技术领域technical field
本发明属于基因组学及基因工程和生物技术领域，具体涉及到一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法。The invention belongs to the fields of genomics, genetic engineering and biotechnology, and specifically relates to a method for editing prokaryotic genomes by using an endogenous CRISPR-Cas system.
背景技术Background technique
CRISPR-Cas系统作为一种原核生物抵御病毒等外源入侵核酸的获得性免疫系统，广泛存在于大约90％的古菌和40％细菌中(VanderOostJetal.,2014；BarrangouRetal.,2014)。CRISPR-Cas系统被划分为三个主要类型：I型，II型和III型；它们分别有一个标志性蛋白：Cas3，Cas9和Cas10(MakarovaKSetal.,2011)。II型CRISPR系统仅需要Cas9一个蛋白与一条crRNA和trans-actingRNA行使DNA干涉活性(DeltchevaEetal.,2011；GasiunasGetal.,2012)。简单的II型CRISPR系统因此被开发成真核生物基因组编辑工具(JinekMetal.,2012；WangHetal.,2013)，并广泛应用于不同的真核生物和细菌(DoudnaJAetal.,2014；SanderJDetal.,2014；HsuPDetal.,2014；SelleKetal.,2015)。CRISPR-Cas system, as an acquired immune system for prokaryotes to resist viruses and other foreign invading nucleic acids, widely exists in about 90% of archaea and 40% of bacteria (VanderOostJetal., 2014; BarrangouRetal., 2014). CRISPR-Cas systems are divided into three main types: type I, type II, and type III; they each have a signature protein: Cas3, Cas9, and Cas10 (MakarovaK Setal., 2011). The type II CRISPR system only requires a Cas9 protein, a crRNA and a trans-acting RNA to perform DNA interference activity (DeltchevaE etal., 2011; Gasiunas Getal., 2012). The simple type II CRISPR system has therefore been developed into a eukaryotic genome editing tool (Jinek Metal., 2012; Wang Hetal., 2013), and is widely used in different eukaryotes and bacteria (DoudnaJAetal., 2014; SanderJDetal., 2014; HsuPD et al., 2014; Selle Ke et al., 2015).
在过去的十年中，各个实验室在一些古菌模式种里建立起了有效的遗传操作体系和工具(LeighJAetal.,2011)。尽管如此，古菌的遗传学研究因其独特的生长条件，生长缓慢以及对大多数抗生素不敏感等因素而仍然显得非常具有挑战性的(ValentineDLetal.,2007)。因此，居于CRISPR系统的基因组编辑方法在古菌中的应用仍然有待研究和开发。In the past decade, various laboratories have established effective genetic manipulation systems and tools in some archaeal model species (LeighJA et al., 2011). Nevertheless, the genetic research of archaea is still very challenging due to factors such as unique growth conditions, slow growth and insensitivity to most antibiotics (ValentineDLetal., 2007). Therefore, the application of genome editing methods based on the CRISPR system in archaea remains to be studied and developed.
目前基于CRISPR系统的基因组编辑工作所利用的均是II型CRISPR系统即CRISPR/Cas9体系，而CRISPR/Cas9系统也存在诸多局限性。首先，CRISPR/Cas9可能存在脱靶效应，已经有研究发现Cas9蛋白可能会容许crRNA与靶标序列之间存在一定程度的错配，而这些错配出现的数量和位置必然会影响到基因编辑的特异性。其次，利用外源的CRISPR/Cas9系统，需要对Cas9蛋白进行适用于宿主细胞的优化改造，并需同时在细胞内表达Cas9蛋白和sgRNA(向导RNA)才能发挥作用，使用程序比较复杂。另外，在一些生长条件极端的生物中，胞内环境可能会影响到Cas9蛋白的活性，从而限制了CRISPR/Cas9系统的应用。At present, the genome editing work based on the CRISPR system uses the type II CRISPR system, that is, the CRISPR/Cas9 system, and the CRISPR/Cas9 system also has many limitations. First, CRISPR/Cas9 may have off-target effects. It has been found that Cas9 protein may allow a certain degree of mismatch between crRNA and target sequence, and the number and position of these mismatches will inevitably affect the specificity of gene editing. . Secondly, to use the exogenous CRISPR/Cas9 system, it is necessary to optimize the Cas9 protein suitable for the host cell, and to express the Cas9 protein and sgRNA (guide RNA) in the cell at the same time to play a role, and the procedure is relatively complicated. In addition, in some organisms with extreme growth conditions, the intracellular environment may affect the activity of Cas9 protein, thus limiting the application of the CRISPR/Cas9 system.
