WO2019184655A1

WO2019184655A1 - Application of crispr/cas system in gene editing

Info

Publication number: WO2019184655A1
Application number: PCT/CN2019/076784
Authority: WO
Inventors: 汪沛; 牛志杰; 林彦妮
Original assignee: 苏州克睿基因生物科技有限公司
Priority date: 2018-03-27
Filing date: 2019-03-02
Publication date: 2019-10-03
Also published as: CN111971389A

Abstract

A genetic engineering expression method for CRISPR/Cas, comprising rapidly constructing an element for the co-expression of a plurality of guide RNAs by means of constructing a plasmid comprising a DNA sequence expressing a plurality of sgRNA-tRNAs.

Description

CRISPR/Cas***在基因编辑中的应用Application of CRISPR/Cas System in Gene Editing

技术领域Technical field

本发明涉及一种基因编辑***的DNA表达体系和基因工程的表达方法，尤其涉及CRISPR/Cas9的表达体系及其基因工程的表达方法。The invention relates to a DNA expression system and a genetic engineering expression method of a gene editing system, in particular to an expression system of CRISPR/Cas9 and a method for expressing the same.

背景介绍Background introduction

CRISPR(clustered,regularly interspaced,short palindromic repeat)/Cas9技术广泛应用于基因组的定点编辑。对比于其它传统基因编辑工具，诸如锌指(Bibikova et al.Science2003.300:764)，转录激活因子样效应物核酸酶(TALEN)(Boch et al.Science 2009.326：1509–1512)，CRISPR/Cas9***更为简单，易操作。该***中，Cas9是一种核酸酶，在与单链引导RNA(sgRNA)结合后，可以在其指引下特异地靶向基因组的双链DNA并造成一个双链断裂(DSB)。这种断裂很容易被识别出并通过以下两种机制进行修复：双链的非同源性连接以及同源引导的重组。在以上的修复过程中会发生不同长度的***和缺失(Indel)以及特异性的序列更改(Cong et al.Science 2013.339：819–823)。CRISPR (clustered, regularlyly interspaced, short palindromic repeat)/Cas9 technology is widely used in fixed-point editing of genomes. Compared to other traditional gene editing tools, such as zinc finger (Bibikova et al. Science 2003. 300: 764), transcription activator-like effector nuclease (TALEN) (Boch et al. Science 2009. 326: 1509–1512), CRISPR/Cas9 The system is simpler and easier to operate. In this system, Cas9 is a nuclease that, when combined with single-stranded guide RNA (sgRNA), can specifically target the double-stranded DNA of the genome and cause a double-strand break (DSB). This fragmentation is readily recognized and repaired by two mechanisms: double-stranded non-homologous linkages and homologously directed recombination. Inserts and deletions of different lengths (Indel) and specific sequence changes occur during the above repair process (Cong et al. Science 2013. 339: 819–823).

核酸酶Cas9之所以能够特异性地靶向DNA是在于它所结合的单链引导RNA(sgRNA)。但如果依赖于一个sgRNA去编辑特定基因，有时会效率不高，而且容易出现等位基因抗性的现象；为了达到对多个基因的高效率编辑，多条不同引导RNA需要同时使用(Ran et al.Cell 2013.154(6):1380–1389)。在这种情况下，Cas9和不同的引导RNA可以在体外预混好，然后导入到细胞或组织中，以便达到多位点编辑的效果(Haoyi et al.Cell 2013.153:1–9)。然而，直接使用Cas9蛋白和sgRNA不仅造价昂贵，而且极大地限制了导入体内的方法，对科研及工业生产都带来极大的不便。Cas9除了蛋白形式，还可以以mRNA或DNA形式转入细胞中，其中mRNA造价非常高。所以以DNA形式转入Cas9表达元件是最为经济有效的方法。另一方面，sgRNA可以以RNA或者DNA的形式转入细胞或组织中。考虑到Cas9是以DNA的形式，如果sgRNA以RNA的形式导入，则极易在Cas9开始表达之前就发生降解，这样当Cas9核酸酶准备好时，可能缺少适用的sgRNA与其配合工作。所以两者都以DNA的形式导入更加符合这个***的作用模式。同时DNA的递送与病毒包装浸染的模式兼容(Malina et al.Methods Enzymol 2014.546:193-213)，这样可以极大的拓展CRISPR/Cas9在基因组中进行编辑的应用。The nuclease Cas9 is capable of specifically targeting DNA in the single-stranded guide RNA (sgRNA) to which it binds. However, if you rely on a sgRNA to edit a specific gene, it is sometimes inefficient and prone to allelic resistance; in order to achieve efficient editing of multiple genes, multiple different guide RNAs need to be used simultaneously (Ran et Al. Cell 2013.154(6): 1380–1389). In this case, Cas9 and different guide RNAs can be pre-mixed in vitro and then introduced into cells or tissues to achieve multi-site editing (Haoyi et al. Cell 2013. 153:1–9). However, the direct use of Cas9 protein and sgRNA is not only expensive, but also greatly limits the method of introduction into the body, which brings great inconvenience to scientific research and industrial production. In addition to the protein form, Cas9 can also be transferred into cells in the form of mRNA or DNA, where the mRNA is very expensive. Therefore, transferring to the Cas9 expression element in the form of DNA is the most cost-effective method. On the other hand, sgRNA can be transferred into cells or tissues in the form of RNA or DNA. Considering that Cas9 is in the form of DNA, if sgRNA is introduced in the form of RNA, it is very easy to degrade before Cas9 begins to express, so that when Cas9 nuclease is ready, there may be a lack of suitable sgRNA to work with it. Therefore, both are introduced in the form of DNA to more closely match the mode of action of this system. At the same time, DNA delivery is compatible with viral packaging dip patterns (Malina et al. Methods Enzymol 2014. 546: 193-213), which greatly expands the use of CRISPR/Cas9 for editing in the genome.

以DNA的形式通过CRISPR/Cas9***靶向基因组多位点已经报导过一些方法。如果把多个不同的引导RNA直接串连起来，通过一个启动子进行转录，这样产物中多个引导RNA就将成为一个整体，其编辑效率都比较低。也有通过多个启动子起到多个引导RNA的合成以达到多位点靶向，但这个在材料上难以构建操作；并且引入了多个启动子序列，对于有长度限制的病毒包装是非常不利的；同时多个启动子的活性难以控制一致，对结果造成很多波动(Edward et al.Methods 2015.91:82-86)。Several methods have been reported for targeting genomic multiple sites in the form of DNA via the CRISPR/Cas9 system. If a plurality of different guide RNAs are directly connected in series, transcription is performed by a promoter, so that a plurality of guide RNAs in the product will become a whole, and the editing efficiency is relatively low. There are also multiple promoters to synthesize multiple guide RNAs to achieve multi-site targeting, but this is difficult to construct in materials; and the introduction of multiple promoter sequences is highly disadvantageous for virus packaging with length limitations. At the same time, the activity of multiple promoters is difficult to control consistently, causing many fluctuations in the results (Edward et al. Methods 2015. 91: 82-86).

