Subject terms: Genetic engineering, Genetic techniques, Neurospora crassa

Design of the CRISPR/Cas9 system forNeurospora crassa

In the presented work we use a step-wise introduction of Cas9 and the gRNA into the fungus. Thecas9 sequence, optimized according to human codon usage, was amplified from the pSpCas9n(BB)-2A-GFP (PX461) vector containing the nucleoplasmin nuclear localization signal (NLS) at its 3′ end. This sequence was cloned into a fungal transformation vector, which was integrated at thehis-3-locus of theN.crassa strain #6103 (see Fig. 1a). The expression ofcas9 is under the control of theccg1-promotor ofN.crassa. The resulting strain was named NcCas9SG.

The successful integration of thecas9 sequence was verified by PCR using oligonucleotides SG3450 and SR2883 for three transformants (see Fig. 1b and c) as well as by Southern blot (see Supplementary Fig.S3). Expression on the transcript level was confirmed for transformants number two and three, both stemming from a microconidia passage of the same independent transformant, by RT-PCR (see Fig. 1d). Transformant number three was chosen for all following experiments. To analyze the expression of Cas9 on the protein level a Western blot using an anti-Cas9 antibody was done, confirming that the Cas9 protein is synthesized in the transformant (see Fig. 1e). To check for undesired adverse effects on growth behavior or conidiation we phenotypically compared NcCas9SG to the host strain #6103 (see Fig. 2a and b). Aerial hyphae growth of NcCas9SG was lower compared to #6103. When grown for 7 days at 25 °C in a glass tube aerial hyphae of strain #6103 grew up to 5.7 cm ± 0.29, carrying spores up to a height of 4.57 cm ± 0.21 in the tube, while the aerial hyphae of NcCas9SG only reached a height of 3.95 cm ± 0.71, carrying spores up to a height of 2.67 cm ± 0.19. The lateral growth behavior on a petri dish containing VMM + S agar on the other hand did not show any significant differences. Also, the number of produced spores did not differ significantly with 5.59 × 10⁸ spores/ml ± 0.55 × 10⁸ for #6103 and 5.38 × 10⁸ spores/ml ± 0.99 × 10⁸ for NcCas9SG. Therefore, we conclude, that the expression of Cas9 does not significantly limit the growth of NcCas9SG, thus making it useful for a mutagenesis system.

After confirming Cas9 expression in NcCas9SG, macroconidia were isolated from the strain and used for the transformation of gRNA. For the introduction of the gRNA into the cell, synthetic crRNA/tracrRNA duplexes instead of plasmid DNA were transformed into macroconidia via electroporation. Thereby avoiding the problems that can occur when the gRNA has to be transcribed from the DNA template¹⁷.

Editing of thecsr-1 gene with CRISPR/Cas9

To test our CRISPR/Cas9 system for its functionality, a selectable marker gene was chosen as a target for editing. Thecyclosporinresistant-1 (csr-1) gene (NCU00726) codes for a peptidyl-prolyl cis–trans isomerase. Mutations in thecsr-1 gene lead to resistance against cyclosporin A (CsA)¹⁸, allowing for an easy identification of putatively mutatedN.crassa colonies on CsA containing medium. Two gRNAs, designated gRNA-c1 and gRNA-c2 (see Fig. 3a and b), targeting different regions of thecsr-1 sequence were used. gRNA-c1 targets position 126–148 of exon 3 and gRNA-c2 targets position 66–88 of exon 4.

When plating transformed macroconidia on VMM + SGF without CsA, an editing efficiency of 7.35% ± 1.38 for gRNA-c1 and 11.89% ± 4.78 for gRNA-c2 was determined. When plating transformed macroconidia on selection media (VMM + SGF + CsA) 100% of the colonies obtained (8/8 colonies, using gRNA-c1 and 24/24 colonies, using gRNA-c2) showed a mutation at the predicted site. 1, 2 or 3 µl of 100 µM gRNA-c2 were used to transform macroconidia. No significant difference in the number of obtained colonies was observed on selection media (see Supplementary Fig.S4). Using 1 µl of gRNA resulted in 223 colonies/plate ± 24, 2 µl resulted in 246.5 colonies/plate ± 2.1, and 3 µl resulted in 209 colonies/plate ± 4.2. Hence, 2 µl of 100 µM of gRNA was considered to saturate the system. The mutations at the predicted site were determined by amplification of the target region via PCR and sequencing. Figure 3c shows examples of different types of mutations at the predicted sites for the two gRNAs. All observed types of mutations are summarized in Table1. These findings are direct proof of the suitability of our newly designedN.crassa CRISPR/Cas9 system.

