Type V (Cas12) systems lie inside unusual bacterial Tn7-like transposons and lack nuclease components altogether. Tn7-like and Tn5053-like transposons that encode nuclease-deficient CRISPR-Cas systems, also known as CRISPR-associated transposons (CRISPR-Tn) or CRISPR-associated transposons (CASTs), catalyze the Insertion of Transposable Elements by Guide RNA-Assisted TargEting (sometimes referred to as INTEGRATE, or INTEGRATE technology). The molecular and sequence determinants of RNA-guided DNA integration for a representative Tn7-like transposon system derived from Vibrio cholerae Tn6677, were previously determined and described (Klompe et al., Nature 571, 219–225 (2019)). Referred to as Tn6677, VchINTEGRATE, VchINT or VchCAST, this representative system is a Type I-F CRISPR-Cas system. A large set of diverse Type I-F CRISPR-associated transposons (CAST) systems were mined and screened to identify a highly active variant derived from Tn7016 found in a Pseudoalteromonas species, hereafter referred to PseINTEGRATE, PseINT, or PseCAST (Klompe et al., Mol Cell 82, 616-628.e5 (2022)). Proteins within any individual CAST system have co-evolved to perform RNA-guided DNA integration only when all proteins and nucleic acid components are derived from a particular CAST system. For example, if specific subunits are sufficiently similar in sequence identity, such as Tn7005 and Tn7013, there is opportunity for cross-reactivity, however this still limits the opportunity to explore unique and divergent CAST combinations. Recently, specific CAST systems that exhibit varied activity in human cells were identified (Lampe, King et al., Nat Biotechnol (2023)). Interestingly, these systems show a dichotomy of DNA targeting and DNA integration activity. While VchCAST exhibits more efficient DNA targeting, PseCAST shows more efficient DNA integration. Herein a strategy to engineer novel chimeric CAST systems is described in which various components of the system were derived from orthogonal CAST systems. These chimeric CASTs were generated through engineered subunits that enable crosstalk between previously orthogonal and unreactive systems. Subunits that were engineered to generate chimeric CAST systems include, but are not limited to: TniQ, TnsC, and TnsB. These chimeric designs enable novel combinations of transposase–targeting machineries to optimize all steps of the CAST pathway. Collectively, these approaches can be applied to generate CAST systems that are Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 derived from orthogonal systems, opening the door to unique combinations of DNA targeting and transposition modules allowing for more optimal combinations, creating opportunities for higher overall integration activity. Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 limited to, an organ, tissue, cell, or tumor, may occur by any means of delivery or administration known to the skilled artisan. The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (e.g., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T<sub>m</sub> of the formed hybrid. Hybridization methods involve the annealing of one nucleic acid to another, Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 complementary nucleic acid, e.g., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and “anneal” or “hybridize” through base pairing interaction is a well-recognized phenomenon. As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub.1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double- stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 amino acid sequence. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T- Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951- 960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, engineered, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the proteins, nucleic acids, and/or systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization to a desired site. Administration by any appropriate route which results in delivery to a desired location in the cell, organism, or subject. A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, guinea pigs, and the like. Examples of non- mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human. A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 Systems Disclosed herein are systems for modification of a target nucleic acid sequence. In some embodiments, the systems comprise: an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CRISPR-Tn or CAST) system or one or more nucleic acids encoding the engineered CRISPR-Tn system. The CRISPR-Tn system comprises at least one or both of: a) one or more Cas proteins and b) one or more transposon- associated proteins. CRISPR-Cas systems are currently grouped into two classes (1-2), six types (I-VI) and dozens of subtypes, depending on the signature and accessory genes that accompany the CRISPR array. The engineered CAST system may include components (e.g., Cas proteins and transposon- associated proteins) derived from a Class 1 CRISPR-Cas system and/or a Class 2 CRISPR-Cas system. Type I CRISPR-Cas systems encode a multi-subunit protein-RNA complex called Cascade, which utilizes a crRNA (or guide RNA) to target double-stranded DNA during an immune response. Cascade itself has no nuclease activity, and degradation of targeted DNA is instead mediated by a trans-acting nuclease known as Cas3. The present system may include components derived from a Type I CRISPR-Cas system (such as subtypes I-B and I-F, including I-F variants). In some embodiments, the components are derived from a Type I-F system. In some embodiments, the components are derived from Type I-F3 system. The present system may include components derived from a Type V CRISPR-Cas system (such as subtype V-K). Type V systems belong to the Class 2 CRISPR-Cas systems, characterized by a single-protein effector complex that is programmed with a gRNA. The transposon-associated Type V CRISPR-Cas systems may be derived from: Anabaena variabilis ATCC 29413 (or Trichormus variabilis ATCC 29413 (see GenBank CP000117.1)), Cyanobacterium aponinum IPPAS B-1202, Filamentous cyanobacterium CCP2, Nostoc punctiforme PCC 73102, and Scytonema hofmannii PCC 7110. In some embodiments, the engineered CAST system comprises Cas5, Cas6, Cas7, Cas8, or any combination thereof. In some embodiments, the engineered CAST system comprises Cas8-Cas5 fusion protein. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 An engineered CAST system of the present invention comprises one or more transposon-associated proteins (e.g., transposases or other components of a transposon). The transposon-associated proteins may facilitate recognition or cleavage of the target nucleic acid and subsequent insertion of a donor nucleic acid into the target nucleic acid. In some embodiments, the transposon-associated proteins are derived, fully or partially, from a Tn7 or Tn7-like transposon. Tn7 and Tn7-like transposons may be categorized based on the presence of the hallmark DDE-like transposase gene, tnsB (also referred to as tniA), the presence of a gene encoding a protein within the AAA+ ATPase family, tnsC (also referred to as tniB), one or more targeting factors that define integration sites (which may include a protein within the tniQ family, also referred to as tnsD, but sometimes includes other distinct targeting factors), and inverted repeat transposon ends that typically comprise multiple binding sites thought to be specifically recognized by the TnsB transposase protein. In Tn7, the targeting factors, or “target selectors,” comprise the genes tnsD and tnsE. Based on biochemical and genetics studies, it is known that TnsD binds a conserved attachment site in the 3’ end of the glmS gene, directing downstream integration, whereas TnsE binds the lagging strand replication fork and directs sequence-non-specific integration primarily into replicating/mobile plasmids. The most well-studied member of this family of transposons is Tn7, hence why the broader family of transposons may be referred to as Tn7-like. “Tn7-like” term does not imply any particular evolutionary relationship between Tn7 and related transposons; in some cases, a Tn7-like transposon will be even more basal in the phylogenetic tree and thus Tn7 can be considered as having evolved from, or derived from, this related Tn7-like transposon. Whereas Tn7 comprises tnsD and tnsE target selectors, related transposons comprise other genes for targeting. For example, Tn5090/Tn5053 encode a member of the tniQ family (a homolog of E. coli tnsD) as well as a resolvase gene tniR; Tn6230 encodes the protein TnsF; and Tn6022 encodes two uncharacterized open reading frames orf2 and orf3; Tn6677 and related transposons encode variant Type I-F and Type I-B CRISPR-Cas systems that work together with TniQ for RNA-guided mobilization; and other transposons encode Type V-U5 CRISPR-Cas systems that work together with TniQ for random and RNA-guided mobilization. Any of the above transposon systems are compatible with the systems and methods described herein. In some embodiments, the one or more transposon-associated proteins comprise TnsA, TnsB, TnsC, or a combination thereof. In some embodiments, the one or more transposon- Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 associated proteins comprise TnsB and TnsC. In some embodiments, the one or more transposon-associated proteins comprise TnsA, TnsB, and TnsC. In some embodiments, the one or more transposon-associated proteins comprise TnsA, TnsB, and TnsC. In some embodiments, the disclosed systems further comprise TnsD, TniQ, or a combination thereof or a nucleic acid encoding TnsD, TniQ, or a combination thereof. Thus, the one or more transposon-associated proteins may comprise TnsD, TniQ, or a combination thereof. In some embodiments, the system comprises Cas5, Cas6, Cas7, Cas8, TnsA, TnsB, TnsC, and TniQ. The different CAST systems from which the one or more Cas protein and the one or more transposon-associated protein may be derived include any known CAST system. For example, the components of the engineered CAST systems described herein may be individually derived (fully or partially) from Vibrio cholerae, Photobacterium iliopiscarium, Vibrio parahaemolyticus, Pseudoalteromonas sp., Pseudoalteromonas ruthenica, Photobacterium ganghwense, Shewanella sp., Vibrio diazotrophicus, Vibrio sp.16, Vibrio sp. F12, Vibrio splendidus, Aliivibrio wodanis, Aliivibrio sp., Endozoicomonas ascidiicola, Parashewanella spongiae or Scytonema hofmannii. The systems may comprise components derived from multiple different CAST systems (e.g., two or more orthogonal systems). In some embodiments, at least one of TnsB, TnsC, or TniQ is a chimeric protein comprising amino acid sequences derived from respective proteins, or homologs thereof, of at least two CAST systems. In some embodiments, the two CAST systems are of same CAST system type or different CAST system types, e.g., Type I or Type V. In some embodiments, the two CAST systems are of same subtype, e.g., the chimeric TnsB, TnsC, or TniQ is derived from two Type I-F CAST systems or two Type I-B CAST systems. In some embodiments, the two CAST systems are of different subtype, e.g., a Type I-F CAST system and a Type I-B CAST system. In some embodiments, the chimeric TnsB, TnsC, or TniQ protein includes amino acid sequences from a protein derived from Tn7016 (PseCAST) and sequences a TnsB homolog from Tn6677 (VchCAST). See Tables 1-3 for exemplary chimeric protein constructs. Wildtype sequences for TnsA, TnsB, TnsC, TniQ, Cas8-Cas5 fusion, Cas7, and Cas6 for PseCAST and VchCAST are provided in SEQ ID NOs: 289-302, respectively (also listed in International Patent Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 Application No. PCT/US2024/015825). TnsB chimeric protein sequences in Table 1 may be provided as a TnsA-TnsB fusion protein, with the TnsB chimera sequence within noted. a. Chimeric TnsB In some embodiments, the system comprises a chimeric TnsB. For example, the TnsB protein may include a sequence in a C-terminal domain which associates with TnsC proteins from one CAST system. However, the N-terminal domain, including the DNA binding region and the sequence which associates with TnsA may be from a different CAST system. The site of grafting, e.g., the sequence location at which the sequence transitions from a first TnsB protein to a second TnsB, may be in the flexible linker. The flexible linker region is largely variable between TnsB proteins and homologs thereof. TnsB protein grafting sites can be determined using similar methods as described in the Examples. Table 1 provides exemplary chimeric TnsB proteins and TnsA-chimeric TnsB fusion protein from other systems with the site of grafting. Accordingly, in some embodiments, the chimeric TnsB has a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a first CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a second at least one second CAST system. In some embodiments, the C-terminal domain comprises the C-terminal hook. In some embodiments, the C-terminal domain comprises the C-terminal hook and at least a portion of the C-terminal linker region of TnsB. In some embodiments, the C-terminal domain comprises at least 10 amino acids. In select embodiments, the C-terminal domain comprises less than 50 amino acids, less than 40 amino acids, or less than 30 amino acids. In some embodiments, the C-terminal domain may be 10-100 amino