如果有一种方法能够在单一启动子控制的转录本里同时表达几个引导RNA，并且在体内可以被精确加工成单个成熟的RNA，这样就可以实现Cas9内切酶多定位编辑的高效性。比如，使用Csy4***(US 2016/031928 A1)，导入一个特定RNA识别及切割的酶，并在引导RNA的设计中加入特定RNA识别位点，这个***虽然可以在生物体里作用，但是会造成一些不便，首先这个***的导入增加生物体的外源DNA的负担，而且加工好的引导RNA比原有结构多出一个RNA识别位点，也是下游反应所不需要的。也有在gRNA序列两端加入可以自切割的ribozyme(核酶)序列(Yangbin et al.JIPB 2014.56：343-349)，这样可以在体内转录后经自切割功能释放出sgRNA，但这种方法受限于ribozyme只能切割一端，这样sgRNA序列两端要连接不同的核酶序列，而且不方便连接多个不同引导RNA对应的DNA片段；还有一个方法就是多个引导RNA通过tRNA进行连接，这样在体内表达后，这个长转录本会经由体内的tRNA加工***释放出多个不同引导RNA，这个***经验证在水稻以及果蝇中都是可以正常作用的(US2016/031928 A1)。If there is a way to simultaneously express several guide RNAs in a single promoter-controlled transcript and can be precisely processed into a single mature RNA in vivo, the efficiency of Cas9 endonuclease multi-localization editing can be achieved. For example, using the Csy4 system (US 2016/031928 A1), introducing a specific RNA recognition and cleavage enzyme, and adding a specific RNA recognition site to the design of the guide RNA, although this system can act in the organism, but it will cause Some inconveniences, first of all, the introduction of this system increases the burden of the exogenous DNA of the organism, and the processed guide RNA has an RNA recognition site more than the original structure, which is not required for the downstream reaction. There are also ribozyme sequences that can be self-cleaved at both ends of the gRNA sequence (Yangbin et al. JIPB 2014.56: 343-349), which can release sgRNA by self-cleavage function after transcription in vivo, but this method is limited. The ribozyme can only cleave one end, so that the sgRNA sequence is ligated with different ribozyme sequences at both ends, and it is not convenient to connect DNA fragments corresponding to different guide RNAs; another method is to connect multiple guide RNAs through tRNA, so that After expression in vivo, this long transcript releases a number of different guide RNAs via the in vivo tRNA processing system, which has been validated in both rice and fruit flies (US2016/031928 A1).

但是，仍然需要制备更为简单通用的，能在哺乳动物细胞内高效表达的CRISPR/Cas9***的多基因编辑方法。However, there is still a need to prepare a more simple and versatile multi-gene editing method for the CRISPR/Cas9 system that can be efficiently expressed in mammalian cells.

发明简述Brief description of the invention

本发明提供了一种在受体细胞中产生多元单链引导RNA(sgRNA)的方法，该方法包括，获得多核苷酸构建体，该构建体包含编码两个或多个sgRNA与tRNA串联单元的DNA序列，将所述构建体引入受体细胞，所述受体细胞的tRNA加工***在tRNA两端切割该异源多核苷酸，产生单链引导RNA(sgRNA)。The invention provides a method for producing a multi-primary single-stranded guide RNA (sgRNA) in a recipient cell, the method comprising obtaining a polynucleotide construct comprising a tandem unit encoding two or more sgRNAs and tRNAs The DNA sequence is introduced into a recipient cell, and the tRNA processing system of the recipient cell cleaves the heterologous polynucleotide at both ends of the tRNA to produce a single-stranded guide RNA (sgRNA).

优选地，本发明的单链引导RNA(sgRNA)是分别针对T细胞受体恒定区基因(TRAC)，组织相容性复合体基因(B2M)，和表皮生长因子受体基因(EGFR)的三个sgRNA。Preferably, the single-stranded guide RNA (sgRNA) of the present invention is directed against the T cell receptor constant region gene (TRAC), the histocompatibility complex gene (B2M), and the epidermal growth factor receptor gene (EGFR), respectively. sgRNA.

优选地，本发明的tRNA是可以形成tRNA特征茎环结构的序列，包括可以转运不同氨基酸的tRNA，以及来源于不同物种的tRNA序列。Preferably, the tRNA of the invention is a sequence which can form a stem-loop structure of a tRNA, including tRNAs which can transport different amino acids, and tRNA sequences derived from different species.

优选地，本发明的受体细胞是哺乳动物细胞，例如293T，K562，Jurkat，Hela细胞等。Preferably, the recipient cell of the invention is a mammalian cell, such as 293T, K562, Jurkat, Hela cells, and the like.

本发明进一步涉及一种在受体细胞中产生多元单链引导RNA(sgRNA)和Cas蛋白的方法，包括表达多元sgRNA元件通用的序列，可变区域的设计指导，以及一步构建的流程方法。The invention further relates to a method of producing a multivariate single-stranded guide RNA (sgRNA) and a Cas protein in a recipient cell, comprising a sequence for expression of a versatile sgRNA element, a design guide for a variable region, and a flow method for one-step construction.

本发明进一步包括一种核酸构建体，该构建体用于产生多元单链引导RNA(sgRNA)，其包含：编码两个或多个sgRNA与tRNA串联单元的DNA序列。The invention further encompasses a nucleic acid construct for producing a multi-primary single-stranded guide RNA (sgRNA) comprising: a DNA sequence encoding two or more sgRNAs in tandem with tRNA.

优选地，在每个单链引导RNA的骨架区的末端加入tRNA序列，然后直接串连起来。Preferably, tRNA sequences are added at the end of the backbone region of each single-stranded guide RNA and then ligated directly.

优选地，以引导RNA的骨架区与tRNA序列融合作为串联单元模板，其中两者连接的区域含有tRNA的前导序列，一般为六个碱基。Preferably, the framework region of the guide RNA is fused to the tRNA sequence as a tandem unit template, wherein the region to which the two are joined contains a leader sequence of the tRNA, typically six bases.

优选地，以多个引导RNA的特异区来设计引物分别对模板进行扩增以形成串联单元。Preferably, primers are designed with a plurality of specific regions of the guide RNA to amplify the template to form a tandem unit.

优选地，以Gibbson连接的方法将串联单元进行顺序组装，连接到启动子之后，更优选地，连接到含启动子序列的线性化质粒中。Preferably, the tandem units are sequentially assembled by Gibbson ligation, ligated to the promoter, and more preferably, ligated into a linearized plasmid containing the promoter sequence.

本发明公开了以下序号所示的技术方案：The invention discloses the technical solution shown by the following serial number:

1.一种在细胞内表达CRISPR-Cas体系的方法，包括：A method of expressing a CRISPR-Cas system in a cell, comprising:

(a)构建包含表达(sgRNA-tRNA)n的DNA片段，其中n为1-8之间的整数；(a) constructing a DNA fragment comprising the expression (sgRNA-tRNA)n, wherein n is an integer between 1 and 8;

(b)将步骤(a)的DNA片段转入细胞内；(b) transferring the DNA fragment of step (a) into the cell;

(c)表达在细胞内的sgRNA与细胞中的Cas蛋白结合，形成CRISPR-Cas体系。(c) The sgRNA expressed in the cell binds to the Cas protein in the cell to form a CRISPR-Cas system.

2.如技术方案1所述的方法，其中n为1或2或3。2. The method of embodiment 1, wherein n is 1 or 2 or 3.

3.如技术方案1或2所述的方法，其中步骤(a)中包含表达(sgRNA-tRNA)n的DNA片段为DNA质粒。3. The method according to claim 1 or 2, wherein the DNA fragment expressing (sgRNA-tRNA)n in step (a) is a DNA plasmid.

4.如技术方案1-3任一项所述的方法，其中细胞中的Cas蛋白是通过电穿孔转染法进入细胞的；或者是通过构建能表达Cas蛋白的DNA质粒，通过电穿孔转染，脂质体转染或病毒侵染在细胞中表达Cas蛋白。4. The method according to any one of claims 1 to 3, wherein the Cas protein in the cell is introduced into the cell by electroporation transfection; or by constructing a DNA plasmid capable of expressing the Cas protein, transfected by electroporation , liposome transfection or viral infection expresses Cas protein in cells.

5.如技术方案4所述的方法，其中表达Cas蛋白的DNA质粒是与步骤(a)的DNA片段属于同一个质粒。5. The method according to claim 4, wherein the DNA plasmid expressing the Cas protein belongs to the same plasmid as the DNA fragment of the step (a).

6.如技术方案4所述的方法，其中表达Cas蛋白的DNA质粒与步骤(a)的DNA片段不属于同一个质粒。6. The method according to claim 4, wherein the DNA plasmid expressing the Cas protein does not belong to the same plasmid as the DNA fragment of the step (a).

7.如前述任一项技术方案所述的方法，其中表达sgRNA的序列为5’-(X)m-恒定区骨架序列-3’，其中X选自A、U、C和G的任一个碱基，m为0-20任一整数，优选m＝17，18，19或20。7. The method according to any one of the preceding claims, wherein the sequence expressing the sgRNA is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from any one of A, U, C and G The base, m is any integer from 0 to 20, preferably m = 17, 18, 19 or 20.