本发明针对以上问题，我们提出了利用原核生物自身的CRISPR系统做基因组编辑。该方法同时利用了CRISPR系统的DNA干涉活性和同源重组，因此极大程度上避免了脱靶效应，增强了编辑特异性。该方法只需要构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，流程简单，操作周期短。该方法利用的是原核生物自身的CRISPR系统，所有含有内源CRISPR-Cas系统的细菌和古菌均适用。In the present invention, aiming at the above problems, we propose to use the CRISPR system of prokaryotic organisms for genome editing. This method utilizes both the DNA interference activity and homologous recombination of the CRISPR system, thus largely avoiding off-target effects and enhancing editing specificity. This method only needs to construct an editing plasmid carrying artificial CRISPR cluster and donor DNA at the same time, the process is simple and the operation period is short. This method utilizes the CRISPR system of prokaryotes themselves, and all bacteria and archaea that contain endogenous CRISPR-Cas systems are applicable.
硫化叶菌是CRISPR研究的模式生物，关于硫磺矿硫化叶菌S.solfataricusP2和冰岛硫化叶菌S.islandicusREY15A中I型和III型CRISPR-Cas系统的研究表明其均具有DNA和(或)RNA干涉活性(ManicaAetal.,2011；ManicaAetal.,2013；ZebecZetal.,2014；GudbergsdottirS.,2011)。我们发现在冰岛硫化叶菌S.islandicusREY15A中I-A型和III-BCmr-α均具有DNA干涉活性(DengLetal.,2013)。“CCN”和“TCN”是冰岛硫化叶菌I-ACRISPR系统DNA干涉活性所需的两个PAMs(protospacer-adjacentmotifs)(LillestolR.Ketal.,2009)。而III-B型CRISPR所介导的DNA干涉活性是不需要PAM序列的，但其依赖于转录，并需确保crRNA5’端repeat序列与protospacer上游序列错配(DengLetal.,2013)。Sulfolobus is a model organism for CRISPR research. Studies on type I and type III CRISPR-Cas systems in S. solfataricusP2 and S. islandicus REY15A have shown that they both have DNA and/or RNA interference Activity (Manica A et al., 2011; Manica A et al., 2013; Zebec Ze et al., 2014; Gudbergsdottir S., 2011). We found that both type I-A and type III-BCmr-α have DNA interference activity in Sulfolobus Iceland S. islandicus REY15A (Deng Letal., 2013). "CCN" and "TCN" are two PAMs (protospacer-adjacentmotifs) required for the DNA interference activity of Sulfolobus Iceland I-ACRISPR system (LillestolR.Ketal., 2009). The DNA interference activity mediated by III-B CRISPR does not require the PAM sequence, but it depends on transcription, and it is necessary to ensure that the repeat sequence at the 5' end of the crRNA is mismatched with the upstream sequence of the protospacer (DengLetal., 2013).
发明内容Contents of the invention
本发明的目的在于提供含有CRISPR-Cas系统的原核生物在内源编辑原核生物基因组中的应用，所述的原核生物为含有内源I型或III型CRISPR-Cas系统，或同时含有内源I型和III型CRISPR-Cas系统的细菌。The object of the present invention is to provide the application of the prokaryote containing the CRISPR-Cas system in the endogenous editing of the prokaryote genome, and the prokaryote contains the endogenous type I or type III CRISPR-Cas system, or contains the endogenous type I Type and Type III CRISPR-Cas systems in bacteria.
本发明的另一个目的在于提供一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，通过构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，在内源CRISPR系统对基因组发生DNA干涉后通过同源重组达到对基因组的编辑。Another object of the present invention is to provide a method for using the endogenous CRISPR-Cas system to edit the genome of prokaryotes. By constructing an editing plasmid that simultaneously carries artificial CRISPR clusters and donor DNA, the endogenous CRISPR system generates DNA on the genome. Genome editing is achieved through homologous recombination after interference.
为了达到上述目的，本发明采取以下技术措施：In order to achieve the above object, the present invention takes the following technical measures:
本发明所要保护的内容包括，含有CRISPR-Cas系统的原核生物在内源编辑原核生物基因组中的应用；内源CRISPR-Cas系统在编辑原核生物基因组中的应用。The content to be protected by the present invention includes the application of prokaryotic organisms containing CRISPR-Cas system in endogenous editing of prokaryotic organism genomes; the application of endogenous CRISPR-Cas system in editing prokaryotic organism genomes.