acids in length. For example, the C-terminal domain may be 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 30 to 100, 30 to 90, 30 to 80, 30 to 70, 30 to 60, 30 to 50, 30 to 40, 40 to 100, 40 to 90, 40 to 80, 40 to 70, 40 to 60, 40 to 50, 50 to 100, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 60 to 100, 60 to 90, 60 to 80, 60 to 70, 70 to 100, 70 to 90, 70 to 80, 80 to 100, 80 to 90, or 90 to 100 amino acids in length. In some embodiments, the chimeric TnsB protein comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any of SEQ ID NOs: 33-35 and 257-288. In some embodiments, the chimeric TnsB protein comprises an amino acid sequence of any of SEQ ID NOs: 33-35 and 257-288. Since TnsA and TnsB associate through the N-terminal portion of TnsB, the TnsA binding site for TnsB will be configured (e.g., with a sequence from that same CAST system as the N-terminal portion of TnsB) to maintain the association between TnsA and TnsB. In the system described in the previous paragraph, the TnsA protein may be derived from the second CAST system. In some embodiments, the chimeric TnsB protein is fused to the TnsA protein. Preferably the C-terminus of TnsA is fused to the N-terminus of TnsB. As described elsewhere herein, the chimeric TnsB protein may be fused to the TnsA protein by a linker oligonucleotide. In some embodiments, the linker oligonucleotide comprises a nuclear localization sequence, as described elsewhere herein. Exemplary TnsA-chimeric TnsB fusion proteins, and their linker oligonucleotides, are provided in Table 1. In some embodiments, the TnsA-chimeric TnsB fusion protein comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any of SEQ ID NOs: 1-32. In some embodiments, the TnsA-chimeric TnsB fusion protein comprises an amino acid sequence of any of SEQ ID NOs: 1-32. Since TnsC and TnsB associate through the C-terminus of TnsC, the C-terminal region of TnsB will be configured to bind the C-terminal region of TnsC. In the system described in the previous paragraph, the TnsC protein may have an amino acid sequence fully or partially derived a TnsC protein or homolog thereof from the first CAST system. In some embodiments, the TnsC protein is a chimeric protein comprising a C-terminal region having an amino acid sequence derived from the first CAST system. TniQ may associate with the N-terminal portion of TnsC. In some embodiments, the system further comprises a TniQ protein derived from the first CAST system. b. Chimeric TnsC In some embodiments, the system comprises a chimeric TnsC. In some embodiments, the chimeric TnsC has a C-terminal domain comprising an amino acid sequence derived from a Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 TnsC protein, or homolog thereof, of a second CAST system and N-terminal domain derived from a TnsC protein, or homolog thereof, of a first CAST system. In some embodiments, the chimeric TnsC protein comprises an amino acid sequence having at least 70% similarity to any of SEQ ID NOs: 48-53. In some embodiments, the chimeric TnsC protein comprises an amino acid sequence of any of SEQ ID NOs: 48-53. In some embodiments, the system further comprises a TnsA and a TnsB protein derived from the second CAST system. In some embodiments, the system further comprises a chimeric TnsB having a C- terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of the second CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, from the first CAST system or a third CAST system. In some embodiments, the system further comprises a TnsA protein derived from same CAST system as the N-terminal domain of the TnsB protein. In some embodiments, the system further comprises a TniQ is derived from the CAST first system. c. Chimeric TniQ In some embodiments, the system comprises a chimeric TniQ. In some embodiments, the chimeric TniQ has an N-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, of a second CAST system and C-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, derived from the first CAST system. In some embodiments, the N-terminal domain sequence of the chimeric TniQ is less than about 200 amino acids. For example, the N-terminal domain sequence of the chimeric TniQ monomer is less than about 200, about 180, about 160, about 140, about 120, about 100, about 80, about 60, about 40, or less amino acids. In some embodiments, the chimeric TniQ comprises any amino acid sequence as shown in Table 2. In some embodiments, the chimeric TniQ is a chimeric TniQ dimer. In some embodiments, the chimeric TniQ dimer comprises a first TniQ monomer having an N-terminal domain sequence from a TniQ of a second CAST system and C-terminal domain sequence from a TniQ of a first CAST system fused to a second TniQ monomer derived from the first CAST system. In some embodiments, the first TniQ monomer in the chimeric TniQ dimer is N-terminal to the second TniQ monomer. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the N-terminal domain sequence of the first TniQ monomer is less than about 75 amino acids. For example, the N-terminal domain sequence of the first TniQ monomer is less than about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, or less amino acids. In some embodiments, the N-terminal domain sequence of the first TniQ monomer is about 40 to about 50 (e.g., about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50) amino acids. In some embodiments, this chimeric TniQ dimer comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any of SEQ ID NOs: 36-47. In some embodiments, chimeric TniQ dimer comprises an amino acid sequence of SEQ ID NOs: 36-47. In some embodiments, the system further comprises a TnsC protein fully or partially derived from the second CAST system. In some embodiments, the system further comprises a TnsA and TnsB protein derived from the second CAST system. In some embodiments, the TnsC protein is a chimeric protein comprising a C-terminal region having an amino acid sequence derived from a TnsC protein, or homolog thereof, of a third CAST system. In some embodiments, the system further comprises a TnsA and a TnsB protein, or a fusion thereof, derived from the third CAST system. In some embodiments, the system further comprises a chimeric TnsB protein having a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or a homolog thereof, of the third CAST system. In some embodiments, the system further comprises a TnsA protein derived from same CAST system as the N-terminal domain of the chimeric TnsB protein. Also provided herein are chimeric TnsB, TnsC, and TniQ proteins as described and exemplified herein. Further provided are nucleic acids encoding the chimeric TnsB, TnsC, and TniQ proteins and compositions comprising the chimeric TnsB, TnsC, and TniQ proteins. In some embodiments, the TnsA and TnsB are provided as a TnsA-TnsB fusion protein. TnsA and TnsB can be fused in any orientation: N-terminus to C-terminus; C-terminus to N-terminus; N-terminus to N-terminus; or C-terminus to C-terminus, respectively. Preferably the C-terminus of TnsA is fused to the N-terminus of TnsB. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the TnsA-TnsB fusion may be fused using an amino acid linker peptide of various lengths to provide greater physical separation and allow more spatial mobility between the fused portions. The linker may comprise any amino acids and may be of any length. In some embodiments, the linker may be less than about 50 (e.g., 40, 30, 20, 10, or 5) amino acid residues. In some embodiments, the linker is a flexible linker, such that TnsA and TnsB have orientation freedom in relationship to each other. For example, a flexible linker may include amino acids having relatively small side chains, and which may be hydrophilic. Without limitation, the flexible linker may contain a stretch of glycine and/or serine residues. In some embodiments, the linker comprises at least one glycine-rich region. For example, the glycine-rich region may comprise a sequence comprising [GS]n, wherein n is an integer between 1 and 10. In some embodiments, the linker further comprises a nuclear localization sequence (NLS). The NLS may be embedded within a linker sequence, such that it is flanked by additional amino acids. In some embodiments, the NLS is flanked on each end by at least a portion of a flexible linker. In some embodiments, the NLS is flanked on each end by a glycine rich region of the linker. In the systems disclosed herein, at least one of the one or more Cas proteins and the one or more transposon-associated protein comprise at least one nuclear localization sequence (NLS). The at least one nuclear localization sequence may be appended to at least one of the one or more Cas protein and the one or more transposon-associated protein at a N-terminus, a C- terminus, embedded in the protein (e.g., inserted internally within the open reading frame (ORF)), or a combination thereof. The nuclear localization sequence may comprise any amino acid sequence known in the art to functionally tag or direct a protein for import into a cell’s nucleus (e.g., for nuclear transport). Usually, a nuclear localization sequence comprises one or more positively charged amino acids, such as lysine and arginine. In some embodiments, the NLS is a monopartite sequence. A monopartite NLS comprises a single cluster of positively charged or basic amino acids. In some embodiments, the monopartite NLS comprises a sequence of K-K/R-X-K/R, wherein X can be any amino acid. Exemplary monopartite NLSs include those from the SV40 large T-antigen, c-Myc, and TUS- proteins. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 In some embodiments, the NLS is a bipartite sequence. Bipartite NLSs comprise two clusters of basic amino acids, separated by a spacer of about 9-12 amino acids. Exemplary bipartite NLSs include the NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 251) and the NLS of EGL-13, MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 252). In some embodiments, the NLS comprises a bipartite SV40 NLS. In certain embodiments, the NLS comprises an amino acid sequence having at least 70% (e.g., having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) similarity to KRTADGSEFESPKKKRKV (SEQ ID NO: 253). In select embodiments, the NLS consists of an amino acid sequence of KRTADGSEFESPKKKRKV (SEQ ID NO: 253). The protein components of the disclosed system (e.g., the Cas proteins or the transposon-associated proteins) may further comprise an epitope tag (e.g., 3xFLAG tag, an HA tag, a Myc tag, and the like). In some embodiments, the epitope tag may be adjacent, either upstream or downstream, to a nuclear localization sequence. The epitope tags may be at the N- terminus, a C-terminus, or a combination thereof of the corresponding protein. In some embodiments, the systems may further comprise a guide RNA (gRNA) or a nucleic acid encoding a gRNA, wherein the gRNA is complementary to at least a portion of a target nucleic acid sequence. In some embodiments, one or more of the at least one Cas protein is part of a ribonucleoprotein (RNP) complex with the gRNA. The gRNA may be a crRNA, crRNA/tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA,” “crRNA,” and “CRISPR guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the binding specificity of the CRISPR-Cas system. A gRNA hybridizes to (complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome in a host cell). In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array. The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be any length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. gRNAs or sgRNA(s) used in the present disclosure can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5960, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9192, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer). To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan 21 (2014)); Heigwer et al. (Nat Methods, 11(2): 122–123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases. In addition to a sequence that binds to a target nucleic acid, in some embodiments, the gRNA may also comprise a scaffold sequence (e.g., tracrRNA). In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA). Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, and Ran, et al. Nature Protocols (2013) 8:2281-2308, incorporated herein by reference in their entireties. In some embodiments, the gRNA sequence does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence. The gRNA can comprise spacer sequence. The spacer sequence can be any length. In some embodiments, the space sequence is 30-40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a target nucleic acid. In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3’ Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3’ end of the target nucleic acid). The gRNA may be a non-naturally occurring gRNA. The system may further comprise a target nucleic acid. The terms “target sequence,” “target nucleic acid,” and “target site” (e.g., a “target genomic DNA sequence”) are used interchangeably herein to refer to a polynucleotide (nucleic acid, gene, chromosome, genome, etc.) to which a guide sequence (e.g., a synthetic guide RNA) is designed to have complementarity, wherein hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex, provided sufficient conditions for binding exist. The target sequence and guide sequence need not exhibit complete complementarity, provided that there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA. Suitable DNA/RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA/RNA binding conditions (e.g., conditions in a cell-free system) are known in the art. The target sequence may or may not be flanked by a protospacer adjacent motif (PAM) sequence. In certain embodiments, a nucleic acid-guided nuclease can only cleave a target sequence if an appropriate PAM is present, see, for example Doudna et al., Science, 2014, 346(6213): 1258096, incorporated herein by reference. A PAM can be 5' or 3' of a target sequence. A PAM can be upstream or downstream of a target sequence. In one embodiment, the target sequence is immediately flanked on the 3' end by a PAM sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In certain embodiments, a PAM is between 2-6 nucleotides in length. The target sequence may or may not be located adjacent to a PAM sequence (e.g., PAM sequence located immediately 3' of the target sequence) (e.g., for Type I CRISPR/Cas systems). In some embodiments, e.g., Type I systems, the PAM is on the alternate side of the protospacer (the 5' end). Makarova et al. describes the nomenclature for all the classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described in by R. Barrangou (Genome Biol.16:247 (2015)). Non-limiting examples of the PAM sequences include: CC, CA, AG, GT, TA, AC, CA, GC, CG, GG, CT, TG, GA, AGG, TGG, T-rich PAMs (such as TTT, TTG, TTC, etc.), Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 NGG, NGA, NAG, NGGNG and NNAGAAW (W=A or T), NNNNGATT, NAAR (R=A or G), NNGRR (R=A or G), NNAGAA, and NAAAAC, where N is any nucleotide. In some embodiments, the PAM may comprise a sequence of CN, in which N is any nucleotide. In select embodiments, the PAM may comprise a sequence of CC. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. There may be mismatches distal from the PAM. The system may further include a donor nucleic acid. The donor nucleic acid may be a part of a bacterial plasmid, bacteriophage, a virus, autonomously replicating extra chromosomal DNA element, linear plasmid, linear DNA, linear covalently closed DNA, mitochondrial or other organellar DNA, chromosomal DNA, and the like. In some embodiments, the donor nucleic acid comprises a cargo nucleic acid sequence. The donor nucleic acid may be flanked by at least one transposon end sequence. In some embodiments, the donor nucleic acid is flanked on the 5’ and the 3’ end with a transposon end sequence. The term “transposon end sequence” refers to any nucleic acid comprising a sequence capable of forming a complex with the transposase enzymes thus designating the nucleic acid between the two ends for rearrangement. Usually, these sequences contain inverted repeats and may be about 10-150 base pairs long, however the exact sequence requirements differ for the specific transposase enzymes. Transposon end sequences are well known in the art. Transposon ends sequences may or may not include additional sequences that promote or augment transposition. The transposon end sequences on either end may be the same or different. The transposon end sequence may be the endogenous CRISPR-transposon end sequences or may include deletions, substitutions, or insertions. The endogenous CRISPR-transposon end sequences may be truncated. In some embodiments, the transposon end sequence includes an about 40 base pair (bp) deletion relative to the endogenous CRISPR-transposon end sequence. In some embodiments, the transposon end sequence includes an about 100 base pair deletion Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 relative to the endogenous CRISPR-transposon end sequence. The deletion may be in the form of a truncation at the distal (in relation to the cargo) end of the transposon end sequences. TnsB recognizes and binds to the transposon end sequences. In the case of the chimeric TnsB proteins, the system from which the DNA binding sites on TnsB are derived dictate the transposon end sequences suitable. In other words, the transposon end sequences, whether natural or engineered, will be derived from the same system as the amino acid sequence of the DNA binding site of TnsB, e.g., the N-terminus, of the chimeric TnsB proteins. The donor nucleic acid, and by extension the cargo nucleic acid, may of any suitable length, including, for example, about 50-100 bp (base pairs), about 100-1000 bp, at least or about 10 bp, at least or about 20 bp, at least or about 25 bp, at least or about 30 bp, at least or about 35 bp, at least or about 40 bp, at least or about 45 bp, at least or about 50 bp, at least or about 55 bp, at least or about 60 bp, at least or about 65 bp, at least or about 70 bp, at least or about 75 bp, at least or about 80 bp, at least or about 85 bp, at least or about 90 bp, at least or about 95 bp, at least or about 100 bp, at least or about 200 bp, at least or about 300 bp, at least or about 400 bp, at least or about 500 bp, at least or about 600 bp, at least or about 700 bp, at least or about 800 bp, at least or about 900 bp, at least or about 1 kb (kilobase pair), or greater. Sequences of exemplary Cas proteins, transposon-associated proteins, gRNAs, and transposon ends can be found in International Patent Publications WO 2020/181264 and WO 2022/261122, incorporated herein by reference. However, the invention is not limited to the disclosed or referenced exemplary sequences. Indeed, genetic sequences can vary between different strains, and this natural scope of allelic variation is included within the scope of the invention. The system may be a cell free system. Also disclosed is a cell comprising the system described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a cell of a non- human primate or a human cell). Thus, in some embodiments, disclosed herein are systems or kits for DNA integration into a target nucleic acid sequence in a eukaryotic cell (e.g., a mammalian cell, a human cell). The one or more Cas proteins, the one or more transposon-associated protein, the at least one gRNA, and the donor nucleic acid may be on the same or different nucleic acids (e.g., vector(s)). In some embodiments, the one or more Cas proteins are encoded by a single nucleic Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 acid. In some embodiments, the one or more transposon-associated proteins are encoded by a single nucleic acid. In some embodiments, the nucleic acid encoding the one or more Cas proteins also encodes the one or more transposon-associated proteins. In some embodiments, the one or more Cas proteins are encoded by a different nucleic acid from the one or more transposon-associated proteins. In some embodiments, the at least one gRNA is encoded by a nucleic acid different from the nucleic acid(s) encoding the one or more Cas proteins and the one or more transposon- associated proteins. In some embodiments, the at least one gRNA is encoded by a nucleic acid also encoding at least one Cas protein, at least one transposon-associated protein, or both. In some embodiments, the one or more Cas proteins, the one or more transposon-associated proteins, and the at least one gRNA are encoded by a single nucleic acid. The gRNA may be encoded anywhere in the nucleic acid encoding the one or more Cas proteins or the one or more transposon-associated proteins. In some embodiments, the gRNA is encoded in the 3’ UTR of a protein coding nucleic acid. In some embodiments, the nucleic acid encoding the one or more Cas proteins, the one or more transposon-associated protein, the at least one gRNA, or any combination thereof further comprises the donor nucleic acid. In some embodiments, the systems further comprise one or more additional genome engineering tools. For example, the systems may further comprise nucleases, such as zinc finger nucleases (ZFNs) and/or transcription activator like effector nucleases (TALENs); transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases. Compositions comprising the systems and chimeric transposon-associated proteins as described herein or a nucleic acid molecule(s) encoding the components of the systems and chimeric transposon-associated proteins are also provided. Nucleic Acids and Delivery The present disclosure also provides for nucleic acids encoding the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins and vectors containing or encoding these nucleic acids. The vectors may be used to propagate the nucleic acid in an appropriate cell and/or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell. The vectors of the present disclosure may be delivered to a eukaryotic cell in a subject. Modification of the eukaryotic cells via the present system can take place in a cell culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to the subject. Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding the disclosed polypeptides or components of the present system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), nucleic acids, and nucleic acids complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the necessary components of the system may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration. Drug selection strategies may be adopted for positively selecting for cells. A donor nucleic acid may contain one or more drug-selectable markers within the cargo. Then presuming that the original donor plasmid is removed, drug selection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events. A variety of viral constructs may be used to deliver the disclosed polypeptides or components of the present system (such as one or more Cas proteins and/or Tns proteins, gRNA(s), donor DNA, etc.) to the targeted cells and/or a subject. Nonlimiting examples of such Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic.7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference. In one embodiment, a nucleic acid encoding the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins is contained in a plasmid vector that allows expression of the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins and subsequent isolation and purification of from the recombinant vector. Accordingly, the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods. To construct cells that express the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins, expression vectors for stable or transient expression of the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins may be constructed via conventional methods as described herein and introduced into host cells. For example, nucleic acids encoding the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors/plasmids/viral vectors should be suitable for integration and replication in eukaryotic cells. In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The systems or the chimeric TnsB, TnsC, and TniQ proteins may be used with various bacterial hosts. In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue- specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- ITXPI #9:*&l$ XZWUW\MZ _Q\P WZ _Q\PW]\ \PM 9:*&l QV\ZWV' 5LLQ\QWVIT XZWUW\MZ[ QVKT]LM IVa constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell. Moreover, inducible and tissue specific expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 promoter/regulatory sequence. Examples of tissue specific or inducible promoter/regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer/promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various commercially available ubiquitous as well as tissue-specific promoters and tumor-specific are available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto. The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5’-and 3’-untranslated regions for mRNA stability and \ZIV[TI\QWV MNNQKQMVKa NZWU PQOPTa&M`XZM[[ML OMVM[ TQSM l&OTWJQV WZ n&OTWJQV4 DF-) XWTaWUI origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae. When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA. In one embodiment, the donor DNA may be delivered using the same gene transfer system (included on the same vector) as used to deliver any one or more of the components of the disclosed systems (e.g., the Cas protein(s) and transposon-associated proteins) or may be delivered using a different delivery system. In another embodiment, the donor DNA may be delivered using the same transfer system as used to deliver gRNA(s). In one embodiment, the present disclosure comprises integration of exogenous DNA into the endogenous gene. Alternatively, an exogenous DNA is not integrated into the endogenous gene. The DNA may be packaged into an extrachromosomal or episomal vector (such as AAV vector), which persists in the nucleus in an extrachromosomal state, and offers donor-template delivery and expression without integration into the host genome. Use of extrachromosomal gene vector technologies has been discussed in detail by Wade-Martins R (Methods Mol Biol.2011; 738:1-17, incorporated herein by reference). The components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins may be delivered by any suitable means. In certain embodiments, the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins are delivered in vivo. In other embodiments, the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins are delivered to isolated/cultured cells (e.g., autologous iPS cells) in vitro to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome. Any of the vectors comprising a nucleic acid sequence that encodes the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding any one or more of the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins is a DNA molecule. In some embodiments, the nucleic acid encoding any one or more of the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding any one or more of the components of the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins is an RNA molecule, which may be electroporated to cells. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res.2012; 1: 27) and Ibraheem et al. (Int J Pharm.2014 Jan 1; 459(1-2):70-83), incorporated herein by reference. Methods of Use Also disclosed herein are methods for nucleic acid modification or integration utilizing the disclosed systems or the chimeric TnsB, TnsC, and TniQ proteins. The methods may comprise contacting a target nucleic acid sequence with a disclosed system or the chimeric TnsB, TnsC, and TniQ protein disclosed herein. The descriptions and embodiments provided above for the systems, compositions, proteins, gRNA, and donor nucleic acid are applicable to the methods described herein. The phrase “modifying a nucleic acid sequence” or “nucleic acid modification” as used herein, refers to modifying at least one physical feature of a nucleic acid sequence of interest. Nucleic acid modifications include, for example, single or double strand breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequence of the nucleic acid sequence. The target nucleic acid sequence may be in a cell. In some embodiments, the contacting a target nucleic acid sequence comprises introducing the system, a composition comprising thereof, or chimeric proteins into the cell. As described above the system, a composition comprising thereof, or chimeric proteins may be introduced into eukaryotic or prokaryotic cells by methods known in the art. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the target nucleic acid is a nucleic acid endogenous to a target cell. In some embodiments, the target nucleic acid is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell. In some embodiments, the target nucleic acid encodes a gene or gene product. The term “gene product,” as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, microRNA (miRNA), and small interfering RNA (siRNA), and coding Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 RNA, such as messenger RNA (mRNA). In some embodiments, the target nucleic acid sequence encodes a protein or polypeptide. Polynucleotides containing the target nucleic acid sequence may include, but is not limited to, purified chromosomal DNA, total cDNA, cDNA fractionated according to tissue or expression state (e.g., after heat shock or after cytokine treatment other treatment) or expression time (after any such treatment) or developmental stage, plasmid, cosmid, BAC, YAC, phage library, etc. Polynucleotides containing the target site may include DNA from organisms such as Homo sapiens, Mus domesticus, Mus spretus, Canis domesticus, Bos, Caenorhabditis elegans, Plasmodium falciparum, Plasmodium vivax, Onchocerca volvulus, Brugia malayi, Dirofilaria immitis, Leishmania, Zea maize, Arabidopsis thaliana, Glycine max, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium tuberculosis, Aquifex, Thermus aquaticus, Pyrococcus furiosus, Thermus littoralis, Methanobacterium thermoautotrophicum, Sulfolobus caldoaceticus, and others. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells, an effective amount of the described system, a composition comprising thereof, or chimeric proteins. In some embodiments, the vector(s) is delivered to the tissue of interest by, for example, an intramuscular, intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. The proteins, composition, components of the present system, or ex vivo treated cells may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the proteins, composition, components of the present system may be mixed, individually or in any combination, with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure. In some embodiments, an effective amount of the proteins, composition, components of the present system as described herein can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “effective amount” Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 refers to that quantity of the proteins, composition, components of the present system such that successful nucleic acid modification (e.g., DNA integration) is achieved. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. For example, in connection with cancer the term “treat” may mean eliminate or reduce a patient's tumor burden, or prevent, delay, or inhibit metastasis, etc. The phrase “pharmaceutically acceptable,” as used in connection with compositions and/or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and/or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and/or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. The methods may be used for a variety of purposes. For example, the methods may include, but are not limited to, inactivation of a microbial gene, RNA-guided DNA integration in a plant or animal cell, methods of treating a subject suffering from a disease or disorder (e.g., KIVKMZ% 8]KPMVVM U][K]TIZ La[\ZWXPa #8?8$% [QKSTM KMTT LQ[MI[M #D78$% n&\PITI[[MUQI% IVL hereditary tyrosinemia type I (HT1)), and methods of treating a diseased cell (e.g., a cell deficient in a gene which causes cancer). The disclosed methods may modify a target DNA sequence in a cell so as to modulate expression of the target DNA sequence, e.g., expression of the target DNA sequence is increased, decreased, or completely eliminated (e.g., via deletion of a gene). The modifications of the target sequence may lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, gene knock- down, etc. In some embodiments, the methods described herein may be used to correct one or more defects or mutations in a gene (referred to as “gene correction”). In such cases, the target sequence encodes a defective version of a gene, and the disclosed compositions and systems further comprise a donor nucleic acid molecule which encodes a wild-type or corrected version of the gene. Accordingly, in some embodiments, the methods described herein may be used to insert a gene or fragment thereof into a cell. In another embodiment, the method of modifying a target sequence can be used to delete nucleic acids from a target sequence in a host cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research. In some embodiments, the methods described herein may be used to genetically modify a plant or plant cell. As used herein, genetically modified plants include a plant into which has been introduced an exogenous polynucleotide. Genetically modified plants also include a plant that has been genetically manipulated such that endogenous nucleotides have been altered to include a mutation, such as a deletion, an insertion, a transition, a transversion, or a combination thereof. For instance, an endogenous coding region could be deleted. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified plant is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region. The genetically modified plant may promote a desired phenotypic or genotypic plant trait. Genetically modified plants can potentially have improved crop yields, enhanced nutritional value, and increased shelf life. They can also be resistant to unfavorable environmental conditions, insects, and pesticides. The present systems and methods have broad applications in gene discovery and validation, mutational and cisgenic breeding, and hybrid breeding. The present methods may facilitate the production of a new generation of genetically modified crops with various improved agronomic traits such as herbicide resistance, herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, disease (e.g. bacterial, fungal, and viral) resistance, high yield, and superior quality. The present methods may also facilitate the production of a new generation of genetically modified crops with optimized fragrance, nutritional value, shelf-life, pigmentations (e.g., lycopene content), starch content (e.g., low- gluten wheat), toxin levels, propagation and/or breeding and growth time. See, for example, CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture (Chen et al., Annu Rev Plant Biol. 2019 Apr 29;70:667-69), incorporated herein by reference. The present method may confer one or more of the following traits to the plant cell: herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 modified oil percent, modified protein percent, resistance to bacterial disease, resistance to fungal disease, and resistance to viral disease. The present disclosure provides for a modified plant cell produced by the present method, a plant comprising the plant cell, and a seed, fruit, plant part, or propagation material of the plant. Transformed or genetically modified plant cells of the present disclosure may be as populations of cells, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, grain, animal feed, a field of plants, and the like. The present disclosure provides a transgenic plant. The transgenic plant may be homozygous or heterozygous for the genetic modification. Also provided by the present disclosure are transformed or genetically modified plant cells, tissues, plants, and products that contain the transformed or genetically modified plant cells. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants. The method may be used to modify a plant stem cell. The present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same genetic modification as the genetically modified cell from which it was derived. The present disclosure further provides a composition comprising a genetically modified cell. In one embodiment, the transformed or genetically modified cells, and tissues and products comprise a nucleic acid integrated into the genome, and production by plant cells of a gene product due to the transformation or genetic modification. Methods of introducing exogenous nucleic acids into plant cells are well known in the art. Such plant cells are considered “transformed.” DNA constructs can be introduced into plant cells by various methods, including, but not limited to PEG- or electroporation-mediated protoplast transformation, tissue culture or plant tissue transformation by biolistic bombardment, or the Agrobacterium-mediated transient and stable transformation. The transformation can be transient or stable transformation. Suitable methods also include viral infection (such as double stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium-mediated transformation, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e., in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium tumefaciens are useful for introducing an exogenous Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 nucleic acid molecule into a vascular plant. The wild-type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. An Agrobacterium-based vector is a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by the nucleic acid sequence of interest to be introduced into the plant host. Agrobacterium-mediated transformation generally employs cointegrate vectors or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety of binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art. See., e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993), incorporated herein by reference. Microprojectile-mediated transformation also can be used to produce a transgenic plant. This method, first described by Klein et al. (Nature 327:70-73 (1987), incorporated herein by reference), relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine, or polyethylene glycol. The microprojectile particles are accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.). In one embodiment, the present methods may be adapted to use in plants. The vectors may be optimized for transient expression of the present system in plant protoplasts, or for stable integration and expression in intact plants via the Agrobacterium-mediated transformation. In certain embodiments, the present methods use a monocot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a monocot plant. In certain embodiments, the present methods use a dicot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a dicot plant. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 The present methods may be used with various microbial species, including human pathogens that are medically important, and bacterial pests that are key targets within the agricultural industry, as well as antibiotic resistant versions thereof. The method may be designed to target any gene or any set of genes, such as virulence or metabolic genes, for clinical and industrial applications in other embodiments. For example, the present methods may be used to target and eliminate virulence genes from the population, to perform in situ gene knockouts, or to stably introduce new genetic elements to the metagenomic pool of a microbiome. The present systems and methods may be used to treat a multi-drug resistance bacterial infection in a subject. The present systems and methods may be used for genomic engineering within complex bacterial consortia. The present methods may be used to inactivate microbial genes. In some embodiments, the gene is an antibiotic resistance gene. For example, the coding sequence of bacterial resistance genes may be disrupted in vivo by insertion of a DNA sequence, leading to non-selective re-sensitization to drug treatment. The methods described here also provide for treating a disease or condition in a subject. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells (e.g., disclosed T cells), a therapeutically effective amount of the present system, polypeptides, or components thereof. In some embodiments, the methods are used to treat a pathogen or parasite on or in a subject by altering the pathogen or parasite. In some embodiments, the methods target a “disease-associated” gene. The term “disease-associated gene,” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. Examples of genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, l&* IV\Q\ZaX[QV% Ka[\QK NQJZW[Q[ \ZIV[UMUJZIVM KWVL]K\IVKM ZMO]TI\WZ #7:EC$% n&PMUWOTWJQV (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial, H. Rare Genetic Disorders: Learning About Genetic Disease Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1):192 (2008); Online Mendelian Inheritance in Man (OMIM); and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (i.e., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip/palate, congenital heart defects, and neural tube defects. In another embodiment, the target DNA sequence can comprise a cancer oncogene. The present disclosure provides for gene editing methods that can ablate a disease-associated gene (e.g., a cancer oncogene), which in turn can be used for in vivo gene therapy for patients. In some embodiments, the gene editing methods include donor nucleic acids comprising therapeutic genes. Kits Also within the scope of the present disclosure are kits that include the chimeric transposon-associated proteins, compositions, or any or all of the components of the present system. The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of administration to a subject to achieve the intended effect. The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub- unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. The kit may further comprise a device for holding or administering the present system, polypeptides, or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and/or a syringe. The present disclosure also provides for kits for performing nucleic acid modification and integration in vitro. Optional components of the kit include one or more of the following: buffer constituents, control plasmid, sequencing primers, cells. Examples The following are examples of the present invention and are not to be construed as limiting. In the below examples: Tn6677 encodes a naturally occurring Cas8-Cas5 fusion protein, as part of the Type I-F CRISPR-Cas system, referred to as Cas8, for simplicity. The Type I-F CRISPR-Cas system encoded within Tn7-like transposons may be more specifically referred to as Type I-F3, Type I-F is used for simplicity. The complex known as TniQ-Cascade, or QCascade, comprises crRNA (one copy), Cas8 (one copy), Cas7 (six copies), Cas6 (one copy), and TniQ (two copies). In some contexts, QCascade subunits have been referred to with other gene and protein naming schemes, e.g., Csy1 or Csy2 or Cas8f instead of Cas8; Csy3 or Cas7f Cas7; Csy4 or Cas6f instead of Cas6. The mini-transposon, also known as a mini-Tn, refers to the mobilizable DNA containing a cargo/payload sequence flanked by conserved left Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 (L) and right (R) ends of the transposon. The mini-Tn may be encoded within a larger donor DNA molecule, for example a plasmid-based donor, or pDonor. CAST systems may also be referred to as INTEGRATE systems; CRISPR-transposon systems; CRISPR-Tn systems; RNA- guided transposase systems; RNA-guided DNA integration system; or a similar set of synonymous terms to refer to the core technology as molecular machinery. RNA-guided DNA integration by CAST systems involve a diverse array of targeting proteins, which include Cascade from Type I-B, Type I-D, and Type I-F CRISPR-Cas systems, and Cas12k from Type V-K CRISPR-Cas systems. Example 1 CryoEM structure of PseCAST QCascade complex VchCAST and PseCAST, two distantly related type I-F CASTs, exhibit distinct DNA binding and integration efficiencies (FIGS. 1A-1C). Given previous mechanistic and structural studies of the QCascade complex from VchCAST, it was hypothesized that structure-guided engineering of the PseCAST QCascade complex might reveal novel interactions and open a path to improve overall integration efficiencies. Recombinant PseQCascade was purified after carefully optimizing the expression vector design (FIG. 11) to determine the cryoEM structure. The purified PseQCascade complex, which was expected to comprise a 1:6:1:2:1 stoichiometry of Cas8:Cas7:Cas6:TniQ:crRNA components (FIG. 1D), was incubated with a LW]JTM&[\ZIVLML 8@5 #L[8@5$ []J[\ZI\M KWV\IQVQVO I ,+&JX \IZOM\ [MY]MVKM IVL .m&77&,m A5?% and then subjected the sample to electron microscopy. Preliminary cryoEM experiments revealed a homogeneous behavior with multiple views and no apparent disassembly (FIG. 12A), and the overall architecture was consistent with other type I-F QCascade complexes, comprising six Cas7 monomers (named hereafter Cas7.1 to Cas7.6) that form a pseudo-helical assembly coating the crRNA molecule (FIG.1E). The Cas8 protein contained two domains: a bulky domain that QV\MZIK\[ _Q\P 7I[0'* IVL JQVL[ \PM KZC@5 .m MVL IVL A5? [MY]MVKM% IVL I [MKWVL p&PMTQKIT LWUIQV \PI\ M`PQJQ\ML I LaVIUQK JMPI^QWZ #:<;' *:$' EW_IZL[ \PM KZC@5 ,m MVL #PMZMIN\MZ PAM-distal region), the RNA hairpin was stabilized by Cas6, which also binds the TniQ dimer. Preliminary maps exhibited greater mobility for the TniQ dimer compared to other QCascade components (FIGS.12B-12C). The quality of the maps approaching the TniQ dimer region degraded rapidly, contrasting the excellent map quality for the PAM-adjacent region (FIG.12D). Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 Multibody approaches in Relion4 improved the overall resolution, with approximately 2.6 Å and 3.0 Å resolution estimates in the PAM-proximal and PAM-distal regions, respectively. To further characterize the dynamics of the system and confirm the existence of novel interactions, multibody analysis in Relion4 was complemented with cryoDRGN, a machine- learning approach for cryoEM analysis (FIG.13). CryoDRGN revealed multiple populations of the complex, with the TniQ dimer populating a wide range of positions relative to the rest of the complex that pivot around Cas6 and Cas7.6. The dimer adopted an ‘open’ conformation that lacked any direct interactions with Cas8, as well as multiple intermediate, ‘closed’ conformations \PI\ IXXZWIKP \PM \QX WN \PM 7I[1 p&PMTQKIT LWUIQV #:<;' *,6$' <V I ZMKMV\ [\Z]K\]ZM WN I PWUWTWOW][ B7I[KILM KWUXTM` JW]VL \W \IZOM\ 8@5% \PM 7I[1 p&PMTQKIT LWUIQV M`PQJQ\ML I different conformation, almost perpendicular to the inner face of the TniQ dimer and aligned with the bulky domain of Cas8. In this dataset, this extended conformation was not observed and instead alternative TniQ-Cas8 interactions that are established between the most distal end of the EVQB LQUMZ IVL \PM IXQKIT XIZ\ WN \PM 7I[1 p&PMTQKIT LWUIQV _MZM LM\MK\ML I[ ZM^MITML \PZW]OP TW_&XI[[ NQT\MZML UIX[ #:<;' *,7$' 6W\P \PM EVQB LQUMZ IVL \PM 7I[1 p&PMTQKIT LWUIQV[ remained in parallel configurations, with only marginal contacts at the periphery of the complex. 8M[XQ\M \PM IXXIZMV\ NTM`QJQTQ\a QV \PQ[ QV\MZIK\QWV% \PM 7I[1 p&PMTQKIT LWUIQV _I[ M[[MV\QIT NWZ RNA-guided DNA integration activity, as revealed by the complete loss of human cell activity when the domain was replaced with a flexible glycine-serine linker (FIG.14). Stabilizing protein-RNA and protein-protein interactions The overall architecture of the TniQ dimer was similar to the VchCAST QCascade dimer, with an antiparallel head-to-tail configuration, forming a compact unit that laterally approaches the interface formed by Cas6 and Cas7.6 (FIG. 2A). The C-terminal domain of one TniQ monomer interacted with Cas6, and the N-terminal domain of the other TniQ monomer interacted with Cas7.6. At the core of this four-fold interface, the crRNA appeared to play an important role, with residues 40–45 establishing multiple RNA-protein stacking interactions (FIG. 2B). It was hypothesized that crRNA interactions with Cas6, Cas7.1, TniQ.1, and TniQ.2 facilitate robust QCascade complex formation, and that disrupting them would decrease transposase recruitment and integration activity. Alanine point mutations were introduced to disrupt nucleobase-side chain stacking interactions and the resulting effects in human genomic Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 DNA integration assays were investigated. Alanine substitutions to Cas6 and TniQ residues contacting the crRNA were well tolerated, whereas a Cas7 R143A mutation (Cas7^R143A) abolished integration activity (FIG.2D). The crRNA trajectory in the hinge region between Cas7.6 and Cas6 differs in PseCAST and VchCAST (FIG. 2E), and PseCAST crRNA residue G41 seemed to play a role as an interaction “hub,” establishing coincident contact with TniQ.1, TniQ.2, and Cas7.6 by adopting a unique, extruded conformation. Protein-protein interactions that may contribute to QCascade function, in part by playing a role in downstream transposase recruitment to the target site were explored. The first of these interactions involved a hydrophobic patch on Cas6 cradling hydrophobic residues in the TWWX KWVVMK\QVO EVQB'* l&PMTQKM[ G+/+g=+0. IVL :,*+gD,+0 #:<;D' ,5&,6$% _PQKP Q[ conserved across homologous QCascade complexes, with minor variations. Specifically, a hydrophobic residue in the TniQ.1 connecting loop (I282 in PseCAST, V270 in VchCAST) inserts deeply into the Cas6 hydrophobic patch to anchor the TniQ monomer to the Cascade module (FIG. 3C). The cradle structure of this interaction potentially acts as a pivot point, facilitating dynamic TniQ movement. Disruption of these hydrophobic interactions via introduction of charged arginine residues in either TniQ or Cas6 led to a marked reduction in integration efficiencies (FIG.3D). The other TniQ monomer (TniQ.2) interacts electrostatically _Q\P 7I[0'/ ^QI l&PMTQ` H,,g>-0 IVL ILRIKMV\ ZM[QL]M[ #:<;' ,9$' ;Q^MV \PM U]T\QUMZQK assembly of Cas7 monomers along the crRNA, loop regions observed to interact with TniQ.2 may have pleiotropic functions, possibly participating in Cas7 monomer-monomer interactions (FIG. 15). With the goal of selectively perturbing Cas7.6-TniQ.2 interactions to investigate its importance, mutagenizing residues that might affect the Cas7 monomer-monomer contacts was avoided and the focus was directed to loops A and B (FIG. 15B). Alanine mutations within the TniQ-interacting regions abolished DNA integration, whereas several mutations within Cas7 had surprisingly little to no impact on overall DNA integration activity (FIG.3F). Protein engineering modulates PAM stringency and improves DNA integration In comparison to other type I-F CASTs, PseCAST exhibits a remarkably flexible PAM preference, with almost no sequence preference at both the -1 and -2 positions in E. coli transposition assays, and this property may lead to a dramatic increase in the effective search space for the 32-bp guide. It was hypothesized that inefficient DNA targeting due to a flexible Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 PAM preference may represent a rate-limiting step in RNA-guided DNA integration, especially within the cellular milieu of human cells, whose genome is ~1000X larger than E. coli. After leveraging the excellent quality of the cryoEM map in the area surrounding Cas8, two hydrophobic alanine residues at the center of the PAM-interacting region were identified. In contrast, systems with stricter PAM preferences, e.g., VchCAST, AsaCAST, and PaeCascade from a type I-F1 CRISPR-Cas system, feature polar residues at the equivalent positions, which allow for hydrogen bonding with specific PAM nucleotides (FIGS.4A-4B, 16A). Mutation of A143 and A144 to residues with greater hydrogen bonding potential might improve PAM stringency, reduce the effective search space, and result in more efficient DNA targeting. It was also decided to mutagenize residues 125–127, as this region also interacts with the PAM (FIGS.4B and 16A). The sequence conservation at these PAM-interacting regions was analyzed and compared to other Cascade homologs that have previously exhibited either robust DNA integration activity or stringent PAM preferences (FIGS.16B-16C). Collectively, fifteen Cas8 variants with PAM-interacting mutations were designed, varying from single point mutations at A243 or A244 to larger mutations in which the entire PAM-interacting region was grafted from a homolog. Changes in PAM preference were quantified by performing an episomal PAM library screen in HEK293T cells, in which a target plasmid (pTarget) contained an AAVS1 target site directly downstream of a randomized 4-bp PAM library (FIG.16D). After transiently transfecting cells with pTarget, pDonor, and all the necessary protein-RNA expression vectors, plasmid DNA was isolated, the PAM motifs from all successful integration products were sequenced, and a consensus motif for each Cas8 variant was constructed; in parallel, absolute QV\MOZI\QWV MNNQKQMVKQM[ I\ \PM OMVWUQK 55FD* [Q\M% _PQKP KWV\IQV[ I .m&77&,m A5?