8.如前述任一项技术方案所述的方法，其中sgRNA能特异性结合B2M基因、TRAC基因或EGFR基因。8. The method of any one of the preceding claims, wherein the sgRNA is capable of specifically binding to a B2M gene, a TRAC gene or an EGFR gene.

9.如前述任一项技术方案所述的方法，其中tRNA选自人-chr17.tRNA2-6-GlyGCC、玉米-chr9.trna85-GlyGCC、人-chr1.tRNA34-GlyGCC和人-chr17.tRNA41-SerCGA中的一种。9. The method according to any one of the preceding claims, wherein the tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.trna85-GlyGCC, human-chr1.tRNA34-GlyGCC and human-chr17.tRNA41- One of the SerCGAs.

10.如前述任一项技术方案所述的方法，其中的细胞是哺乳动物细胞。10. The method of any of the preceding claims, wherein the cells are mammalian cells.

11.如技术方案10所述的方法，其中的细胞是293T或K562细胞。11. The method of embodiment 10, wherein the cell is a 293T or K562 cell.

12.如前述任一项技术方案所述的方法，其中的Cas蛋白是Cas9蛋白。12. The method of any of the preceding claims, wherein the Cas protein is a Cas9 protein.

13.如技术方案11所述的方法，其中的Cas9蛋白是来源于化脓链球菌或金黄色葡萄球菌的Cas9蛋白。13. The method of embodiment 11, wherein the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes or Staphylococcus aureus.

14.一种质粒，包含了技术方案1中的表达(sgRNA-tRNA)n的DNA片段，其中n为1-8之间的整数。A plasmid comprising the DNA fragment expressing (sgRNA-tRNA)n in the first aspect, wherein n is an integer between 1 and 8.

15.如技术方案14所述的质粒，其中n为1或2或3。15. The plasmid of embodiment 14, wherein n is 1 or 2 or 3.

16.如技术方案14或15所述的质粒，还包含了表达Cas蛋白的DNA序列。16. The plasmid according to claim 14 or 15, which further comprises a DNA sequence which expresses the Cas protein.

17.如技术方案14-16任一项所述的质粒，其中表达sgRNA的序列为5’-(X)m-恒定区骨架序列-3’，其中X选自A、T、C和G的任一个碱基，m为0-20任一整数，优选m＝17，18，19或20。The plasmid according to any one of claims 14-16, wherein the sequence expressing the sgRNA is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from the group consisting of A, T, C and G Any base, m is any integer from 0 to 20, preferably m = 17, 18, 19 or 20.

18.如技术方案14-17任一项所述的方法，其中tRNA选自人-chr17.tRNA2-6-GlyGCC、玉米-chr9.tRNA85-GlyGCC、人-chr1.tRNA34-GlyGCC和人-chr17.tRNA41-SerCGA中的一种。18. The method of any one of claims 14-17, wherein the tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.tRNA85-GlyGCC, human-chr1.tRNA34-GlyGCC, and human-chr17. One of tRNA41-SerCGA.

19.如技术方案14-18任一项所述的质粒，其中的Cas蛋白是Cas9蛋白。The plasmid according to any one of claims 14 to 18, wherein the Cas protein is a Cas9 protein.

20.如技术方案19所述的方法，其中的Cas9蛋白是来源于化脓链球菌或金黄色葡萄球菌的Cas9蛋白。20. The method of embodiment 19, wherein the Cas9 protein is a Cas9 protein derived from S. pyogenes or S. aureus.

本发明的CRISPR/Cas蛋白的表达体系能快速实现多个sgRNA的元件在质粒中组装，进而可以在体内同时靶向多靶点进行高效编辑，本发明还能提供一个通用平台用于检验不同连接元件体内被加工释放sgRNA的效率。The CRISPR/Cas protein expression system of the invention can quickly realize the assembly of multiple sgRNA elements in a plasmid, and can simultaneously target multiple targets for efficient editing in vivo, and the invention can also provide a universal platform for testing different connections. The efficiency of processing the sgRNA in the component body.

附图说明DRAWINGS

图1：STS-pUC57质粒示意图；Figure 1: Schematic diagram of the STS-pUC57 plasmid;

其中sgRNA1靶向基因TRAC，sgRNA2靶向基因B2M，tRNA为人-chr17.tRNA2-6-GlyGCC(下文用tRNA ^gly代替) Among them, sgRNA1 targets gene TRAC, sgRNA2 targets gene B2M, and tRNA is human-chr17.tRNA2-6-GlyGCC (hereinafter replaced by tRNA ^gly )

图2：对比不同构建方式对双基因的编辑效率；Figure 2: Comparison of editing efficiency of double genes by different construction methods;

其中STS、STST、TSTS和TSTST表示在质粒U6启动子和终止子之间连有不同的元件；STS元件为sgRNA1-tRNA ^gly-sgRNA2；STST元件为sgRNA1-tRNA ^gly-sgRNA2-tRNA ^gly；TSTS元件为tRNA ^gly-sgRNA1-tRNA ^gly-sgRNA2；TSTST元件为tRNA ^gly-sgRNA1-tRNA ^gly-sgRNA2-tRNA ^gly；S2TS1T元件为sgRNA2-tRNA ^gly-sgRNA1-tRNA ^gly；+Cas9为共转可以表达SpCas9的质粒；STST-Cas9为可以同时表达STST元件以及Cas9蛋白的单质粒。 Among them, STS, STST, TSTS and TSTST indicate that there are different elements between the promoter of the plasmid U6 and the terminator; the STS element is sgRNA1-tRNA ^gly- sgRNA2; the STST element is sgRNA1-tRNA ^gly- sgRNA2-tRNA ^gly ; TSTS element For tRNA ^gly -sgRNA1-tRNA ^gly -sgRNA2; TSTST element is tRNA ^gly -sgRNA1-tRNA ^gly -sgRNA2-tRNA ^gly ; S2TS1T element is sgRNA2-tRNA ^gly -sgRNA1-tRNA ^gly ; +Cas9 is co-transformed plasmid expressing SpCas9 ;STST-Cas9 is a single plasmid that can simultaneously express STST elements as well as Cas9 proteins.

图3：单质粒***与传统共转对多基因编辑效率对比；Figure 3: Comparison of the efficiency of single-plasmid system with traditional co-transformation for multi-gene editing;

其中S1-Cas9：可以表达sgRNA1和Cas9的质粒，编辑靶点TRAC基因；Wherein S1-Cas9: a plasmid capable of expressing sgRNA1 and Cas9, editing the target TRAC gene;

S2-Cas9：可以表达sgRNA2和Cas9的质粒，编辑靶点B2M基因；S2-Cas9: a plasmid capable of expressing sgRNA2 and Cas9, editing the target B2M gene;

S3-Cas9：可以表达sgRNA3和Cas9的质粒，编辑靶点EGFR基因；S3-Cas9: a plasmid capable of expressing sgRNA3 and Cas9, editing the target EGFR gene;

+Cas9为共转可以表达SpCas9的质粒；STST-Cas9为可以同时表达STST元件以及Cas9蛋白的单质粒；STSTST-Cas9：为可以同时表达STSTST(sgRNA1-tRNA ^gly-sgRNA2--tRNA ^gly–sgRNA3-tRNA ^gly)元件以及Cas9蛋白的单质粒。 +Cas9 is a plasmid that expresses SpCas9; STST-Cas9 is a single plasmid that can simultaneously express STST elements and Cas9 protein; STSTST-Cas9: can simultaneously express STSTST (sgRNA1-tRNA ^gly- sgRNA2--tRNA ^gly- sgRNA3- The tRNA ^gly ) element and the single plasmid of the Cas9 protein.

图4：自切割元件合成示意图；Figure 4: Schematic diagram of the synthesis of self-cutting elements;

其中单链合成中的一对引物(通用正向引物及可变反向引物)及元件合成中N对引物需要通过序列合成得到，其余产物均可以通过PCR扩增得到。Among them, a pair of primers (general forward primer and variable reverse primer) in single-strand synthesis and N-pair primers in element synthesis need to be synthesized by sequence, and other products can be obtained by PCR amplification.