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:
1)构建基因组编辑质粒：1) Construct genome editing plasmid:
在原核生物基因组上拟编辑区域选取一段序列作为protospacer即靶标位点，根据protospacer设计两条反向互补的引物，其序列分别为正向引物：5’-AAAG-Nn-3’，反向引物：5’-TAGC-N’n-3’，其中Nn和N’n为反向互补序列，N和N’表示碱基A、T、G或C，n表示protospacer的碱基个数；将上述两条引物退火形成具有粘性末端的双链DNA，即spacer片段；人工CRISPR载体pSe-Rp经过限制性内切酶BspMI酶切处理，然后与具有粘性末端的spacer片段酶连，得到能产生成熟的crRNA的人工CRISPR质粒(pAC)；再将包含突变序列和与宿主细胞基因组上靶标位点两端同源的供体DNA片段插入到上述pAC质粒上，得到基因组编辑质粒(pGE)；Select a sequence in the region to be edited on the prokaryotic genome as the protospacer or the target site, and design two reverse complementary primers according to the protospacer, and their sequences are forward primers: 5'-AAAG-Nn -3', reverse Primer: 5'-TAGC-N'n -3', where Nn and N'n are reverse complementary sequences, N and N' represent bases A, T, G or C, n represents the number of bases of protospacer ; the above two primers are annealed to form a double-stranded DNA with sticky ends, that is, the spacer fragment; the artificial CRISPR vector pSe-Rp is digested with the restriction endonuclease BspMI, and then ligated with the spacer fragment with sticky ends to obtain energy An artificial CRISPR plasmid (pAC) that produces a mature crRNA; then insert a donor DNA fragment containing a mutant sequence and homologous to both ends of the target site on the host cell genome into the above pAC plasmid to obtain a genome editing plasmid (pGE);
所述的供体DNA片段由左右两条同源片段与中间设计的突变序列通过SOE-PCR的方法扩增而成。The donor DNA fragment is amplified by the SOE-PCR method from the left and right homologous fragments and the mutant sequence designed in the middle.
2)突变株的获得：pGE质粒电转入原核生物感受态细胞后，质粒上的人工CRISPR簇转录出pre-crRNA，pre-crRNA在细胞内被加工成成熟的crRNA；crRNA与细胞内源的CRISPR-Cas蛋白形成crRNP复合体，通过crRNA与宿主细胞基因组上的目标DNA链配对来识别靶标位点进行切割；随后质粒上的供体DNA片段与靶标位点两侧序列发生同源重组，进而得到基因组编辑突变株。2) Acquisition of mutant strains: After the pGE plasmid is electrotransferred into prokaryotic competent cells, the artificial CRISPR cluster on the plasmid transcribes pre-crRNA, and the pre-crRNA is processed into mature crRNA in the cell; The CRISPR-Cas protein forms a crRNP complex, which recognizes the target site for cutting by pairing the crRNA with the target DNA strand on the host cell genome; then the donor DNA fragment on the plasmid undergoes homologous recombination with the sequences on both sides of the target site, and then Genome editing mutants were obtained.
以上所述的步骤中，所述的原核生物为含有内源I型或III型CRISPR-Cas系统，或同时含有内源I型和III型CRISPR-Cas系统的细菌或古菌，包括但不限于冰岛硫化叶菌(优选原核冰岛硫化叶菌S.islandicusREY15A)，为所有含有内源CRISPR-Cas系统的细菌和古菌，例如：大肠杆菌Escherichiacoli，表皮葡萄球菌Staphylococcusepidermidis，腐败希瓦菌Shewanellaputrefaciens，嗜热细菌Thermusthermophilus，嗜热链球菌Streptococcusthermophilus，强烈火球菌Pyrococcusfuriosus，嗜酸热硫化叶菌Sulfolobusacidocaldarius，硫磺矿硫化叶菌Sulfolobussolfataricus，热自养甲烷热杆菌Methanothermobacterthermautotrophicus，盐富饶菌Haloferaxvolcanii，黑胫病菌Pectobacteriumatrosepticum，白喉棒状杆菌Corynebacteriumdiphtheriae等。In the steps described above, the prokaryotes are bacteria or archaea containing endogenous Type I or Type III CRISPR-Cas systems, or both endogenous Type I and Type III CRISPR-Cas systems, including but not limited to Sulfolobus Iceland (preferably prokaryotic Sulfolobus Icelandicus S. islandicus REY15A), for all bacteria and archaea containing endogenous CRISPR-Cas systems, such as: Escherichia coli, Staphylococcus epidermidis, Shewanella putrefaciens, thermophiles Bacteria Thermusthermophilus, Streptococcus thermophilus, Pyrococcus furiosus, Sulfolobus acidocaldarius, Sulfolobus solfataricus, Methanothermobacterthermautotrophicus, Haloferaxvolcanii, Pectobacterium atros Corynebacterium diphtheriae et al.