Editing of a non-selectable gene with CRIPSR/Cas9

Next, a non-selectable gene encoding N-acylethanolamine amidohydrolase-2 (naa-2; NCU04092) involved in the auxin biosynthesis pathway inN.crassa¹⁹, was targeted. Again, two different gRNAs (see Fig. 4a and b) were designed: gRNA-n1 (targeting sequence position 211–233 of exon 3) and gRNA-n2 (targeting sequence position 683–705 of exon 3). After the transformation of NcCas9SG macroconidia with the respective gRNA, the conidia were transferred on VMM + SGF plates and 19 randomly picked colonies for each gRNA were screened for a putative mutation. In case of gRNA-n1 5.26% (1/19 colonies) of the analyzed colonies carried a mutation at the predicted site while no mutations (0/19 colonies) were found for gRNA-n2. These findings show, that mutations may be found even without a selective marker, albeit it is laborious and time consuming.

Editing of more than one gene at a time using CRIPSR/Cas9

Typically, the CRISPR/Cas9 system allows for editing of multiple genes at the same time by introducing several gRNAs to a cell that target different sequences at once. This technique was shown to work in several filamentous fungi such asAspergillusfumigatus²⁰,A.niger²¹ andBeauveriabassiana²². We set out to use this technique inN.crassa, using a combination of two gRNAs at the same time (gRNA-c2/gRNA-n1 and gRNA-c2/gRNA-n2) to transform NcCas9SG macroconidia. These gRNA combinations seemed promising to minimize the expenditure of time when screening for the mutation since one gRNA targeted thecsr-1 gene, allowing the selection for a CRISPR event via CsA. The combination of gRNA-c2 with either gRNA-n1 or gRNA-n2 increased the efficiency of observingnaa-2 mutations significantly (summarized in Table2). In case of gRNA-n1 55.5% (11/18) of the colonies carried a mutation at the predicted site. This is a tenfold increase compared to the previous transformation without the gRNA-c2 where only 5.26% of the analyzed colonies carried a mutation. The same increase was observed for gRNA-n2 where 16.7% (1/6) colonies carried a mutation at the predicted site. Figure 4c shows examples of different types of mutations at the predicted sites in thenaa-2 sequence for both gRNAs. All observed types of mutations are summarized in Table3. Analysis of the target site for gRNA-c2 showed again, that 100% of the colonies carried the mutation at the predicted site, meaning that 50% (12/24) of the analyzed colonies carried a mutation at both targeted genes. Therefore, by using this approach, it becomes much easier to detect the individuals carrying the desired mutation.

Table 4.

Strains

The histidine auxotrophicN.crassa strain FGSC #6103 (his-3 (Y234M723) mat A) provided by the Fungal Genetics Stock Center (FGSC; Kansas City, MO, USA) was used for transformation with vector pSG897.

The bacterial strainE.coli XL1-Blue (recA1,endA1,gyrA96,thi-1,hsdR17,supE44,relA1, andlacF’proABlacIqZΔM15Tn10(Tetr)) (200249, Stratagene) was used for the propagation of vector constructs.

Media and growth conditions

For the cultivation ofN.crassa, Vogel’s minimal medium³⁶ with 2% sucrose (VMM + S) (supplemented with 0.02% histidine in case of thehis-3N.crassa strain FGSC #6103) was used and for maintaining single colonies on plates Vogel’s minimal medium with 1% sorbose, 0.05% glucose, and 0.05% fructose (VMM + SGF) was used.N.crassa microconidia production was induced on a synthetic cross medium (SC) supplemented with 0.06% iodoacetate as described in³⁷. Fungi were cultivated in a climate chamber at 25 °C under long-day conditions.

TheE.coli strains were cultivated in a Luria Broth culture medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, pH 7.5) supplemented with the required antibiotics at 37 °C and 250 rpm.