图5：起始质粒及质粒最终构建体示意图；Figure 5: Schematic diagram of the starting plasmid and the final construct of the plasmid;

其中，线性化质粒引物设计示意(A)及线性化质粒产物示意(B)；线性化质粒与图4中合成的多片段连接后最终形成质粒构建体(C)；其中质粒骨架(Plasmid scaffold)可以多变，除了包含质粒复制，抗性基因等质粒基本元件外，还可以含有Cas9表达元件或是应用于慢病毒包装的元件。Among them, the linearized plasmid primer design is shown (A) and the linearized plasmid product is shown (B); the linearized plasmid is ligated with the multi-fragment synthesized in Figure 4 to finally form a plasmid construct (C); wherein the plasmid skeleton (Plasmid scaffold) It can be varied, and may contain Cas9 expression elements or elements for lentiviral packaging, in addition to plasmid basic elements including plasmid replication, resistance genes, and the like.

图6：tRNA二级结构示意图；图A，B，C和D分别指示T(人-chr17.tRNA2-6-GlyGCC)；T1(玉米-chr9.tRNA85-GlyGCC)；T2(人-chr1.tRNA34-GlyGCC)；T3(人-chr17.tRNA41-SerCGA)Figure 6: Schematic diagram of secondary structure of tRNA; panels A, B, C and D indicate T (human-chr17.tRNA2-6-GlyGCC); T1 (corn-chr9.tRNA85-GlyGCC); T2 (human-chr1.tRNA34, respectively) -GlyGCC); T3 (human-chr17.tRNA41-SerCGA)

图7：以图6中不同的tRNA(T，T1，T2或T3)作为连接元件，所构建的多sgRNA表达元件在293T细胞里，对靶点的编辑效率对比。Figure 7: Comparison of the editing efficiency of the target multiplexed sgRNA expression elements in 293T cells using the different tRNAs (T, T1, T2 or T3) in Figure 6 as the ligation elements.

图8：以图6中不同的tRNA(T1，T2或T3)作为连接元件，所构建的多sgRNA表达元件在K562细胞里，对靶点的编辑效率对比。Figure 8: Comparison of the editing efficiency of the target multi-sgRNA expression elements in K562 cells with different tRNAs (T1, T2 or T3) as the ligation elements in Figure 6.

发明详述Detailed description of the invention

CRISPR/Cas体系CRISPR/Cas System

CRISPR/Cas***是存在于原核生物的一种免疫***，主要是来针对于外来入侵的基因元件，比如质粒或是病毒等。该***可以从外来的基因元件中获得免疫，即将其DNA片段以一定的格式保留下来。而保留下来的DNA可以转录加工成引导RNA，并与该***的单一蛋白或蛋白复合体结合，定向识别并切割再次入侵的基因元件。其中CRISPR/Cas9***是其中一个广为研究的分支，由于该***的效应部分只涉及到单个蛋白(Cas9蛋白)，所以在科研中的应用更为简便广泛。The CRISPR/Cas system is an immune system found in prokaryotes, mainly for genetic components that are invasive, such as plasmids or viruses. The system can be immunized from foreign genetic elements by retaining its DNA fragments in a certain format. The retained DNA can be transcribed into a guide RNA and combined with a single protein or protein complex of the system to directionally recognize and cleave the re-invading genetic elements. Among them, the CRISPR/Cas9 system is one of the widely studied branches. Since the effect part of the system involves only a single protein (Cas9 protein), it is more convenient and widely used in scientific research.

Cas蛋白Cas protein

成簇规律间隔短回文重复序列(CRISPR)和CRISPR结合蛋白(Cas蛋白)组成了一套强大的核酸酶***。CRISPR相关内切酶Cas蛋白可以通过单链引导RNA(sgRNA)靶向特定的基因组序列。The clustered regular interval palindrome repeat (CRISPR) and the CRISPR binding protein (Cas protein) constitute a powerful nuclease system. The CRISPR-associated endonuclease Cas protein can target specific genomic sequences via single-stranded guide RNA (sgRNA).

Cas蛋白的非限制性实例包括：Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8，Cas9(也称为Csn1或Csx12)、Cas10、Cas12、Cas13、Cas14、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4其在不同物种中的同源蛋白、核酸内切酶失活的突变蛋白、或其修饰形式。Non-limiting examples of Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3 , Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3 , Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologous proteins in different species, mutant proteins inactivated by endonucleases, or modified forms thereof.

在一个具体实施方式中，本发明的Cas蛋白是Cas9蛋白。In a specific embodiment, the Cas protein of the invention is a Cas9 protein.

Cas9，也称为Csn1或Csx12，是既参与crRNA生物合成又参与摧毁入侵DNA的巨型蛋白质。已经在不同的细菌物种如嗜热链球菌(S.thermophilus)、无害利斯特氏菌 (Listeria innocua)(Gasiunas，Barrangou et al.2012；Jinek，Chylinski et al.2012)和化脓性链球菌(S.Pyogenes)(Deltcheva，Chylinski et al.2011)中描述了Cas9。本发明的Cas9包括但不限于以下类型：化脓链球菌(Streptococcus pyogenes)Cas9蛋白，其氨基酸序列见SwissProt数据库登录号Q99ZW2；脑膜炎奈瑟氏菌(Neisseria meningitidis)Cas9蛋白，其氨基酸序列见UniProt数据库编号A1IQ68；嗜热链球菌(Streptococcus thermophilus)Cas9蛋白，其氨基酸序列见UniProt数据库编号Q03LF7；金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白，其氨基酸序列见UniProt数据库编号J7RUA5。Cas9, also known as Csn1 or Csx12, is a giant protein involved in both crRNA biosynthesis and in the destruction of invading DNA. Already in different bacterial species such as S. thermophilus, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes Cas9 is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011). The Cas9 of the present invention includes, but is not limited to, the following types: Streptococcus pyogenes Cas9 protein, the amino acid sequence of which is described in SwissProt database accession number Q99ZW2; Neisseria meningitidis Cas9 protein, the amino acid sequence of which is described in the UniProt database. No. A1IQ68; Streptococcus thermophilus Cas9 protein, the amino acid sequence of which is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, the amino acid sequence of which is shown in UniProt database number J7RUA5.

单链引导RNA(sgRNA)Single-stranded guide RNA (sgRNA)

在CRISPR/Cas9***中，引导RNA由两部分组成，一部分是crRNA，另一部分是trans-crRNA(tracrRNA)，两者由碱基对互补的作用形成复合体。在实际使用中，这两者可以融合在一起，以单链引导RNA的形式存在，它依然能够形成与之前双链相似的结构，行使引导蛋白识别及切割外来基因的作用。In the CRISPR/Cas9 system, the guide RNA consists of two parts, one is the crRNA and the other is the trans-crRNA (tracrRNA), which forms a complex by the complementation of the base pair. In practical use, the two can be fused together in the form of a single-stranded guide RNA, which is still capable of forming a structure similar to the previous double strand, and functions to guide the recognition and cleavage of foreign genes.

sgRNA为单链引导RNA，一般包含引导序列、tracr配对序列和tracr序列。sgRNA的结构包括开始的～20碱基的可变区(即引导序列)以及接连的约80个碱基的恒定区骨架(包括tracr配对序列和tracr序列，在sgRNA中以融合单链形式的出现)。The sgRNA is a single-stranded guide RNA and generally comprises a leader sequence, a tracr pairing sequence and a tracr sequence. The structure of the sgRNA includes the initial -20 base variable region (ie, the leader sequence) and the successive constant region backbone of approximately 80 bases (including the tracr pairing sequence and the tracr sequence, which appear as fused single-stranded forms in the sgRNA). ).

在本发明具体实施方式中，所述的单链引导RNA序列为5’-(X)m-恒定区骨架序列-3’，其中X选自A、U、C和G的任一个碱基，m为0-20任一整数，优选m＝17，18，19或20，更优选m＝20；其中骨架序列是连接在(X)m后的约80个碱基序列。In a specific embodiment of the present invention, the single-stranded guide RNA sequence is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from any one of A, U, C, and G, m is any integer from 0 to 20, preferably m = 17, 18, 19 or 20, more preferably m = 20; wherein the backbone sequence is about 80 base sequences attached after (X)m.