以上所述的基因组编辑包括但不限于缺失、插入和点突变。Genome editing as described above includes, but is not limited to, deletions, insertions, and point mutations.
与现有技术相比，本发明具有以下优点：Compared with the prior art, the present invention has the following advantages:
本发明提供的利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，只需构建一个同时携带人工CRISPR簇和供体DNA的编辑质粒，在内源CRISPR系统对基因组发生DNA干涉后通过同源重组达到对基因组的编辑。其最大的优点在于：应用宿主范围广，所有含有内源CRISPR-Cas系统的细菌和古菌均可操作；可用于多种编辑方式，缺失、插入和点突变等均可操作；更高的编辑效率，筛选阳性率高，背景低；流程简单，时间周期短，大大减轻原核生物基因组编辑的工作量。The method of using the endogenous CRISPR-Cas system to edit the genome of prokaryotic organisms provided by the present invention only needs to construct an editing plasmid carrying artificial CRISPR cluster and donor DNA at the same time. Recombination achieves editing of the genome. Its biggest advantage is: it has a wide range of hosts, and all bacteria and archaea containing endogenous CRISPR-Cas systems can be operated; it can be used in a variety of editing methods, including deletion, insertion and point mutation; higher editing Efficiency, high screening positive rate, low background; simple process, short time period, greatly reducing the workload of prokaryotic genome editing.
附图说明Description of drawings
图1为本发明利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法的原理示意图。Figure 1 is a schematic diagram of the principle of the method for editing the genome of prokaryotes using the endogenous CRISPR-Cas system of the present invention.
图2中A为实施例1中利用冰岛硫化叶菌S.islandicusREY15A内源CRISPR系统对其lacS基因进行精准缺失突变设计示意图；A in Figure 2 is a schematic diagram of the precise deletion mutation design of the lacS gene using the endogenous CRISPR system of Sulfolobus Icelandicus REY15A in Example 1;
图2中B为实施例1中X-gal染色分析比较野生型与转化子示意图；B among Fig. 2 is that X-gal staining analysis compares the schematic diagram of wild type and transformant in embodiment 1;
图2中C为实施例1中PCR检测转化子是否为43bp缺失突变株示意图；In Fig. 2, C is a schematic diagram of detecting whether the transformant is a 43bp deletion mutant by PCR in embodiment 1;
图2中D为实施例1中DNA测序分析比较野生型和突变型lacS示意图。D in FIG. 2 is a schematic diagram of comparing wild-type and mutant lacS in DNA sequencing analysis in Example 1.
图3中A为本发明实施例2中利用冰岛硫化叶菌S.islandicusREY15A内源III-B型CRISPR系统对其Cmr-2α蛋白的C端进行6×His标签插入突变设计示意图；A in Figure 3 is a schematic diagram of the design of 6×His tag insertion mutation at the C-terminus of the Cmr-2α protein using the endogenous III-B type CRISPR system of Sulfolobus Icelandicus REY15A in Example 2 of the present invention;
图3中B为实施例2中DNA测序比较野生型cmr-2α和携带6×His标签突变型cmr-2α示意图；B in Figure 3 is a schematic diagram of comparing wild-type cmr-2α and mutant cmr-2α carrying a 6×His tag by DNA sequencing in Example 2;
图3中C为实施例2中用His鼠抗westernblot鉴定携带6×His标签的Cmr-2α蛋白示意图；C in Figure 3 is a schematic diagram of identifying the Cmr-2α protein carrying the 6×His tag using His mouse anti-westernblot in Example 2;
图3中D为实施例2中在Cmr2α-His菌株中用携带6×His标签的Cmr-2α蛋白做共纯化示意图。D in Fig. 3 is a schematic diagram of co-purification with Cmr-2α protein carrying 6×His tag in the Cmr2α-His strain in Example 2.
图4为实施例2中pGE-2α-His电转Δcas3/cas7和Δcas3感受态细胞，并PCR检验转化子示意图；Figure 4 is a schematic diagram of electroporation of Δcas3/cas7 and Δcas3 competent cells with pGE-2α-His in Example 2, and PCR detection of transformants;
图4中A为实施例2中PCR所用引物的设计示意图；Among Fig. 4, A is the schematic diagram of the design of the primers used in PCR in Example 2;
图4中B为实施例2中两个缺失菌株各12个转化子PCR验证电泳图。B in FIG. 4 is the PCR verification electrophoresis of 12 transformants of each of the two deletion strains in Example 2.