Generation ofN. crassa Cas9 strain NcCas9SG

The strain NcCas9SG was created by transformingN.crassa strain FGSC #6103 (his-3 (Y234M723) mat A) with the vector construct pSG897 carrying theSpcas9 sequence with a 3′ nucleoplasmin NLS. TheSpcas9 sequence was amplified from the plasmid pSPCas9(BB)-2A-GFP (Addgene plasmid ID: 48138) using the oligonucleotides SG3449 and SG3444 adding anAscI andSwaI recognition site to the PCR product. Blunt-end PCR products were subcloned using a CloneJet PCR cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation. After hydrolysis of the subcloned product and the vector pMM536³⁸ with the corresponding restriction endonucleases from NEB (Ipswich, MA, USA), according to the standard protocol, gel elution was performed with NucleoSpin Gel and PCR Clean-Up Kit (Macherey–Nagel), according to the manufacturer’s instructions. Followed by ligation with T4 DNA-ligase by NEB (Ipswich, MA, USA) according to the standard protocol, creating vector pSG897. The transformation process was carried out as described in³⁹ and the transformants were selected using VMM + SGF plates without histidine.

gRNA design and synthesis

The gRNAs were designed using the CHOPCHOP v3 website (https://chopchop.cbu.uib.no)⁴⁰. For gRNA synthesis two RNA oligonucleotides (crRNA and tracrRNA) synthesized by IDT (integrated DNA technologies, Coralville, Iowa, USA) were ordered (https://eu.idtdna.com/pages). For synthesis of the gRNA equimolar amounts of crRNA and tracrRNA were combined in the IDT-Duplex buffer followed by a 5 min incubation at 95 °C, yielding 100 µM gRNA. After cooling the formed gRNA to room temperature, it can be used for electroporation or stored at − 20 °C.

Transformation of gRNA

TheN.crassa transformation method for plasmids as described in³⁹ was adapted to transform macroconidia of NcCas9SG with the synthesized gRNA(s). 40 µl of macroconidia (2.5 × 10⁹ conidia/ml) were mixed with 2 µl of 100 µM gRNA and kept on ice for 5 min. After transferring the mixture to a cuvette, electroporation was performed using the following parameter: 25 µF, 600 Ω and 1.5 kV. After the pulse the macroconidia were mixed with 1 ml of 1 M sorbitol and incubated at room temperature for 10 min. Without selection pressure the mixture was diluted 1:10,000 or 1:1000 and 100 µl of the dilution were mixed with 8 ml VMM + SGF top-agar and plated on VMM + SGF plates. With selection pressure undiluted 200 µl of the mixture were mixed with top agar. When targeting thecsr-1 gene 5 µg/ml cyclosporine-A was added to the media for selection. Plates were incubated in a climate chamber at 25 °C under long-day conditions for 5–7 days and grown colonies were further analyzed for mutations at the target sites.

DNA and RNA isolation

DNA isolation was performed either from 3 days old mycelium grown at 25 °C in liquid VMM + S by using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research Europe, Freiburg, Germany) according to the manufacturer’s instructions or by lysing macroconidia in 50 µl TE buffer for 10 min at 99 °C, followed by a 10 min incubation on ice and a centrifugation step (15,000 × g for 5 min). 40 µl of the supernatant were then transferred to a clean tube and used as PCR template.

Bacterial plasmid DNA was isolated using the NucleoSpin Plasmid Easypure Kit, according to the manufacturer’s instructions (Macherey–Nagel, Düren, Germany).

RNA isolation from 3–4 day old mycelium was performed with peqGold TriFAST (Peqlab, Erlangen, Germany), according to the manufacturer’s instructions, followed by a DNase treatment step using DNase I (ThermoFisher Scientific) according to the standard protocol.

PCR, RT-PCR and sanger sequencing

PCR was performed as previously described in³⁸ using the Taq polymerase from NEB (Ipswich, MA, USA).

For analyzing transcripts, RT-PCR was performed using the OneTaq One-Step RT-PCR Kit from NEB (Ipswich, MA, USA) according to the manufacturer’s instructions.

Sanger sequencing was performed by the Institute of Clinical Molecular Biology (IKMB) in Kiel.

The oligonucleotides used in our study were synthesized by Eurofins MWG Operon (Ebersberg, Germany) and are listed in Table5.