在本发明具体实施方式中，sgRNA是能特异性结合B2M基因、TRAC基因或EGFR基因的sgRNA。In a specific embodiment of the invention, the sgRNA is an sgRNA capable of specifically binding to a B2M gene, a TRAC gene or an EGFR gene.

转运RNA(tRNA)Transport RNA (tRNA)

转运RNA是可以转运氨基酸的聚核糖核酸(RNA)分子，它的成熟形式通常由70-90个核苷酸组成的单链形式。这些单链通常由分子内的碱基配对可以进一步形成三叶草形式的二级结构。转运RNA存在于各种生命体中，包括细菌、植物、动物等等。它在生命体内是连接信使RNA和蛋白的工具。转运RNA一端通过碱基配对与信使RNA结合，翻译其携带的遗传信息，另一端则连接特异的氨基酸，将其按照信使RNA的指引顺序运输到蛋白质合成机器上。这样生物体的遗传信息才会体现在蛋白的氨基酸序列上，再由各种蛋白质作为执行生命活动的主力。所以转运RNA存在广泛，在进化中保持着稳定且基本一致的结构。tRNA是由基因组tRNA基因转录出的转录本(从5’端到3’端)进一步加工而来。这些加工包括去除部分内含子序列(intron)，tRNA 5’和3’两端的剪切，3’端的加尾以及碱基的修饰等等。其中tRNA两端的剪切是本专利中所利用到的性质：在真核生物中(包括植物和动物)主要依靠RNaseP去除5’端多余序列，和RNaseZ去除3’端多余序列。A transport RNA is a polyribonucleic acid (RNA) molecule that can transport amino acids, and its mature form is usually in the form of a single strand consisting of 70-90 nucleotides. These single strands can be further formed into a secondary structure in the form of clover, usually by base pairing within the molecule. Transport RNA is present in a variety of living organisms, including bacteria, plants, animals, and the like. It is a tool for connecting messenger RNA and proteins in life. One end of the transfer RNA binds to the messenger RNA through base pairing, translates the genetic information it carries, and the other end links specific amino acids and transports them to the protein synthesis machine in the order of messenger RNA. The genetic information of the organism will be reflected in the amino acid sequence of the protein, and then the various proteins will be the main force for performing life activities. Therefore, the transport RNA is widely existed and maintains a stable and basically consistent structure in evolution. The tRNA is further processed from a transcript transcribed from the genomic tRNA gene (from the 5' end to the 3' end). These processing include removal of a portion of the intron, cleavage at the 5' and 3' ends of the tRNA, tailing at the 3' end, and modification of the base and the like. The cleavage at both ends of the tRNA is a property utilized in this patent: in eukaryotes (including plants and animals), RNaseP is mainly used to remove the 5'-end redundant sequence, and RNaseZ is used to remove the 3'-end redundant sequence.

在具体实施方式中，本发明的tRNA基因来源于人(Homo sapiens)或玉米种属(Zea_mays)。在另一具体实施方式中，本发明的tRNA基因是人-chr17.tRNA2-6-GlyGCC(tRNA序列chr17:8029064-8029134)；人-chr1.tRNA34-GlyGCC(chr1:161413094-161413164)；人-chr17.tRNA41-SerCGA(chr17:8042199-8042280)；玉米-chr9.tRNA85-GlyGCC(chr9:152440635-152440705)；优选为人-chr1.tRNA34-GlyGCC。上述基因信息可从tRNA网站查得gtrnadb.ucsc.edu/。In a specific embodiment, the tRNA gene of the invention is derived from human (Homo sapiens) or maize species (Zea_mays). In another specific embodiment, the tRNA gene of the invention is human-chr17.tRNA2-6-GlyGCC (tRNA sequence chr17:8029064-8029134); human-chr1.tRNA34-GlyGCC (chr1:161413094-161413164); human- chr17.tRNA41-SerCGA (chr17:8042199-8042280); maize-chr9.tRNA85-GlyGCC (chr9: 152440635-152440705); preferably human-chr1.tRNA34-GlyGCC. The above gene information can be found from the tRNA website gtrnadb.ucsc.edu/.

质粒构建体Plasmid construct

本发明运用的CRISPR/Cas***均是以DNA的形式，涉及到质粒的构建。质粒是细菌染色体外，存在于细胞质中的遗传物质。通常是闭合环状双链DNA分子，具有自我复制的功能，并且表达所携带的遗传物质。本发明主要利用质粒导入细胞内，在细胞里发挥以下两个作用：质粒携带的Cas表达***(包括启动子，Cas人源化序列，入核信号等)可以在细胞体内经过转录翻译形成Cas蛋白；质粒携带的引导RNA表达盒(cassette)可以在细胞体内经过转录和加工得到成熟的单链引导RNA。The CRISPR/Cas system used in the present invention is in the form of DNA and involves the construction of a plasmid. A plasmid is a genetic material that is extrachromosomally present in the cytoplasm. It is usually a closed circular double-stranded DNA molecule that functions as a self-replicating agent and expresses the genetic material it carries. The invention mainly uses a plasmid to introduce into a cell, and plays the following two functions in the cell: the Cas expression system carried by the plasmid (including a promoter, a Cas humanized sequence, a nuclear signal, etc.) can be transcribed and translated into a Cas protein in the cell body. The plasmid-carrying guide RNA expression cassette (cassette) can be transcribed and processed in the cell body to obtain mature single-stranded guide RNA.

可以表达(sgRNA-tRNA)n的DNA序列可以和Cas蛋白的DNA序列构建在同一个质粒中，也可以分别构建在两个质粒中。该质粒也可以是用于病毒制备的穿梭质粒。The DNA sequence which can express (sgRNA-tRNA)n can be constructed in the same plasmid as the DNA sequence of the Cas protein, or can be constructed separately in two plasmids. This plasmid may also be a shuttle plasmid for virus preparation.

多元单链引导RNA(sgRNA)与tRNA构建体的表达方法Expression method of multiple single-stranded guide RNA (sgRNA) and tRNA construct

将tRNA和sgRNA串连起来，可以利用tRNA在生物体内的加工***将其连接的sgRNA在体内加工释放出来。参考之前对于tRNA连接引导RNA的研究，我们总结有以下几种模式。在果蝇模型实验中，研究人员倾向于在两端加上tRNA以提高编辑效率；而在水稻模型中，研究人员采用tRNA-sgRNA为基本单位进行多个串连以达到多位点靶向编辑。本发明对于各种sgRNA和tRNA连接方式和顺序进行了比较，其中的构建包括序列直接合成，通过酶切连接，Gibbson组装以及Golden Gate方法来进行分子克隆，以达到不同的构建体。By connecting tRNA and sgRNA in tandem, the tRNA can be processed and released in vivo using the processing system of tRNA in vivo. With reference to previous studies on tRNA-linked guide RNA, we have summarized the following patterns. In the Drosophila model experiment, the researchers tend to add tRNA to both ends to improve editing efficiency. In the rice model, the researchers used tRNA-sgRNA as the basic unit to perform multiple concatenations to achieve multi-site targeted editing. . The present invention compares the manner and sequence of various sgRNA and tRNA linkages, including direct sequence synthesis, molecular cloning by restriction enzyme ligation, Gibbson assembly, and the Golden Gate method to achieve different constructs.

优选地，我们将每个可供体内切割的单位定义为一个sgRNA接连一个指定的tRNA，形成多个sgRNA-tRNA的DNA序列构建体，即(sgRNA-tRNA)n，n为1-8之间的整数，优选n＝1，2或3。每个单位只有前约20个碱基根据所靶向序列的不同而有所改变。其后连接的tRNA可视为原始sgRNA的骨架延伸部分，与之并为恒定区。Preferably, we define each unit that can be cleaved in vivo as a sgRNA followed by a specified tRNA to form a DNA sequence construct of multiple sgRNA-tRNAs, ie (sgRNA-tRNA)n, n between 1-8 An integer, preferably n = 1, 2 or 3. Only about 20 bases in the front of each unit vary depending on the sequence being targeted. The tRNA that is subsequently ligated can be considered as the backbone extension of the original sgRNA, which is also the constant region.