图5为本发明实施例3中Cmr-2α(SiRe-0894),Cmr-2β(SiRe-0598)和它们的同源蛋白PyrococcusfuriosusPF1129进行N端序列比对示意图；Figure 5 is a schematic diagram of the N-terminal sequence alignment of Cmr-2α (SiRe-0894), Cmr-2β (SiRe-0598) and their homologous protein Pyrococcus furiosus PF1129 in Example 3 of the present invention;
图5中A为实施例3中四个保守的氨基酸选为拟突变位点示意图；In Fig. 5, A is a schematic diagram of four conserved amino acids selected as pseudo-mutation sites in Example 3;
图5中B为实施例3中利用冰岛硫化叶菌S.islandicusREY15A内源I-A型CRISPR系统对其Cmr-2α蛋白HDdomain进行多位点突变设计示意图；B in Figure 5 is a schematic diagram of multi-site mutation design of the Cmr-2α protein HDdomain using the endogenous Type I-A CRISPR system of Sulfolobus Icelandicus REY15A in Example 3;
图5中C为实施例3中DNA测序显示Cmr-2α蛋白HDdomain三种突变型示意图。C in FIG. 5 is a schematic diagram of three mutant types of Cmr-2α protein HDdomain shown by DNA sequencing in Example 3.
具体实施方式detailed description
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别说明，实施例中的实验操作均按常规实验条件或按照材料、试剂制造厂商说明书建议的条件。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental operations in the examples were carried out according to conventional experimental conditions or according to the conditions suggested by the manufacturer's instructions of materials and reagents.
实施例1：Example 1:
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:
本实施例利用冰岛硫化叶菌S.islandicusREY15A内源(I-A型CRISPR系统和III-B型CRISPR系统)CRISPR系统对其lacS基因进行精准缺失突变为例进行说明：This example uses the endogenous (Type I-A CRISPR system and Type III-B CRISPR system) CRISPR system of S. islandicus REY15A to carry out precise deletion mutation of its lacS gene as an example to illustrate:
1.编辑质粒的构建1. Construction of Editing Plasmids
(1)在冰岛硫化叶菌S.islandicusREY15Aβ-半乳糖苷酶基因lacS(SIRE_RS11295)上选取+933至+972共40个碱基作为protospacer，其反向互补序列为5’-AGTGTAGTAATTAACACCAATCCAGTCTAACCTACCCCTT-3’，其紧邻一个CCT-PAM(ProtospacerAdjacentMotif)，因此能被I-A型CRISPR系统target；同时又由于crRNA5’端序列与对应的targetsite序列错配，所以也能同时被III-B型CRISPR系统target。基于这个protospacer设计两条引物(LacS-E-SpF/LacS-E-SpR)(表1)，两条引物通过退火生成两端含有粘性末端的spacer片段；(1) A total of 40 bases from +933 to +972 were selected from the S. islandicus REY15Aβ-galactosidase gene lacS (SIRE_RS11295) as the protospacer, and its reverse complementary sequence was 5'-AGTGTAGTAATTAACACCAATCCAGTCTAACCTACCCTT-3', It is next to a CCT-PAM (Protospacer Adjacent Motif), so it can be targeted by the Type I-A CRISPR system; at the same time, because the 5' end sequence of crRNA is mismatched with the corresponding target site sequence, it can also be targeted by the Type III-B CRISPR system at the same time. Based on this protospacer, two primers (LacS-E-SpF/LacS-E-SpR) (Table 1) were designed, and the two primers were annealed to generate spacer fragments with sticky ends at both ends;
表1.本发明所用到引物序列Table 1. The primer sequences used in the present invention
上表中黑色下划线标识的为限制性酶切位点，形成spacer粘性末端的四个碱基被黑色加粗显示；Cmr-2αHDdomain突变位点被斜体字体标识。In the above table, the black underline marks are the restriction enzyme cutting sites, and the four bases forming the cohesive end of the spacer are shown in bold black; the Cmr-2αHDdomain mutation site is marked in italics.