Southern blot

N.crassa was cultivated in 50 ml VMM + S liquid medium for 3 days at 25 °C (80 rpm), for isolating DNA. After filtering with sterile mull and washing the mycelium with water it was ground in liquid nitrogen. The powder was resuspended in 50% (v/v) DNA lysis buffer (10 mM Tris–HCl pH 8.0; 1 mM EDTA; 100 mM NaCl; 2% (v/v) SDS) followed by phenol extraction with 1 vol phenol. The aqueous phase was extracted twice with 1 vol phenol–chloroform–isoamyl alcohol (25:24:1) and 100 µg of RNase A was added to the aqueous phase, followed by additional phenol extraction and ethanol precipitation. The resulting DNA pellets were resuspended in TE buffer (20 mM Tris–HCl pH 7.5, 0.1 mM EDTA). The isolated DNA was used for southern blotting as previously described⁴¹. The probes were digoxigenin-labeled generated with the PCR digoxigenin labeling Mix (Roche, Mannheim, Germany), following the manufacturer’s protocol. Oligonucleotides used for the probes are found in Table5.

Protein analysis

For isolating proteins,N.crassa was cultivated in 50 ml VMM + S liquid medium for 3 days at 25 °C (80 rpm). After filtering with sterile mull and washing the mycelium with water it was ground in liquid nitrogen. The powder was resuspended in 50% (v/v) protein extraction buffer (20 mM HEPES–KOH pH 7.9; 100 mM KCl; 20% (v/v) glycerol; 0.2 mM EDTA and 10 µl/ml buffer of protease inhibitor cocktail (P9599) from SIGMA-Aldrich (St. Louis, MO, USA)) and incubated on ice for 10 min. To remove the cell debris, the samples were centrifuged at 12,000 × g for 20 min (4 °C). The supernatant was used for SDS-PAGE and Western-Blot.

Total proteins were separated on 10% SDS–PAGE gels (TGX stain-free FastCast Acrylamide Kit 10%; Bio-Rad, Hercules, CA, USA) and electro transferred to PVDF membrane using the Trans-Blot Turbo™ Transfer System according to the manufacturer’s recommendations (Bio-Rad, Hercules, CA, USA). Protein immunodetection was performed according to the standard procedure using the primary antibody Cas9 (7A9-3A3) Mouse mAb (1:3000 dilution; Cell Signaling, Danvers, MA, USA) and a secondary anti-mouse IgG horseradish peroxidase-conjugated antibody (1:5000 dilution, Cell Signaling, Danvers, MA, USA). The membrane was cut before antibody detection. Signals were detected by chemiluminescence using the GelDoc Go gel imaging system (BioRad, Hercules, CA, USA).