本发明中将恒定区作为模板，而可变区的序列可以通过不同引物经由PCR反应引入。模板可以直接合成双链(～160nt)，也可以合成部分互补的单链通过PCR补平来形成双链。由于恒定区中sgRNA原骨架已经被优化，对于同一种的Cas9不需要在这一部分进行改变(对于不同来源的Cas9蛋白，这个骨架序列一般不同)，而tRNA的选择有许多空间，包括对于不同物种不同种类的tRNA，还可以选择其他非tRNA的可以两端切割的RNA元件。本发明中对于每个模板，合成了两条单链，一条只包括sgRNA骨架部分，另一条包括不同tRNA序列及与第一条少量重叠的部分。对于两条单链进行退火处理和PCR延伸，即得到双链模板；然后根据多个靶点设计引物进行PCR扩增，得到多个sgRNA-tRNA元件。对质粒载体进行线性化，与sgRNA-tRNA元件进行无缝连接，得到所需构建的质粒。The constant region is used as a template in the present invention, and the sequence of the variable region can be introduced via a PCR reaction through different primers. The template can be directly synthesized into a double strand (~160 nt), or a partially complementary single strand can be synthesized by PCR to form a double strand. Since the sgRNA protoskeleton in the constant region has been optimized, it is not necessary to change this part for the same Cas9 (this skeletal sequence is generally different for different sources of Cas9 protein), and there are many spaces for tRNA selection, including for different species. Different types of tRNA can also select other non-tRNA RNA elements that can be cleaved at both ends. In the present invention, for each template, two single strands are synthesized, one comprising only the sgRNA backbone portion and the other comprising different tRNA sequences and portions that overlap a small amount with the first strip. The two single strands are annealed and PCR extended to obtain a double-stranded template; then PCR amplification is performed according to multiple target design primers to obtain multiple sgRNA-tRNA elements. The plasmid vector was linearized and ligated seamlessly with the sgRNA-tRNA element to obtain the plasmid of the desired construction.

在细胞内构建CRISPR/Cas体系的方法Method for constructing CRISPR/Cas system in cells

构建(sgRNA-tRNA)n的DNA质粒，与可以表达Cas蛋白的质粒，或者构建可同时表达(sgRNA-tRNA)n和Cas蛋白的单质粒。本发明有如下三种方式在细胞内构建CRISPR/Cas体系。转染方法可以选用脂质体转染，电穿孔转染(电转)和病毒感染的方法。A DNA plasmid of (sgRNA-tRNA)n is constructed, together with a plasmid which can express the Cas protein, or a single plasmid which can simultaneously express (sgRNA-tRNA) n and Cas protein. The present invention has the following three ways to construct a CRISPR/Cas system in a cell. Transfection methods can be selected from liposome transfection, electroporation transfection (electroporation) and viral infection.

方法一：对于293T细胞进行脂质体转染：293T细胞复苏后，培养至第三代或以上，于转染前一天进行细胞传代，分细胞于24孔板，每个孔5万个细胞。待细胞生长约培养板底面积40％-60％时，用Lipo2000(Life 11668-027)作为转染试剂，每个孔转染总量为500ng的质粒。细胞转染后48小时，测定编辑效率。Method 1: Liposomal transfection of 293T cells: After 293T cells were resuscitated, cultured to the third generation or above, cells were passaged one day before transfection, and cells were plated in 24-well plates at 50,000 cells per well. When the cells were grown at about 40%-60% of the bottom area of the plate, Lipo2000 (Life 11668-027) was used as a transfection reagent, and a total of 500 ng of plasmid was transfected per well. Editing efficiency was determined 48 hours after cell transfection.

方法二：对于K562细胞进行电转，细胞复苏后，培养至第三代或以上，于转染当天进行细胞计数，混合细胞(200,000)和质粒(500ng)电转条件如下：电压1450V，10毫秒宽度，3脉冲数。细胞电转48小时，测定编辑效率。Method 2: K562 cells were electroporated, and after cell resuscitation, cultured to the third generation or above, cell counts were performed on the day of transfection, and the mixed cells (200,000) and plasmid (500 ng) were subjected to the following conditions: voltage 1450 V, 10 msec width, 3 pulses. The cells were electroporated for 48 hours and assay efficiency was determined.

方法三：将可以表达(gRNA-tRNA)n和Cas蛋白的DNA片段克隆进病毒载体，对293T细胞进行转染：转染前一天，将293T细胞消化传代至100mm培养皿中，密度为70-80％，培养24小时(完全培养基：DMEM(Gibco C11995500BT),10％血清(Corning 35010155)后，将培养基置换为Opti-MEM；将待转染的病毒载体质粒32μg和转染试剂分别溶于500μl Opti-MEM(Gibco 31985-070)，混匀静置5分钟，将转染试剂滴加到质粒中，混匀室温放置20分钟，将混合物加到细胞培养基中。培养6-8小时换新鲜完全培养基。转染48小时和72小时，分别收集培养上清液，于3500rpm离心10分钟去沉淀，上清经由0.22μm的滤膜过滤。病毒上清液通过置换培养基的方法进行感染其他原代细胞或细胞系，感染后6-8小时换新鲜完全培养基，继续培养40小时后分析被感染细胞的靶点基因编辑效率。Method 3: DNA fragments capable of expressing (gRNA-tRNA) n and Cas proteins were cloned into a viral vector, and 293T cells were transfected: one day before transfection, 293T cells were digested into 100 mm culture dishes at a density of 70- 80%, cultured for 24 hours (complete medium: DMEM (Gibco C11995500BT), 10% serum (Corning 35010155), the medium was replaced with Opti-MEM; 32 μg of the viral vector plasmid to be transfected and the transfection reagent were separately dissolved In 500 μl Opti-MEM (Gibco 31985-070), the mixture was allowed to stand for 5 minutes, and the transfection reagent was added dropwise to the plasmid, and the mixture was allowed to stand at room temperature for 20 minutes, and the mixture was added to the cell culture medium for 6-8 hours. The fresh complete medium was changed. The culture supernatant was collected for 48 hours and 72 hours, and the supernatant was collected by centrifugation at 3500 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter. The virus supernatant was subjected to a medium replacement method. Infect other primary cells or cell lines, change the fresh complete medium 6-8 hours after infection, and analyze the target gene editing efficiency of the infected cells after 40 hours of incubation.

基因编辑效率Gene editing efficiency

基因编辑效率测定有多种方法：对于被编辑的细胞进行二代测序，分析编辑位点的效率；对于编辑的细胞群进行单克隆培养，分析每个单克隆的基因型，与野生型进行对比得出编辑的比率等等。There are several methods for gene editing efficiency determination: second-generation sequencing of edited cells, analysis of editing site efficiency; monoclonal culture of edited cell populations, analysis of each monoclonal genotype, comparison with wild type Get the ratio of editing and so on.

本发明优选TIDE分析：细胞进行脂质体转染，电转或是病毒侵染后一般48-72小时，将细胞反复吹打，形成细胞悬液，收集在EP管里。将细胞悬液离心，弃上清。加入80微升DNA提取试剂QE(Lucigen:QE09050)，混匀细胞。98℃加热2分钟，再置于65℃6分钟。以此细胞裂解液为模板，进行目标基因片段的PCR扩增。扩增的产物进行测序，通过分析编辑位点的套峰情况来确定编辑的效率。Preferably, the TIDE assay is performed in the present invention: the cells are subjected to lipofection, electroporation or virus infection for 48-72 hours, and the cells are repeatedly blown to form a cell suspension, which is collected in an EP tube. The cell suspension was centrifuged and the supernatant was discarded. 80 μl of DNA extraction reagent QE (Lucigen: QE09050) was added and the cells were mixed. Heat at 98 ° C for 2 minutes and then at 65 ° C for 6 minutes. PCR amplification of the target gene fragment was carried out using this cell lysate as a template. The amplified products were sequenced and the editing efficiency was determined by analyzing the peaks of the editing sites.