(2)人工CRISPR载体pSe-Rp(Peng,2015)经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；(2) The artificial CRISPR vector pSe-Rp (Peng, 2015) was digested with BspMI, and the digested product was enzyme-ligated with the above spacer fragment to obtain the artificial CRISPR plasmid;
(3)设计两条在lacS基因上缺失43bp的SOEPCR引物(LacS-E-SOEF/LacS-E-SOER)和分别带有SalI和NotI酶切位点的两条引物(LacS-E-SalIF/LacS-E-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；(3) Design two SOEPCR primers (LacS-E-SOEF/LacS-E-SOER) with a 43bp deletion in the lacS gene and two primers (LacS-E-SalIF/LacS-E-SalIF/ LacS-E-NotIR) (Table 1), using the SOEPCR method to obtain donor DNA fragments for homologous recombination;
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-lacS1；(4) The donor DNA fragment and the above-mentioned artificial CRISPR plasmid were digested with SalI and NotI respectively, and then enzyme-linked and transformed to obtain the editing plasmid pGE-lacS1;
2.突变株的筛选2. Screening of mutant strains
(1)500ngpGE-lacS1质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；(1) 500ngpGE-lacS1 plasmid was electrotransferred into Sulfolobus Icelandicus S.islandicusREY15A competent cells, coated with SCV (0.2% sucrose, 0.2% casaminoacidsplus1% vitaminsolution) solid medium, cultured at 78°C for 7 days, and picked transformants in 5ml SCV liquid culture medium for three days;
(2)各取90μl菌液分别添加10μlX-gal(20mg/ml)78℃孵育一个小时，观察反应体系颜色；(2) Add 10 μl X-gal (20 mg/ml) to 90 μl bacterial solution and incubate at 78°C for one hour, observe the color of the reaction system;
(3)然后分别用(F1/R1和F2/R2)(表1)进行PCR检测，发现挑取的17个转化子均含有pGE-lacS1质粒，同时其中16个转化子的lacS基因发生了43bp缺失，1个为逃逸突变株，并通过引物(LacS-Seq-F/LacS-Seq-R)(表1)扩增基因组DNA测序验证，结果显示该方法的编辑效率在90％以上。(3) Then use (F1/R1 and F2/R2) (Table 1) to perform PCR detection respectively, and find that the 17 picked transformants all contain pGE-lacS1 plasmid, and the lacS gene of 16 transformants has 43bp One was deleted, and one was an escape mutant, which was verified by amplifying genomic DNA sequencing with primers (LacS-Seq-F/LacS-Seq-R) (Table 1). The results showed that the editing efficiency of this method was above 90%.
实施例2：Example 2:
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:
本发明实施例以冰岛硫化叶菌S.islandicusREY15A内源III-B型CRISPR系统对其Cmr-2α蛋白的C端进行6×His标签插入突变为例，进行说明。In the embodiment of the present invention, the C-terminus of the Cmr-2α protein of S. islandicus REY15A endogenous III-B type CRISPR system is used as an example to illustrate the 6×His tag insertion mutation.
1.编辑质粒的构建1. Construction of Editing Plasmids
(1)在冰岛硫化叶菌S.islandicusREY15A基因组上选取cmr-2α基因(SIRE_RS04505)的最后25个碱基和cmr-3α基因的前7个碱基共32个碱基作为protospacer，其反向互补序列为5’-AGTGTAGTAATTAACACCACAATCCAGTCTAACCTACCCCTT-3’，其前面没有CCN-PAM，因而只具有III-B型CRISPR的DNA干涉活性。基于这个protospacer设计两条引物(2α-His-SpF/2α-His-SpR)(表1)，两条引物通过退火生成两端含有粘性末端的spacer片段；(1) Select the last 25 bases of the cmr-2α gene (SIRE_RS04505) and the first 7 bases of the cmr-3α gene on the S. islandicus REY15A genome as a protospacer, and its reverse complement The sequence is 5'-AGTGTAGTAATTAACACCACAATCCAGTCTAACCTACCCCT-3', without CCN-PAM in front of it, so it only has the DNA interference activity of type III-B CRISPR. Based on this protospacer, two primers (2α-His-SpF/2α-His-SpR) (Table 1) were designed, and the two primers were annealed to generate spacer fragments with sticky ends at both ends;
(2)人工CRISPR载体pSe-Rp经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；(2) The artificial CRISPR vector pSe-Rp is digested with BspMI, and the digested product is enzyme-ligated with the above spacer fragment to obtain an artificial CRISPR plasmid;