• 1.Roche, C. M., Loros, J. J., McCluskey, K. & Louise Glass, N.Neurosporacrassa: Looking back and looking forward at a model microbe.Am. J. Bot.101, 2022–2035 (2014).10.3732/ajb.1400377 [DOI] [PubMed] [Google Scholar]
• 2.Collopy, P. D.et al. High-throughput construction of gene deletion cassettes for generation ofNeurosporacrassa knockout strains.Methods Mol. Biol.638, 33–40 (2010).10.1007/978-1-60761-611-5_3 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 3.Ninomiya, Y., Suzuki, K., Ishii, C. & Inoue, H. Highly efficient gene replacements inNeurospora strains deficient for nonhomologous end-joining.Proc. Natl. Acad. Sci. U. S. A.101, 12248–12253 (2004).10.1073/pnas.0402780101 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 4.Colot, H. V.et al. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.Proc. Natl. Acad. Sci. U. S. A.103, 10352–10357 (2006).10.1073/pnas.0601456103 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 5.Park, G.et al. Global analysis of serine-threonine protein kinase genes inNeurosporacrassa.Eukaryot Cell10, 1553–1564 (2011).10.1128/EC.05140-11 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Park, G.et al. High-throughput production of gene replacement mutants inNeurosporacrassa.Methods Mol. Biol.722, 179–189 (2011).10.1007/978-1-61779-040-9_13 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 7.Dunlap, J. C.et al. Enabling a community to dissect an organism: Overview of the Neurospora functional genomics project.Adv. Genet.57, 49–96 (2007).10.1016/S0065-2660(06)57002-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Wang, S.et al. Molecular tools for gene manipulation in filamentous fungi.Appl. Microbiol. Biotechnol.101, 8063–8075 (2017).10.1007/s00253-017-8486-z [DOI] [PubMed] [Google Scholar]
• 9.Barrangou, R.et al. CRISPR provides acquired resistance against viruses in prokaryotes.Science315, 1709–1712 (2007).10.1126/science.1138140 [DOI] [PubMed] [Google Scholar]
• 10.Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.Nature507, 62–67 (2014).10.1038/nature13011 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 11.Jinek, M.et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science337, 816–821 (2012).10.1126/science.1225829 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Dicarlo, J. E.et al. Genome engineering inSaccharomycescerevisiae using CRISPR-Cas systems.Nucleic Acids Res.41, 4336–4343 (2013).10.1093/nar/gkt135 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 13.Liu, R., Chen, L., Jiang, Y., Zhou, Z. & Zou, G. Efficient genome editing in filamentous fungusTrichodermareesei using the CRISPR/Cas9 system.Cell Discov.1, 15007 (2015).10.1038/celldisc.2015.7 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 14.Nødvig, C. S., Nielsen, J. B., Kogle, M. E. & Mortensen, U. H. A CRISPR-Cas9 system for genetic engineering of filamentous fungi.PLoS One10, e0133085 (2015).10.1371/journal.pone.0133085 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 15.Schuster, M. & Kahmann, R. CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.Fungal Genet. Biol.130, 43–53. 10.1016/j.fgb.2019.04.016 (2019).10.1016/j.fgb.2019.04.016 [DOI] [PubMed] [Google Scholar]
• 16.Matsu-ura, T., Baek, M., Kwon, J. & Hong, C. Efficient gene editing inNeurosporacrassa with CRISPR technology.Fungal Biol. Biotechno.l10.1186/s40694-015-0015-1 (2015). 10.1186/s40694-015-0015-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 17.Song, R.et al. CRISPR/Cas9 genome editing technology in filamentous fungi: Progress and perspective.Appl. Microbiol. Biotechnol.103, 6919–6932 (2019).10.1007/s00253-019-10007-w [DOI] [PMC free article] [PubMed] [Google Scholar]
• 18.Bardiya, N. & Shiu, P. K. T. Cyclosporin A-resistance based gene placement system forNeurosporacrassa.Fungal Genet. Biol.44, 307–314 (2007).10.1016/j.fgb.2006.12.011 [DOI] [PubMed] [Google Scholar]
• 19.Sardar, P. & Kempken, F. Characterization of indole-3-pyruvic acid pathway-mediated biosynthesis of auxin inNeurosporacrassa.PLoS One13, e0192293 (2018).10.1371/journal.pone.0192293 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Zhang, C., Meng, X., Wei, X. & Lu, L. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining inAspergillusfumigatus.Fungal Genet. Biol.86, 47–57 (2016).10.1016/j.fgb.2015.12.007 [DOI] [PubMed] [Google Scholar]
• 21.Kuivanen, J., Wang, Y. M. J. & Richard, P. EngineeringAspergillusniger for galactaric acid production: Elimination of galactaric acid catabolism by using RNA sequencing and CRISPR/Cas9.Microb. Cell Fact.15, 1–9 (2016).10.1186/s12934-016-0613-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Chen, J.et al. CRISPR/Cas9-mediated efficient genome editing via blastospore-based transformation in entomopathogenic fungusBeauveriabassiana.Sci. Rep.7, 1–10 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Pohl, C., Kiel, J. A. K. W., Driessen, A. J. M., Bovenberg, R. A. L. & Nygård, Y. CRISPR/Cas9 based genome editing ofPenicilliumchrysogenum.ACS Synth. Biol.5, 754–764 (2016).10.1021/acssynbio.6b00082 [DOI] [PubMed] [Google Scholar]