B2M基因B2M gene

β-2微球蛋白，即B2M，是I类MHC分子的轻链，因此是主要组织相容性复合体(MHC)不可缺少的部分。在人类基因组中，B2M被位于15号染色体上的b2m基因编码，而其他MHC基因在6号染色体上以基因簇的形式存在。人类的B2M蛋白有119个氨基酸(见UniProt数据库编码P61769)。在缺乏β-2微球蛋白的小鼠模型中可以说明，B2M对I类MHC分子在细胞表面的呈递、多肽结合槽的稳定性是必须的。I类MHC分子存在于人体所有的有核细胞表面，MHC的错配会引起免疫排斥，导致移植物的破坏，而通过敲除B2M基因来去除细胞表面的I类MHC分子可以防止错配的发生。Beta-2 microglobulin, or B2M, is the light chain of class I MHC molecules and is therefore an integral part of the major histocompatibility complex (MHC). In the human genome, B2M is encoded by the b2m gene located on chromosome 15, while other MHC genes are present as gene clusters on chromosome 6. The human B2M protein has 119 amino acids (see UniProt database code P61769). In a mouse model lacking β-2 microglobulin, it can be demonstrated that B2M is essential for the presentation of class I MHC molecules on the cell surface and the stability of the polypeptide binding groove. Class I MHC molecules are present on all nucleated cell surfaces in the human body. Mismatches in MHC can cause immune rejection and cause graft destruction. Elimination of mismatches can be achieved by knocking out B2M genes to remove Class I MHC molecules on the cell surface. .

TRAC基因TRAC gene

T细胞受体(T cell receptor,TCR)是在T细胞表面的受体，参与T细胞与被呈递的抗原接触后的T细胞激活过程。一般情况下，TCR由α和β两条链组成，形成一个异二聚体，并与CD3分子一起在细胞表面形成T细胞受体复合物。每条α或β链都含有可变区和恒定区，其中α链的恒定区由位于14号染色体上的TRAC基因编码。TCR能识别与MHC分子结合的经过处理的多肽片段，由于识别需要MHC分子的呈递，也称为MHC限制性。当供者和受者的MHC分子不同时，TCR能够识别MHC的不同，并导致T细胞的激活和扩增，可能引起移植物抗宿主疾病(GvHD)。敲除TRAC基因可以去除TCRα链的表达，从而可以将TCR从T细胞表面除去，因此能够防止TCR识别同种异体抗原而引起的移植物抗宿主疾病。T cell receptor (TCR) is a receptor on the surface of T cells involved in the activation of T cells after contact of T cells with the presented antigen. In general, TCR consists of two chains, alpha and beta, forming a heterodimer and forming a T cell receptor complex on the cell surface along with the CD3 molecule. Each of the alpha or beta strands contains a variable region and a constant region, wherein the constant region of the alpha chain is encoded by a TRAC gene located on chromosome 14. TCR recognizes a processed polypeptide fragment that binds to an MHC molecule and is also referred to as MHC restriction because recognition requires the presentation of MHC molecules. When the MHC molecules of the donor and recipient are different, the TCR is able to recognize the difference in MHC and cause activation and expansion of T cells, possibly causing graft versus host disease (GvHD). Knocking out the TRAC gene removes the expression of the TCR alpha chain, thereby removing the TCR from the surface of the T cell, thereby preventing graft-versus-host disease caused by TCR recognition of allogeneic antigen.

EGFR基因EGFR gene

EGFR(epidermal growth factor receptor，简称为EGFR、ErbB-1或HER1)是表皮生长因子受体(HER)家族成员之一。该家族包括HER1(erbB1，EGFR)、HER2(erbB2，NEU)、HER3(erbB3)及HER4(erbB4)。HER家族在细胞生理过程中发挥重要的调节作用。EGFR广泛分布于哺乳动物上皮细胞、成纤维细胞、胶质细胞、角质细胞等细胞表面，EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥重要的作用。EGFR (epidermal growth factor receptor, hereinafter referred to as EGFR, ErbB-1 or HER1) is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). The HER family plays an important regulatory role in the process of cell physiology. EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc. EGFR signaling pathway plays an important role in the physiological processes such as cell growth, proliferation and differentiation.

具体实施方式detailed description

实施例1.tRNA介导sgRNA的不同表达构建体在细胞内的编辑效率比较Example 1. Comparison of editing efficiency of different expression vectors of tRNA-mediated sgRNA in cells

实验目的：验证多个sgRNA同时表达的有效性，tRNA的数目和位置对整个表达元件的影响，以及sgRNA在表达元件中的顺序影响，单质粒***的有效性。测试***选用哺乳动物细胞系293T，由每个sgRNA对应位点的编辑效率来比较不同构建体的有效性，同时结合操作上的简易性，最后得出高效的通用模型。EXPERIMENTAL OBJECTIVE: To verify the validity of simultaneous expression of multiple sgRNAs, the effect of the number and location of tRNAs on the entire expression element, and the sequence of sgRNA in the expression elements, the validity of the single plasmid system. The test system uses the mammalian cell line 293T, and the editing efficiency of each sgRNA corresponding site is used to compare the effectiveness of different constructs. At the same time, combined with the ease of operation, a highly efficient general model is obtained.

方法及材料：Method and materials:

293T细胞用于此研究293T cells were used in this study

1.sgRNA的元件通过基因合成获得1. The components of sgRNA are obtained by gene synthesis

基因合成如下，其中顺序包括U6启动子(SEQ ID No:1)，表达sgRNA1的DNA片段(SEQ ID No:2)，表达tRNA ^gly的DNA片段(SEQ ID No:3)，表达sgRNA2的DNA片段(SEQ ID No:4)和U6终止子(DNA片段：tttttt)。以下简称sgRNA1-tRNA ^gly-sgRNA2为STS元件，其中S代表sgRNA，T代表tRNA。 The gene was synthesized as follows, wherein the sequence includes a U6 promoter (SEQ ID No: 1), a DNA fragment expressing sgRNA1 (SEQ ID No: 2), a DNA fragment expressing tRNA ^gly (SEQ ID No: 3), and a DNA fragment expressing sgRNA2. (SEQ ID No: 4) and U6 terminator (DNA fragment: tttttt). Hereinafter, sgRNA1-tRNA ^gly- sgRNA2 is an STS element, wherein S represents sgRNA and T represents tRNA.

该基因片段被亚克隆进pUC57载体中，所形成的质粒STS-pUC57(序列图谱见图1)转化入感受态大肠杆菌(DH5a)，以菌体的形式由合成公司(GeneWiz)交付。The gene fragment was subcloned into the pUC57 vector, and the resulting plasmid STS-pUC57 (sequence map is shown in Figure 1) was transformed into competent E. coli (DH5a) and delivered as a bacterial form by the synthesis company (GeneWiz).

2.元件改造2. Component transformation

2.1 STST及TSTS表达元件的构建2.1 Construction of STST and TSTS expression components

通过对上述步骤获得的质粒STS-pUC57做序列片段增加的方式实现进一步改造，分别在STS元件的后端和前端各加上一个tRNA序列，进而分别得到STST表达元件和TSTS表达元件。所需要合成的引物如下。Further modification was carried out by increasing the sequence fragment of the plasmid STS-pUC57 obtained in the above step, and a tRNA sequence was added to the rear end and the front end of the STS element, respectively, to obtain an STST expression element and a TSTS expression element, respectively. The primers to be synthesized are as follows.

表1Table 1

F代表正向引物，R代表反向引物。F represents the forward primer and R represents the reverse primer.

以质粒STS-pUC57为模板，用以上合成的每对引物分别进行PCR反应，得到各个PCR片段进行胶纯化回收(QIGEN公司的试剂盒)。PCR was carried out using each of the primers synthesized above using the plasmid STS-pUC57 as a template, and each PCR fragment was subjected to gel purification (QIGEN kit).

PCR反应设置：模板10ng；正向引物(F)：2.5μl；反向引物(R)：2.5μl；2X Q5(PCR预混液，NEB,MO494S)：25μl；H ₂O：补平50μl。程序设置：(98℃，30秒；((98℃，20秒；58℃，20秒；72℃，20秒(P1T,P3T)或2分钟(P1V,P3V))34循环)；72℃，5分钟)。PCR产物经1％琼脂糖凝胶电泳分离，所得产物经试剂盒回收(QIAGEN28706)，最终产物溶于水并进行浓度测定。 PCR reaction setup: template 10 ng; forward primer (F): 2.5 μl; reverse primer (R): 2.5 μl; 2X Q5 (PCR premix, NEB, MO494S): 25 μl; H ₂ O: 40 μl. Program setting: (98 ° C, 30 seconds; ((98 ° C, 20 seconds; 58 ° C, 20 seconds; 72 ° C, 20 seconds (P1T, P3T) or 2 minutes (P1V, P3V)) 34 cycles); 72 ° C, 5 minutes). The PCR product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706), and the final product was dissolved in water and subjected to concentration measurement.