(3)设计两条包含六个组氨酸编码子的SOEPCR引物(2α-His-SOEF/2α-His-SOER)和分别带有SalI和NotI酶切位点的两条引物(2α-His-SalIF/2α-His-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；(3) Design two SOEPCR primers (2α-His-SOEF/2α-His-SOER) containing six histidine codes and two primers (2α-His-SOER) with SalI and NotI restriction sites respectively (2α-His- SalIF/2α-His-NotIR) (Table 1), using the SOEPCR method to obtain donor DNA fragments for homologous recombination;
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-2α-His；(4) The donor DNA fragment and the above-mentioned artificial CRISPR plasmid were respectively digested with SalI and NotI, and then enzyme-linked and transformed to obtain the editing plasmid pGE-2α-His;
2.突变株的筛选2. Screening of mutant strains
(1)500ngpGE-2α-His质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；(1) 500ngpGE-2α-His plasmid was electrotransferred into S. islandicus REY15A competent cells, coated with SCV (0.2% sucrose, 0.2% casaminoacidsplus1% vitamin solution) solid medium, cultured at 78°C for 7 days, and picked for transformation Subculture in 5ml SCV liquid medium for three days;
(2)用引物(2α-Seq-F/2α-Seq-R)(表1)扩增cmr-2α基因进行测序验证，结果显示转化子为成功插入6×His标签的突变株；(2) Using primers (2α-Seq-F/2α-Seq-R) (Table 1) to amplify the cmr-2α gene for sequencing verification, the results showed that the transformant was a mutant strain with a 6×His tag successfully inserted;
(3)用含5-FOA的SCV培养基对插入突变株进行反筛，以消除编辑质粒pGE-2α-His；(3) Back-screening the insertion mutants with SCV medium containing 5-FOA to eliminate the editing plasmid pGE-2α-His;
3.His标签融合蛋白的共纯化与检测3. Co-purification and detection of His tag fusion protein
3LSCV+U(尿嘧啶)培养基培养Cmr2α-His菌株，待OD600达到0.4时，收集菌体并重悬于50mMPBS缓冲液，超声波破碎后高速离心取上清，与500μlNi琼脂糖珠充分混合一小时后过柱，然后分别用20mM咪唑PBS，60mM咪唑PBS洗柱，最后用2ml200mM咪唑PBS重复洗脱Ni柱两次，SDS-PAGE后分别银染分析和用His鼠抗做westernblot检测。Cmr2α-His strain was cultured in 3LSCV+U (uracil) medium. When theOD600 reached 0.4, the bacteria were collected and resuspended in 50mMPBS buffer. After ultrasonic crushing, the supernatant was taken by high-speed centrifugation and mixed with 500μl Ni agarose beads for one hour. After passing through the column, wash the column with 20mM imidazole PBS and 60mM imidazole PBS respectively, and finally wash the Ni column twice with 2ml 200mM imidazole PBS. After SDS-PAGE, silver staining analysis and western blot detection were performed with His mouse antibody respectively.
检测了7个转化子，7个均为插入突变株。Seven transformants were detected, all of which were insertion mutants.
4.为了进一步排除I-A型CRISPR系统在本实施例中的影响，我们用编辑质粒pGE-2α-His电转两个缺失了I型CRIPSR活性的冰岛硫化叶菌S.islandicusREY15A感受态细胞：Δcas3/cas7和Δcas3，各挑取12个转化子进行PCR验证，得到的插入突变株比例分别为11/12和12/12(图4)，进一步说明该方法成功率达到90％以上。4. In order to further exclude the influence of the Type I-A CRISPR system in this example, we used the editing plasmid pGE-2α-His to electroporate two Sulfolobus Icelandicus S. islandicus REY15A competent cells that lack Type I CRIPSR activity: Δcas3/cas7 and Δcas3, 12 transformants were selected for PCR verification, and the ratios of the obtained insertion mutants were 11/12 and 12/12 respectively (Fig. 4), further illustrating that the success rate of this method reached over 90%.
实施例3：Example 3:
一种利用内源CRISPR-Cas系统进行原核生物基因组编辑的方法，包括以下步骤：A method for editing a prokaryotic genome using an endogenous CRISPR-Cas system, comprising the following steps:
本实施例以利用冰岛硫化叶菌S.islandicusREY15A内源I-A型CRISPR系统对其Cmr-2α蛋白HDdomain进行多位点突变为例，进行说明。This example uses the endogenous Type I-A CRISPR system of Sulfolobus Icelandicus REY15A to carry out multi-site mutation of its Cmr-2α protein HDdomain as an example to illustrate.