% _MZM IT[W quantified (FIG.4C). The results revealed that certain mutations led to improvements in integration efficiencies by as much as 3.5-fold, but without a clear correlation between PAM stringency and overall genomic integration activity (FIG.4C). For example, the variant with the greatest improvement in integration activity, Cas8<sup>R241K,A244S</sup>, actually exhibited a reduced PAM preference, compared to the stronger preference for cytidine in the -2 position with WT Cas8 (FIGS.4C and 16E). Interestingly, Cas8<sup>A243Q,A244N</sup> exhibited decreased PAM preference, whereas when the entire PAM region was grafted from a type I-F1 system (<sub>241</sub>RPAAV<sub>245</sub> (SEQ ID NO: 255)>KPQNI (SEQ ID NO: 256)), the resulting mutant restored a strong preference for Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 cytidine at the -2. Mutations within the upstream PAM-interacting region (residues 125-127) resulted in moderate improvements on integration activity, with either unchanged or moderately reduced PAM stringency (FIG.4C). A Cas8<sup>R241A</sup> mutant with disrupted ‘R-wedge,’ which normally forms stacking interactions with the -1 PAM position to help unwind dsDNA, unexpectedly exhibited both WT integration efficiencies and PAM stringency (FIG.4C). Together, mutational profiling of the PAM-interacting region revealed key residues whose mutation improved integration efficiencies, but the combination of PAM specificity and integration activity results failed to support the hypothesis that PAM promiscuity is a key bottleneck towards achieving higher efficiency PseCAST integration activity in human cells (FIGS.4C and 16E). Previous work investigating canonical type I-F defense systems revealed important interactions between the N-terminal region of Cas8 and the dsDNA backbone; the N-terminal, positively charged vise domain undergoes a conformational change that results in a “clamping” of the complex onto dsDNA. When comparing PseCas8 to canonical type I-F PaeCascade Cas8 (FIG. 17A), a markedly different conformation of the N-terminus in which the vise domain is completely absent was observed. This vise domain may have been lost in PseCas8 and replacing the N-terminal sequence of PseCas8 with PaeCas8 may restore the vise domain, thus improving DNA binding; however, a thorough screening of chimeric Cas8 constructs revealed a clear intolerance of PseCas8 to sequence perturbations in this region (FIG. 17B). Synthetic strategies to improve DNA binding of PseQCascade were pursued by fusing a variety of DNA-binding domains (FIG.17C). However, fusions of a variety of small archaeal DNA-binding proteins or other motifs showed no improvement, and in some cases a reduction, in overall integration efficiencies (FIG.17C). The combination of complete ablation of activity with chimeric N- terminal designs and no improvements with DNA-binding domains suggests that the DNA- binding affinity of PaeCas8 is not a critical bottleneck in the overall transposition pathway. Unfavorable nucleobase positioning along the RNA-DNA heteroduplex Cascade complexes bind the target DNA by forming a discontinuous RNA-DNA heteroduplex in 6-bp segments, and RNA-DNA base pairs for the first 4 segments engaged by Cas7 monomers were clearly resolved within the PseQCascade complex, but the remaining two segments featured weaker RNA density and no DNA density. Density for the RNA-DNA heteroduplex across the first 3 segments (crRNA residues 9 to 26) was exceptionally good, with Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 clear separation within base pairs and features compatible with a local resolution beyond 3 Å. It was therefore possible to accurately model RNA-DNA interactions to a high level of confidence in these regions of the map. The resulting view revealed peculiarities in the base-pair geometry, with acute divergence from ideal values in some base pairs. The third and fourth base pair within each segment exhibited severe deviation from ideal planarity values (buckling), while the first and fifth base pair exhibited exacerbated propeller twist deviations. Only the second base pair across distinct segments exhibited geometric and hydrogen-bonding distance values closer to energetically favored conditions (FIGS.4D-4H). EaXM <&: 7I[KILM KWUXTM`M[ JQVL \PM \IZOM\ 8@5% []KP \PI\ \PM \_W&[\ZIVLML n&[PMM\ ‘finger’ motif of each Cas7 monomer engages the crRNA to flip out every sixth nucleotide of the 32-nt spacer, thereby preventing RNA-DNA basepairing. Finger motif residues involved in this nucleotide dislocation might promote the consistent distortion of adjacent base pairs, and to explore this effect, Cas7 mutations intended to relax this distortion were introduced, to attempt to promote energetically favorable hydrogen-bonding geometries and stabilize the RNA-DNA heteroduplex. Taking advantage of the high local resolution around this region (FIGS.18A-18B), numerous bulky hydrophobic residues were identified, including I69, L70, and L224, that were not highly conserved across nearby homologs (FIG.18C), and subjected to site-directed mutagenesis. After generating the desired Cas7 mutations, genomic DNA integration experiments were performed in HEK293T cells at the AAVS1 locus (FIG.4H). Intriguingly, the Cas7 heteroduplex-interacting residues, though not highly conserved, appeared to have low tolerance for mutations. While Cas7^L224F and various valine mutations exhibited near-WT integration efficiencies, all other mutations, including Cas7^I69P, resulted in detrimental impacts on DNA integration (FIG.4H). Intriguingly, L70H, which would theoretically recapitulate a stacking interaction observed in the previous VchCAST structure, completely ablated integration activity (FIG. 4H). Together, the intolerance to perturbations in the Cas7 finger domain suggests these kinking interactions may in fact facilitate proper successful DNA integration. Structure-based engineering of chimeric CAST systems. Rational engineering of PseQCascade yielded only moderate improvements in integration activity, suggesting a non-trivial path forward to overcome the apparently weak DNA binding activity in human cells. Although recent studies shed light on the kinetics of Cascade Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 target search and recognition, the intermediate steps of Cascade complex formation, TniQ- Cascade association, and 3D-diffusion remain poorly understood, particularly in human cells. PseCAST was originally identified through a homolog screen that investigated both overall integration activity and several subunit-specific properties: crRNA processing, TnsB-donor DNA interactions, and QCascade and TnsC-mediated transcriptional activation. Through this screening process, VchCAST (Tn6677) and PseCAST (Tn7016) were the only two systems that yielded detectable DNA integration in human cells, despite exhibiting distinct subunit-specific activities. Based on these results, chimeric CAST systems that would enable ‘crosstalk’ between otherwise orthogonal components were pursued. The first goal was to combine the most active DNA targeting and DNA integration machineries derived from divergent CASTs (FIGS. 5A). To identify robust DNA targeting homologs, DNA binding activity was tested across 20 type I-F CASTs via transcriptional repression in E. coli (FIG. 19A). Surprisingly, QCascade complexes from only two systems, VchCAST and Tn7005, exhibited RFP repression under the tested conditions, with only weak activity from PseCAST and Tn7000 (FIG.19B). Yet when the overall DNA integration activity of VchCAST and PseCAST was tested at the exact same sites used for transcriptional repression, greater integration activity was observed for PseCAST, mirroring results in human cells (FIG. 19C). This reinforced the conclusion that the weak DNA targeting activity of PseCAST may impose a lower ceiling on achievable DNA integration efficiencies in diverse cell types, despite having co-evolved with a highly active transposition (TnsABC) module. To address this potential bottleneck by the TnsABC machinery, PseCAST was combined with the QCascade machinery from VchCAST. Intrinsic CAST modularity precludes simply mixing and matching components from evolutionary diverse systems, but did not rule out a more nuanced approach by taking advantage of recent high-resolution structures, predicted structures via structural alignments, and AlphaFold-multimer predicted structures. (FIGS.5B and 19). In particular, a model for the putative TnsABC co-complex from PseCAST featured the heptameric arrangement of TnsC, similar to empirical structures for VchCAST, while also revealing predicted interactions between PseTnsC and the C-terminus of PseTnsB that were reminiscent of the TnsB ‘hook’ described for type V-K ShCAST (FIGS.5B and 20A). This model, in conjunction with experimentally determined type V-K structures and biochemical studies of Tn7, led to speculation that the C-terminal tail of TnsB functions a key mediator of Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 TnsC interactions, and that the specificity of CAST transpososome assembly would be dictated in part by cognate TnsB-TnsC interactions. Reengineering this interaction may enable the TnsAB and donor DNA components from one CAST system to be combined with the QCascade and TnsC components from an orthogonal CAST system. Sixteen chimeric TnsAB constructs were designed in which different lengths of the PseTnsB C-terminus were substituted with corresponding residues from the VchTnsB C- terminus (FIG.5C). These variants were then screened for RNA-guided DNA integration activity in E. coli, in conjunction with VchQCascade and VchTnsC, but with a pDonor containing transposon ends compatible with PseTnsB (FIG.5D). WT PseTnsAB, lacking any chimeric substitutions, showed undetectable activity when combined with VchCAST DNA targeting machinery (FIG. 5E). Remarkably, however, several chimeric TnsAB designs were able to robustly rescue activity, showing up to ~10% integration efficiencies (FIG.5E). These designs, which only reprogrammed 20 – 29 amino acids in the C-terminus of PseTnsAB exhibited graft points between the Pse and VchTnsB sequence in an unstructured region that links the “hook” region of the C-terminus to the remainder of the protein sequence (FIG.5C); furthermore, when comparing this region to solved type V-K complexes, it is located in a similar region as the 52- residue long “flexible linker” that was unresolved. Together, substitutions in this region minimized disruptions to the overall protein fold, while nonetheless providing a chimeric hook that is compatible for cognate interactions with VchTnsC. After designing and cloning similar constructs, integration activity was detected with the converse combination, combining PseQCascade and PseTnsC with a chimeric VchTnsAB design (FIG.21A). Furthermore, when these chimeric designs were applied to a broader range of homologous TnsAB variants and their cognate mini-Tn donor substrates, integration activity was also observed for chimeric designs derived from additional transposon variants, denoted Tn7005 and Tn7015. Intriguingly, TnsAB chimeras derived from Tn7010 and Tn7011 showed no evidence of activity (FIG.21B), suggesting that some CASTs may require targeted screening to identify tolerable chimeric graft points. Next, it was explored whether this engineering approach could also generate compatible chimeras between divergent CRISPR-associated transposons, candidate Type I-F (VchCAST) and Type V-K (ShCAST) systems, each of which comprise distinct transposase architectures and likely arose from unique domestication events. TnsB variants derived from ShCAST exhibited low, but detectable levels of activity as well (FIGS. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 21B-21C). When investigating the transposon insertion orientation preference for type I/V CAST chimeras, it was observed that chimeras in which the TnsB was derived from ShCAST exhibited a “T-LR” insertion preference, as observed in previous ShCAST studies, while type I-F CASTs exhibited a “T-RL” preference. Together, these results reveal that rational, structure-guided engineering of precise regions of CAST systems can overcome the natural orthogonality of diverse systems, enabling novel genome editing designs. Example 2 Design of chimeric TnsAB proteins Using Alpahfold, theoretical structures of PseCAST transposase components TnsA, TnsB, and seven copies of TnsC were generated to predict how these proteins interact (FIG.6A). Although the transposon end nucleic acids were not included, the C-terminal region of TnsB from PseCAST forms a similar hook interaction as previously observed in Type V-K ShCAST systems (Park et al., Nature 613, 775-782 (2023)). Furthermore, when analyzing the sequence conservation of the C-terminus of TnsB homologs at both the sequence and structural levels, a region of extremely low conservation, followed by a short terminal region with moderate conservation was observed (FIGS.6A and 6B). To test if recoding the C-terminal region of TnsB would enable previously incompatible CAST proteins to interact and carry out RNA-guided DNA transposition, 17 TnsB variants were constructed in which different regions of the C-terminus of TnsB from PseCAST were recoded to match the same region of VchCAST TnsB (Table 1, FIG. 7A). The integration efficiencies of these constructs were tested when co-transformed with a PseCAST pDonor and VchCAST proteins. Integration efficiencies for various TnsAB chimeras with VchCAST QCascade and TnsC were measured using the workflow for the integration assay shown in FIG. 5D, in which E. coli BL21 cells are transformed with a pEffector (expressing TniQ-Cascade and TnsC), alongside a pDonor-TnsAB plasmid encoding the mini-transposon and a fused TnsAB fusion protein. These fusion proteins contained candidate chimeric TnsB polypeptide sequences, in which the C-terminus of TnsB from Tn7016 (PseCAST) is replaced with the corresponding sequence from the TnsB homolog from Tn6677 (VchCAST), see FIG. 5E. Integration efficiencies were measured using junction quantitative PCR and normalized against a reference locus in the E. coli genome. The bar graph (FIG.5E) reports the genomic integration activity for chimeric TnsAB fusion proteins, containing chimeras of the TnsB polypeptide at the indicated Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 amino acid junction point (residue listed to the left of the construct cartoon). These chimeras contain the Tn7016 (PseCAST) TnsB polypeptide sequence (dark salmon) from the N-terminus up until the junction point, followed by the Tn6677 (VchCAST) TnsB polypeptide (blue) past the junction point until the C-terminus. Integration efficiencies were measured across two replicate experiments, and positive and negative controls are shown at the bottom, using an all- Tn6677 (VchCAST) TnsB polypeptide, which is not a chimera, or an all-Tn7016 (PseCAST) TnsB polypeptide, which cannot function with the TniQ-Cascade and TnsC components from the VchCAST system. Of note, all chimeric designs were tested with a mini-transposon containing Tn7016 (PseCAST)-specific transposon left end and right end sequences. A particular region of the C-terminus was particularly amenable to generating functional, chimeric TnsB proteins. Three successful graft points were along the highly variable “linker region” of the C-terminus, connecting the “hook” region of TnsB with the remainder of the protein (FIG.7B). In some embodiments, chimeric TnsB variants may be designed in which multiple regions of TnsB are derived from an orthogonal homolog. Chimeric TnsB variants were also derived from other CAST systems beyond Tn6677 (VchCAST) and Tn7016 (PseCAST) (Table 1). These chimeric TnsB variants may be used in other cells, cell types, or organisms, such as eukaryotic cells, in order to design optimal DNA targeting and transposition systems. TnsB chimeras may enable Type I-F and Type V-K CASTs to function together, enabling novel interactions across divergent CAST systems and types. Example 3 Design of chimeric TnsC proteins TnsC may also harbor a C-terminal region that performs sequence-specific interactions with TnsB that enables homolog-specific interactions. Given the unstructured region of recently solved Type I-F TnsC structures (Hoffman, Kim, Beh et al., Nature 609, 384-393 (2022)) (FIG. 8A), combined with analysis of TnsC sequence conservation across homologs (FIG.8B), several TnsC variants were designed to mediate orthogonal TnsB-TnsC interactions (Table 3). These TnsC chimeras may be combined with chimeric TnsAB proteins, or chimeric proteins of other CAST subunits, such as TniQ. Chimeras may also be derived from additional CAST systems beyond Tn6677 and Tn7016. Example 4 Design of chimeric TniQ proteins Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 An additional opportunity to generate chimeric CAST systems lies at the Cascade:TniQ:TnsC interface. As TniQ bridges the gap between the DNA targeting module (CRISPR-Cascade) and the transposase module (TnsABC), it is a prime target to generate chimeric CAST systems with orthogonal Cascade and TnsABC components. There have been several structures of Type I-F TniQ-Cascade complexes (Halpin-Healy et al., Nature 577, 271- 274 (2019), Park et al., Mol Cell 83, 1827-1838 (2023)), a Type I-B TnsC-TniQ-Cascade complex (Wang et al., Cell 186, 4204-4215 (2023)), and several Type V-K CAST structures solved (Schmitz et al., Cell 185, 4999-5010 (2022), Park et al., Nature 613, 775-782 (2023)). Given that both Type I-B and Type V-K employ a singular TniQ that interacts with TnsC, the TniQ dimer may present in Type I-F CAST systems is structurally relevant, but that only one monomer of TniQ may functionally interact with TnsC to recruit the TnsAB transposase machinery. Thus, with small, N-terminal peptide modifications in one of the two TniQ monomers, divergent Cascade and TnsABC modules can function to coordinate RNA-guided DNA transposition (Figure 10A). Two TniQ monomers were fused together to be translated as a single-chain dimer and engineered TniQ chimeras in which only one N-terminus of TniQ dimer is recoded to an orthogonal system (Table 2). These variants, alongside WT TniQ homodimers from PseCAST and VchCAST, were tested to determine if any chimeras were functional for RNA-guided transposition (Figure 10B); the shortest modification tested, where only 43 amino acids were grafted from the N-terminus of PseTniQ to VchTniQ, showed clear evidence of targeted DNA integration (Figure 10B). Modification of only TniQ₁ was tolerated, suggesting that the TniQ₂ N-terminus in this design may interact with Cascade, thus requiring sequence derived from VchCAST. In some embodiments, chimeric TniQ sequences are derived from additional CAST systems, or unrelated transposase systems to recruit divergent transposase proteins. In some embodiments, these chimeric TniQs can be expressed in eukaryotic systems. In some embodiments, hundreds of TniQ variants can be generated and screened at library-scale. Materials and Methods Protein purification. PseCAST QCascade was overexpressed and purified as previously described (Halpin-Healy, T. S., et al. Nature 577, 271–274 (2020)) with the following modifications. All proteins were codon optimized, placed downstream of consensus RBS Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 sequences, and TniQ contained an N-terminal 10xHis-TEV tag. The minimal CRISPR-array was placed upstream of Cas7 and contained a 32 bp spacer targeting the AAVS1 locus (see Table 4 for detailed plasmid sequences). After overnight expression at .5 mM IPTG, cell pellets were resuspended in QCascade lysis buffer (50 mM Tris-Cl, pH 7.5, 700 mM NaCl, 0.5 mM PMSF, EDTA-free Protease Inhibitor Cocktail tablets (Roche), 1 mM dithiothreitol (DTT), 5% glycerol) and lysed by sonication. Lysates were clarified by centrifugation at 15,000g for 30 min at 4 °C. Initial purification was performed by immobilized metal-ion affinity chromatography with NiNTA Agarose (Qiagen) using NiNTA wash buffer (50 mM Tris-Cl, pH 7.5, 700 mM NaCl, 10 mM imidazole, 1 mM DTT, 5% glycerol) and NiNTA elution buffer (50 mM Tris-Cl pH 7.5, 700 mM NaCl, 300 mM imidazole, 1 mM DTT, 5% glycerol). The sample was further purified by size exclusion chromatography over a Superose 6 Increase 10/300 column (GE Healthcare) equilibrated with QCascade storage buffer (20 mM Tris-Cl, pH 7.5, 700 mM NaCl, 1 mM DTT, 5% glycerol). Fractions were pooled, concentrated, snap frozen in liquid nitrogen, and stored at k1) d7' E9F KTMI^IOM _I[ VW\ XMZNWZUML' CryoEM structure determination. Purified PseCAST QCascade was incubated with 5 times excess of target DNA for 10 minutes at room temperature. The complex, in the 2-4 µM range, was initially imaged in a Talos L120C (Thermo Fisher) electron microscope equipped with a LaB6 electron source and a Ceta-M camera. Negative staining experiments were carried out using uranyl-formate solution at 0.75% (w/v) in water. CF-400 (EMS) continuous carbon grids were activated for 30 seconds using an Ar/O₂ gas mix plasma at 25 W using a Solarus2 plasma cleaner (Gatan). Immediately after plasma activation, 3 µL of the PseCAST QCascade/DNA complex at concentrations of 1, 2 and 4 µM were applied to the activated grids. After 1 minute incubation, the excess solution was gently blotted away and 3 µL of 0.75% uranyl-formate solution was added for an additional 1-minute incubation. Excess staining solution was blotted away, and the grids were left on the bench drying for 5 minutes. Grid screening revealed well stained, homogeneous and dispersed particles with a circular shape compatible in dimensions and shape with the estimated molecular size of the complex as well as showing similarities with previously reported images of other Cascade complexes (FIG. 12A). The 1 µM range concentration grid was chosen for manual collection of 10 negative staining QUIOM[ #XQ`MT [QbM +'. u(XQ`MT% * [MKWVL M`XW[]ZM% &+ \W &, eU LMNWK][$ NWZ M`XTWZI\WZa KTI[[&+8 analysis in Relion4. The resulting negative staining C2D averages confirmed the homogeneity of Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 the sample and its potential for high-resolution (FIG.12A, left). Next, the behavior of the complex was explored under cryogenic conditions using the negative stain conditions as a reference starting point. UltraAu foil 1.2/1.3 “Gold” grids (Quantifoil) were vitrified using a FQ\ZW6W\ ?IZS <F #EPMZUW :Q[PMZ$ [M\ ]X \W *))" P]UQLQ\a IVL - t' EPM [IUXTM KWVKMV\ZI\QWV was in the 2 and 4 µM range. Grids were plasma cleaned with the same protocol described for the negative staining grids and after application of 3 µL solution, the grids were blotted and plunged frozen in liquid ethane. Vitrobot settings were: blot force -5, drain and waiting time 0 with blotting times varying between 2.5 to 3.5 seconds. Following these parameters, 8 grids were frozen, 4 grids at 2 µM concentration and 4 grids at 4 µM concentration.2 grids, one at 2 µM and another at 4 µM concentration were transferred to a cooled 910 side entry holder (Gatan) for screening under cryogenic conditions in the same Talos L120C microscope used for negative staining using similar imaging conditions. Both grids showed good ice distribution with the 2 µM grid showing better particle distribution and contrast in ice. Using SerialEM, 10 images were collected with similar settings as in negative staining experiments for exploratory reference-free C2D analysis in Relion4 under cryogenic conditions (FIG. 12A, middle). The resulting C2D averages were promising, with distinctive and multiple views of the complex. The grid was recovered and stored for high resolution data collection in a Titan Krios G3i electron microscope equipped with a BioQuantum/K3 energy filter and direct detection. High resolution data was KWTTMK\ML I\ PQOP UIOVQNQKI\QWV _Q\P +` PIZL_IZM JQVVQVO QV \PM =, LM\MK\WZ #)'/-1. u(XQ`MT pixel size after binning) at a fluence of ~20e-/pixel/second and 1 second exposure time for a total dose of ~50 e-(u². Defocus range was adjusted to variate between -0.8 to -2 µm and the total number of K3 fractions was adjusted to 50. 24 hours collection on the recovered grid yielded ~22,000 images which were on-the-fly motion corrected in Relion4 with ctf estimation in ctffind4. Image processing was integrally done in Relion 4 and cryoDRGN. First, 100 images were manually selected for Laplacian picking, which yielded ~4,000 particles that were normalized and extracted with 8 times binning. Fast C2D analysis using the VDAM algorithm generated C2D averages in multiple orientations that were selected and used as training set for Topaz, used through the Relion wrapper. Using the optimized trained model from Topaz, the full dataset of ~22,000 images yielded ~1.5 million particles that after two C2D steps using T parameters of 3 and then 6 was reduced to ~667,000 particles. ArnA contamination accounted for the bulk of the eliminated particles. Next, the reduced dataset was refined using a filtered Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 map of VchCAST QCascade as reference. Alignments were not performed with this initial classification (K20, tau fudge T=6). Multiple classes were identified with damaged or poorly aligned particles, a class without the TniQ dimer, and a dominating class with better features. A re-extraction step was then performed with the recenter option activated and at 4x binning (2.594 u(XQ`MT$' 5N\MZ [MTMK\QWV WN +8 KTI[[ I^MZIOM[ [PW_QVO [MKWVLIZa [\Z]K\]ZM NMI\]ZM[% IV IJ&QVQ\QW 3D model was reconstructed using the Stochastic Gradient Descent (SGD) algorithm with all selected particles from the class 2D job (K4, tau fudge T=3). A second 3D refinement produced a KWV[MV[][ ZMNQVMUMV\ QV \PM . u ZIVOM \PI\ ]XWV QV[XMK\QWV [PW_ML KTMIZ [MKWVLIZa NMI\]ZM[ IVL substantial heterogeneity at the PAM distal region hosting the TniQ dimer. A soft-mask (10 pixel extension, 8 pixel soft edge and initial threshold of 0.002) was used for 3D classification without alignment using 20 classes and T parameters 3, 6 and 8. A minor population (~8 % of the particles) of Cascade without TniQ was identified and removed from the dataset as well as poorly aligned or damaged particles, reducing the total dataset to ~128,000 particles. Re- ZMNQVMUMV\ WN \PQ[ LI\I[M\ IN\MZ ZM&M`\ZIK\QWV \W JQVVQVO + #c*'+ u(XQ`MT$ XZWL]KML I []J ,u UIX but exacerbated heterogeneity of the TniQ dimer region was evident. Using focused classification of this region of the map produced multiple classes without clear discrete states suggesting continuous heterogeneity. Previous to applying a multibody approach, the ~128,000 particle dataset was re-refined after refining the ctf parameters (defocus values per particle and astigmatism per micrograph) followed by bayesian particle polishing for signal decay and local particle movement correction. three rigid body groups were defined via soft masking (6 pixel mask extension, 6 pixel soft edge decay, initial threshold 0.002): the first body included Cas8, and the first Cas7 monomer (Cas7.1), the second body contained Cas7 monomers 2 to 5 and the third body included the TniQ dimer, Cas6, Cas7.6 and the crRNA 3’-proximal hairpin. Residual rotation priors were defined to 10 degrees with translation offset of 2 pixels. Two wide masks were defined: one (body 1) covering the best part of the map and including Cas8, the first five Cas7 proteins, and surrounding densities including the corresponding sections of the crRNA- DNA heteroduplex; and a second soft mask (body 2) covering Cas7.6, Cas6 and the TniQ dimer. ?]T\QJWLa ZMNQVMUMV\ XZWL]KML UIX[ _Q\P M`KMX\QWVIT Y]ITQ\a NWZ MIKP JWLa% _Q\P KTMIZ []J ,u features for the Cas8 and the Cas7 regions. The maps for the PAM distal body including the TniQ dimer, improved substantially but residual heterogeneity remained specially at the distal end of the TniQ dimer. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 ModelAngelo was used for initial model building using the improved maps from the multibody analysis. With default options and using sequence information from the cloned constructs ModelAngelo built correctly approximately 90% of the residues. Manual inspection of the built model corrected limited errors and completed areas where the resolution did not allow accurate placement of side chains. The built models were refined against the multibody maps independently first with phenix refine (secondary structure restrain activated) and then with Refmac5, adjusting the experimental/ideal geometry weights manually to avoid overfitting. CryoDRGN analysis was performed with the final set of ~128,000 particles used for multibody analysis in Relion. This set of particles was re-extracted to a box size of 128 pixels and an initial training in 1 dimension (Zdim=1) was performed. After assessing the homogeneity of this set of particles 3 different training were performed with 2, 4 and 8 dimensions (Zdim=2, 4 and 8). Principal component analysis (PCA), UMAP and K-means clustering dimensionality reduction techniques were used to explore the derived latent spaces, producing similar results irrespective of the Zdim used. A final training was performed with particle re-extracted to 256 pixels size and Zdim 2 and 8. Exploration of the latent space derived from these training revealed multiple conformation of the TniQ dimer as shown in FIG.13. Plasmid construction Bacterial expression plasmids for PseCAST QCascade were E. coli codon optimized and synthesized by GenScript. For human cell transfections, genetic components encoding PseCAST CAST proteins were human codon optimized, synthesized by GenScript, and cloned in pcDNA3.1 expression vectors. All CAST constructs were cloned into plasmids using a combination of restriction digestion, ligations, Gibson assembly, and Golden Gate assembly. All PCR fragments for cloning were generated in house using Q5 DNA Polymerase (New England Biolabs (NEB)) and gel purified using a Qiagen Gel Extraction Kit. To clone the 4N PAM library used for HEK293T episomal integration assays, two overlapping oligos containing ‘NNNN’ were phosphorylated with T4 PNK (NEB) and PaJZQLQbML I\ 2.j7 NWZ + UQV]\M[ JMNWZM KWWTQVO \W ZWWU \MUXMZI\]ZM' EPM ZM[]T\QVO WTQOWL]XTM` _I[ TQOI\ML QV\W I \IZOM\ XTI[UQL ^MK\WZ XZMLQOM[\ML _Q\P 6[U6< #..j7 NWZ + PW]Z[$ ][QVO E- DNA ligase (NEB). Cloning reactions were transformed into chemically competent NEB Turbo E. coli, plated on agar plates with the appropriate antibiotic to grow overnight, and inoculated in 5 uL LB media and antibiotic for approximately 7 hours. Colony counting was then performed to ensure sufficient library diversity. Plasmids were then extracted using Qiagen Miniprep columns Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 and verified by a combination of Sanger sequencing (Azenta/Genewiz) and whole-plasmid nanopore sequencing (Plasmidsaurus). Mammalian cell culture and transfections HEK293T cells were cultured at 37 qC and 5% CO2 and maintained in DMEM media with 10% FBS and 100 U/ml of penicillin and streptomycin (Thermo Fisher Scientific).24 hours before transfection, a 48-well plate was coated with poly-D-lysine (Thermo Fisher Scientific) and seeded with 10,000 cells per well. Cells were \ZIV[NMK\ML _Q\P 8@5 UQ`\]ZM[ IVL * oT WN >QXWNMK\IUQVM +))) #EPMZUW :Q[PMZ DKQMV\QNQK$ XMZ the manufacturer’s instructions. Transcriptional activation and integration assays were performed as previously described Lampe, G. D. et al. Nat. Biotechnol. 42, 87–98 (2023). For plasmid- based PAM library assays, cells were co-transfected with the following PseCAST CAST plasmids: 200ng pTnsAB, 50ng pTnsC, 75ng pQCascade, 100ng pCRISPR (crRNA), 200ng pDonor, and 100ng pTarget (4N PAM library). Cells were harvested 4 days after transfection. Analysis of HEK293T integration assays Genomic integration assays were analyzed as previously described. In brief, 5µL of genomic lysate (10% of total lysate volume) was used for 2 rounds of PCR. In the first PCR, a forward primer was used that anneals to the AAVS1 locus, and a reverse primer was used that anneals to both the AAVS1 locus and a primer binding site in the donor DNA. These oligos included 5’ overhangs encoding read 1 and read 2 Illumina adapters. In the second PCR, “universal” primers were used which anneal to the read 1 and read 2 sequences, and appended unique index sequences and the remaining Illumina adapter sequences for next generation sequencing. Samples were then pooled, gel purified, and sequenced on a NextSeq 500/550 with at least 75 cycles in read 1. The relative abundance of reads that contain a PseCAST transposon end sequence (representing an integration read) vs downstream AAVS1 sequence (unintegrated read) was calculated. For the episomal PAM library assay, samples were prepared as above except a different forward oligo was used that anneals directly upstream of the degenerate PAM library in PCR 1, such that both the PAM sequence and the presence of the transposon end sequence would be captured with the forward read. PCR 1 cycles were reduced to 15 cycles. After Illumina sequencing, reads were filtered to have a transposon end sequence, thus representing a PAM library member which was successfully targeted by PseCAST for transposition. The input library was sequenced as well to calculate enrichment and depletion scores. Library members were then ranked by their enrichment values (proportion of output library / proportion of input library). The Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 top 10% of library members were used to generate a consensus WebLogo (Version 2.8.2, 2005- 09-08, weblogo.berkeley.edu) for the PAM preference of each Cas8 variant. All library members and their associated enrichment values were used to generate PAM wheels using Krona. E. coli repression and integration assays. E. coli transcriptional repression assays were performed as previously described with some minor modifications. In brief, an E. coli strain expressing mRFP from the chromosome, a gift from L. S. Qi, was transformed with pQCascade. Due to toxicity with strong expression of PseCAST Qcascade utilizing a strong J23119 promoter, a weaker J23101 promoter was used for all pQCascade constructs. crRNA sequences were designed to target the template strand of mRFP proximal to the 5’ end of the coding region (60 bp downstream of the mRFP start codon). For each unique transformation, two replicates were performed and relative mRFP repression was analyzed as previously described (Hoffmann, F. T. et al. Nature 609, 384–393 (2022)). Integration assays were performed as previously described (Klompe, S. E., et al., Nature 571, 219–225 (2019) and Hoffmann, F. T. et al. Nature 609, 384–393 (2022)) with the following modifications. Although J23101 promoters were used for QCascade, J23119 promoters were still used for constitutive expression of all TnsABC cassettes, as there was no observed toxicity. In brief, TnsABC expression vectors harboring donor DNA (pDonor- TnsABC) encoded a tnsA-tnsB-tnsC operon downstream of a strong constitutive promoter (J23119), as well as a mini-transposon donor DNA of 0.9 and 1.2 kb in length for VchCAST and PseCAST, respectively, all on a pUC19 backbone. Strains harboring medium-strength J23101 promoter-controlled pQCascade constructs were first made chemically competent, followed by duplicate transformations with pDonor-TnsABC and lysate generation for qPCR after an 18h incubation at 37°C. Lysates were analyzed via qPCR as previously performed. Data availability Cryo-EM maps and models will be deposited on EMDB and PDB. Sequences Table 1. Chimeric TnsB/TnsAB protein sequences

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Table 2. Chimeric TniQ dimer sequences

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Table 3. Chimeric TnsC protein sequences Description Protein Sequence SEQ ID NO

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Table 4. Plasmid Sequences

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The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 C^LAIMS What is claimed is: 1. A system comprising an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated transposon (CAST) system or one or more nucleic acids encoding the engineered CAST system, wherein the engineered CAST system comprises: a) one or more Cas proteins selected from: Cas5, Cas6, Cas7, Cas8, Cas12, and combinations thereof; and b) one or more transposon-associated proteins selected from TnsA, TnsB, TnsC, TnsD, TniQ, and combinations thereof, wherein at least one of TnsB, TnsC, or TniQ is a chimeric protein comprising amino acid sequences derived from respective TnsB, TnsC, or TniQ proteins, or homolog thereof, of at least two CAST systems, and, optionally c) at least one guide RNA (gRNA) complementary to at least a portion of a target nucleic acid sequence, or at least one nucleic acid encoding thereof. 2. The system of claim 1, comprising a chimeric TnsB, wherein the chimeric TnsB has a C- terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a first CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of a second at least one second CAST system. 3. The system of claim 2, wherein the C-terminal domain comprises the C-terminal hook and at least a portion of C-terminal linker region of TnsB. 4. The system of claim 2 or 3, further comprising: a TnsA protein derived from the second CAST system; a TnsC protein having an amino acid sequence fully or partially derived a TnsC protein or homolog thereof from the first CAST system; and/or a TniQ protein derived from the first CAST system. 5. The system of claim 4, wherein the TnsC protein is a chimeric protein comprising a C- terminal region having an amino acid sequence derived from the first CAST system. Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 6. The system of claim 1, comprising a chimeric TnsC, wherein the chimeric TnsC has a C- terminal domain comprising an amino acid sequence derived from a TnsC protein, or homolog thereof, of a second CAST system and N-terminal domain derived from a TnsC protein, or homolog thereof, of a first CAST system. 7. The system of claim 6, further comprising: a TnsA and a TnsB protein derived from the second CAST system or a chimeric TnsB having a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, of the second CAST system and an N-terminal domain comprising an amino acid sequence derived from a TnsB protein, or homolog thereof, from the first CAST system or a third CAST system, and a TnsA protein derived from same CAST system as the N-terminal domain of the TnsB protein; and/or a TniQ is derived from the CAST first system. 8. The system of claim 1, comprising a chimeric TniQ having an N-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, of a second CAST system and C-terminal domain comprising an amino acid sequence derived from a TniQ protein, or homolog thereof, derived from the first CAST system. 9. The system of claim 8, wherein the chimeric TniQ is fused to a second TniQ derived from the first CAST system. 10. The system of claim 8 or 9, further comprising: a TnsC protein fully or partially derived from the second CAST system, and, optionally, a TnsA and TnsB protein derived from the second CAST system. 11. The system of claim 10, wherein the TnsC protein is a chimeric protein comprising a C- terminal region having an amino acid sequence derived from a TnsC protein, or homolog thereof, of a third CAST system, and the system further comprises: a TnsA and TnsB protein derived from the third CAST system; or Attny Docket No. COLUM-42515.601 Client Ref No. CU24119 a chimeric TnsB protein having a C-terminal domain comprising an amino acid sequence derived from a TnsB protein, or a homolog thereof, of the third CAST system, and a TnsA protein derived from same CAST system as the N-terminal domain of the chimeric TnsB protein. 12. The system of any of claims 2-11, wherein the one or more Cas proteins are derived from the first CAST system. 13. The system of any of claims 1-12, wherein TnsB is a chimeric protein comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 33-35 and 257-288; TnsC is a chimeric protein comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 48-53; and/or TniQ is a chimeric protein comprising comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 36-47. 14. A method for modifying a nucleic acid comprising contacting a target nucleic acid sequence with a system of any of claims 1-13 or one or more components thereof. 15. A cell comprising a system of any of claims 1-13 or one or more components thereof.