• 24.Shi, T. Q.et al. CRISPR/Cas9-based genome editing in the filamentous fungusFusariumfujikuroi and its application in strain engineering for gibberellic acid production.ACS Synth. Biol.8, 445–454 (2019).10.1021/acssynbio.8b00478 [DOI] [PubMed] [Google Scholar]
• 25.Rozhkova, A. M. & Kislitsin, V. Y. CRISPR/Cas genome editing in filamentous fungi.Biochemistry (Mosc)86, S120–S139 (2021).10.1134/S0006297921140091 [DOI] [PubMed] [Google Scholar]
• 26.Dhawale, S. S. & Marzluf, G. A. Transformation ofNeurosporacrassa with circular and linear DNA and analysis of the fate of the transforming DNA.Curr. Genet.10, 205–211 (1985).10.1007/BF00798750 [DOI] [PubMed] [Google Scholar]
• 27.Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.Genome Res.24, 1012–1019 (2014).10.1101/gr.171322.113 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 28.Al Abdallah, Q., Ge, W. & Fortwendel, J. R. A simple and universal system for gene manipulation inAspergillusfumigatus: In vitro-assembled Cas9-guide RNA ribonucleoproteins coupled with microhomology repair templates.mSphere2, e00446 (2017).10.1128/mSphere.00446-17 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 29.Wang, Q., Cobine, P. A. & Coleman, J. J. Efficient genome editing inFusariumoxysporum based on CRISPR/Cas9 ribonucleoprotein complexes.Fungal Genet. Biol.117, 21–29 (2018).10.1016/j.fgb.2018.05.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 30.Fu, Y.et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol.31, 822–826 (2013).10.1038/nbt.2623 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 31.Garrigues, S., Peng, M., Kun, R. S. & De Vries, R. P. Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.mBio14, e00668 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
• 32.Schuster, M., Schweizer, G., Reissmann, S. & Kahmann, R. Genome editing inUstilagomaydis using the CRISPR–Cas system.Fungal Genet. Biol.89, 3–9 (2016).10.1016/j.fgb.2015.09.001 [DOI] [PubMed] [Google Scholar]
• 33.Abdallah, Q. A., Souza, A. C. O., Martin-Vicente, A., Ge, W. & Fortwendel, J. R. Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes inAspergillusfumigatus.Fungal Biol. Biotechnol.5, 1–8 (2018).10.1186/s40694-018-0057-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 34.Rhoades, N.et al. Recombination-independent recognition of DNA homology for meiotic silencing inNeurosporacrassa.Proc. Natl. Acad. Sci. U. S. A118, e2108664118 (2021).10.1073/pnas.2108664118 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 35.Gladyshev, E. & Kleckner, N. Direct recognition of homology between double helices of DNA inNeurosporacrassa.Nat. Commun.5, 3509 (2014).10.1038/ncomms4509 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Vogel, H. J. A convenient growth medium forNeurospora (Medium N).Microbial. Genet. Bull.13, 42–43 (1956). [Google Scholar]
• 37.Ebbole, D. & Sachs, M. S. A rapid and simple method for isolation ofNeurosporacrassa homokaryons using microconidia.Fungal Genet. Rep.10.4148/1941-4765.1472 (1990). 10.4148/1941-4765.1472 [DOI] [Google Scholar]
• 38.Mercker, M., Kollath-Leiß, K., Allgaier, S., Weiland, N. & Kempken, F. The BEM46-like protein appears to be essential for hyphal development upon ascospore germination inNeurosporacrassa and is targeted to the endoplasmic reticulum.Curr. Genet.55, 151–161 (2009).10.1007/s00294-009-0232-3 [DOI] [PubMed] [Google Scholar]
• 39.Margolin, B. S., Freitag, M. & Selker, E. U. Improved plasmids for gene targeting at thehis-3 locus ofNeurosporacrassa by electroporation.Fungal Genet. Rep.44, 34–36 (1997). 10.4148/1941-4765.1281 [DOI] [Google Scholar]
• 40.Labun, K.et al. CHOPCHOP v3: Expanding the CRISPR web toolbox beyond genome editing.Nucleic Acids Res.47, W171–W174 (2019).10.1093/nar/gkz365 [DOI] [PMC free article] [PubMed] [Google Scholar]
• 41.Southern, E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis.J. Mol. Biol.98, 503–517 (1975).10.1016/S0022-2836(75)80083-0 [DOI] [PubMed] [Google Scholar]

Supplementary Materials

Data Availability Statement

The datasets generated and/or analysed during the current study are available in the opendata@uni-kiel the research data repository of the Christian-Albrechts-University Kiel. It can be accessed via the DOI: 10.57892/100-39101.57892/100-39 or the following link:https://opendata.uni-kiel.de/receive/fdr_mods_00000039.