1.编辑质粒的构建1. Construction of Editing Plasmids
(1)在冰岛硫化叶菌S.islandicusREY15A基因组上选取cmr-2α基因N端HDdomain四个保守氨基酸H/D/K/I处选取40个碱基作为protospacer，其序列为5’-CGACCCTCCTTGGAAGGCATGGGTAATTACAAGGAATATT-3’，其前面有CCA-PAM。基于这个protospacer设计两条引物(2α-HDmut-SpF/2α-HDmut-SpR)，两条引物通过退火生成两端含有粘性末端的spacer片段；(1) On the S. islandicus REY15A genome, select 40 bases at the four conserved amino acids H/D/K/I of the N-terminal HDdomain of the cmr-2α gene as the protospacer, and its sequence is 5'-CGACCCTCCTTGGAAGGCATGGGTAATTACAAGGAATATT-3 ', preceded by CCA-PAM. Based on this protospacer, two primers (2α-HDmut-SpF/2α-HDmut-SpR) were designed, and the two primers were annealed to generate a spacer fragment with sticky ends at both ends;
(2)人工CRISPR载体pSe-Rp经BspMI酶切处理，酶切产物与上述spacer片段酶连，得到人工CRISPR质粒；(2) The artificial CRISPR vector pSe-Rp is digested with BspMI, and the digested product is enzyme-ligated with the above spacer fragment to obtain an artificial CRISPR plasmid;
(3)设计两条包含四个突变氨基酸编码子的SOEPCR引物(3) Design two SOEPCR primers comprising four mutated amino acid coding subunits
(2α-HDmut-SOEF/2α-HDmut-SOER)和分别带有SalI和NotI酶切位点的两条引物(2α-HDmut-SOEF/2α-HDmut-SOER) and two primers with SalI and NotI restriction sites respectively
(2α-HDmut-SalIF/2α-HDmut-NotIR)(表1)，用SOEPCR的方法得到用于同源重组的供体DNA片段；(2α-HDmut-SalIF/2α-HDmut-NotIR) (Table 1), using the SOEPCR method to obtain donor DNA fragments for homologous recombination;
(4)供体DNA片段和上述人工CRISPR质粒分别用SalI和NotI酶切，然后酶连转化，得到编辑质粒pGE-2αHD；(4) The donor DNA fragment and the above-mentioned artificial CRISPR plasmid were respectively digested with SalI and NotI, and then enzyme-linked and transformed to obtain the editing plasmid pGE-2αHD;
2.突变株的筛选2. Screening of mutant strains
(1)500ngpGE-2αHD质粒电转入冰岛硫化叶菌S.islandicusREY15A感受态细胞，涂布SCV(0.2％sucrose,0.2％casaminoacidsplus1％vitaminsolution)固体培养基，78℃培养7天，挑取转化子于5mlSCV液体培养基培养三天；(1) 500ngpGE-2αHD plasmid was electrotransferred into Sulfolobus Iceland S.islandicusREY15A competent cells, coated with SCV (0.2% sucrose, 0.2% casaminoacidsplus1% vitaminsolution) solid medium, cultured at 78°C for 7 days, and picked transformants in 5ml SCV liquid culture medium for three days;
(2)用引物(2α-Seq-F/2α-Seq-R)(表1)扩增cmr-2α基因进行测序验证，挑取的7转化子均为突变株，说明编辑效率为100％，由于同源重组位置的多样性，结果显示出三种突变类型，分别为只突变H/D两个氨基酸，突变H/D/K三个氨基酸以及H/D/K/I四个氨基酸均发生突变，比例为1:2:4。(2) Use the primers (2α-Seq-F/2α-Seq-R) (Table 1) to amplify the cmr-2α gene for sequencing verification. The 7 transformants picked are all mutant strains, indicating that the editing efficiency is 100%. Due to the diversity of homologous recombination positions, the results showed three types of mutations, namely, only two amino acids of H/D were mutated, three amino acids of H/D/K were mutated, and all four amino acids of H/D/K/I were mutated. mutation, the ratio is 1:2:4.
实施例4：Example 4:
我们利用此方法在冰岛硫化叶菌S.islandicusHVE10/4(一个广泛应用于病毒研究的菌株)中利用其I-A型CRISPR系统成功进行了lacS基因的编辑。pGE质粒的构建及操作步骤同上实施例1。值得指出的是，S.islandicusHVE10/4由于转化效率很低，在实施传统基因敲除方法时很难成功，而利用本发明提供的内源CRISPR系统基因组编辑方法，挑取的转化子均为突变株，编辑效率达到了100％。We used this method to successfully edit the lacS gene in S. islandicus HVE10/4 (a strain widely used in virus research) using its type I-A CRISPR system. The construction and operation steps of the pGE plasmid are the same as in Example 1 above. It is worth pointing out that, due to the low transformation efficiency of S. islandicusHVE10/4, it is difficult to successfully implement the traditional gene knockout method, but using the endogenous CRISPR system genome editing method provided by the present invention, the selected transformants are all mutations strain, the editing efficiency reached 100%.
本发明保护范围不限于上述实施例。The protection scope of the present invention is not limited to the above-mentioned embodiments.
尽管上文已经用一般性说明及具体实施例对本发明做了详尽的描述，但在本发明基础上作一些修改或改进，这对本领域技术人员而言是一望而知的，毋庸赘述。因此，在未偏离本发明主旨的基础上做的或多或少的修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention, so there is no need to repeat them. Therefore, more or less modifications or improvements made on the basis of not departing from the gist of the present invention all belong to the protection scope of the present invention.
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