Gibson组装：P1V：100ng；P1T：30ng；2X Gibson预混液(NEB 2611S):10μl； H2O：补平20μl。反应混合物放置50℃，2小时。转化进感受态细胞TOP10(天根CB104)。挑取单克隆培养并测序鉴定。序列正确的单克隆进行扩大培养，并通过去内毒素的质粒抽提试剂盒(QIAGEN 12362)进行提取质粒，即为构建含TSTS表达元件的质粒TSTS-PUC57。将组装中的P1V和P1T分别换成P3V和P3T，以同样的方法构建出含STST表达元件的质粒STST-pUC57。Gibson assembly: P1V: 100 ng; P1T: 30 ng; 2X Gibson master mix (NEB 2611S): 10 μl; H2O: 20 μl. The reaction mixture was placed at 50 ° C for 2 hours. Transform into competent cell TOP10 (Tiangen CB104). Monoclonal cultures were picked and sequenced. The sequence-corrected monoclonal was expanded and cultured, and the plasmid was extracted by the endotoxin-free plasmid extraction kit (QIAGEN 12362) to construct a plasmid TSTS-PUC57 containing the TSTS expression element. The plasmid STST-pUC57 containing the STST expression element was constructed in the same manner by replacing P1V and P1T in the assembly with P3V and P3T, respectively.

2.2 TSTST表达元件的构建2.2 Construction of TSTST expression components

在STS表达元件的两端同时加上tRNA得到TSTST的表达元件。具体构建方法如下：以已构建出的STST-pUC57为模板，P1V-F、P1V-R为引物进行PCR来线性化质粒。反应设置及程序设置同2.1。PCR产物经凝胶电泳纯化后测定浓度，进行下一步组装。Gibson组装：2.2中PCR产物：100ng；P1T：30ng；2X Gibson预混液(NEB 2611S):10μl；H ₂O：补平20μl。其后反应条件，转化条件，测序鉴定及纯化均同2.1，以这样的方法构建出质粒TSTST-pUC57。 TRNA is simultaneously added to both ends of the STS expression element to obtain an expression element of TSTST. The specific construction method is as follows: using the constructed STST-pUC57 as a template, P1V-F and P1V-R are primers for PCR to linearize the plasmid. The reaction settings and program settings are the same as 2.1. The PCR product was purified by gel electrophoresis, and the concentration was measured, and the next assembly was carried out. Gibson assembly: PCR product in 2.2: 100 ng; P1T: 30 ng; 2X Gibson master mix (NEB 2611S): 10 μl; H ₂ O: 20 μl. Thereafter, the reaction conditions, transformation conditions, sequencing identification and purification were the same as 2.1, and the plasmid TSTST-pUC57 was constructed in this way.

2.3 sgRNA逆序表达元件的构建2.3 Construction of sgRNA reverse expression elements

S2TS1T是指sgRNA2-tRNA ^gly-sgRNA1-tRNA ^gly形式的构建：以构建的STST-pUC57为模板，分别用以下的引物来进行质粒线性化以及片段元件的扩增。 S2TS1T refers to the construction of the sgRNA2-tRNA ^gly- sgRNA1-tRNA ^gly form: using the constructed STST-pUC57 as a template, the following primers were used for plasmid linearization and fragment element amplification.

表2Table 2

以STST-PUC57为模板，PCR反应及产物回收参见2.1。Using STST-PUC57 as a template, PCR reaction and product recovery are shown in 2.1.

对PCR产物设置BbsI的酶切反应：模板(PV，S1T，S2T)：1μg；BbsI(NEB R0539V)：1μl；NEB Buffer 2.1(NEB B7202V)：3μl；H ₂O：补平至30μl。酶切在37℃水浴中进行2小时。酶切产物经1％琼脂糖凝胶电泳分离，所得产物经试剂盒回收(QIAGEN 28706)最终产物溶于水并进行定量。 The digestion reaction of BbsI was set for the PCR product: template (PV, S1T, S2T): 1 μg; BbsI (NEB R0539V): 1 μl; NEB Buffer 2.1 (NEB B7202V): 3 μl; H ₂ O: flattened to 30 μl. The digestion was carried out in a 37 ° C water bath for 2 hours. The digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.

设置连接反应：上一步得到的酶切片段(PV：1μg；S1T：500ng；S2T：500ng)T4连接酶：0.6μl；T4连接酶缓冲液1μl；H ₂O补平10μl。连接在16℃中进行2小时。 The ligation reaction was set: the digested fragment obtained in the previous step (PV: 1 μg; S1T: 500 ng; S2T: 500 ng) T4 ligase: 0.6 μl; T4 ligase buffer 1 μl; H ₂ O was filled in 10 μl. The ligation was carried out at 16 ° C for 2 hours.

连接产物转化感受态细胞TOP10(天根CB104)。挑取单克隆培养并测序。单克隆扩大培养及质粒抽提如2.1。The ligation product transformed competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it. Monoclonal expansion culture and plasmid extraction as in 2.1.

2.4单质粒***构建2.4 Single plasmid system construction

所有之前构建的表达元件均以pUC57为质粒骨架，并且元件的上下游序列均一致。SpCas9-pX330质粒(Addgene 71707)带有真核里表达Cas9所需的元件，以此为质粒骨架，将引导RNA表达元件亚克隆到这个质粒中，则可以通过单质粒***实现多位点编辑。按以下引物通过PCR的方法线性化质粒pX330，并扩增表达元件(统称(ST)n)：All previously constructed expression elements have pUC57 as the plasmid backbone, and the upstream and downstream sequences of the elements are identical. The SpCas9-pX330 plasmid (Addgene 71707) carries the elements required for expression of Cas9 in eukaryotic cells, and as a plasmid backbone, subcloning of the guide RNA expression element into this plasmid enables multi-site editing by a single plasmid system. The plasmid pX330 was linearized by PCR according to the following primers, and the expression elements (collectively referred to as (ST) n) were amplified:

表3table 3

以GTGT-pUC57为PCR模板，PCR反应及产物回收参见2.1GTGT-pUC57 as PCR template, PCR reaction and product recovery see 2.1

设置酶切反应：模板(pX330L，(ST)n)1μg；AflIII(NEB：R0541V)1μl；XbaI(NEB R0145V)1μl；NEB Cutsmart缓冲液3μl；H ₂O补平30μl。酶切在37℃水浴中进行2小时。酶切产物经1％琼脂糖凝胶电泳分离，所得产物经试剂盒回收(QIAGEN 28706)最终产物溶于水并进行定量。 The digestion reaction was set: template (pX330L, (ST)n) 1 μg; AflIII (NEB: R0541V) 1 μl; XbaI (NEB R0145V) 1 μl; NEB Cutsmart buffer 3 μl; H ₂ O to fill 30 μl. The digestion was carried out in a 37 ° C water bath for 2 hours. The digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.

设置连接反应：上一步得到的酶切片段(pX330L 500ng；(ST)n 400ng)T4连接酶：0.6μl；T4连接酶缓冲液1μl；H ₂O补平10μl。连接反应在16℃中进行2小时。 The ligation reaction was set: the cleavage fragment obtained in the previous step (pX330L 500 ng; (ST) n 400 ng) T4 ligase: 0.6 μl; T4 ligase buffer 1 μl; H ₂ O to fill 10 μl. The ligation reaction was carried out at 16 ° C for 2 hours.

连接产物转化感受态细胞TOP10(天根CB104)。挑取单克隆培养并测序。单克隆扩大培养及质粒抽提如2.1，最终质粒产物为STST-pX330，可以同时表达STST元件和Cas9蛋白。The ligation product transformed competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it. Monoclonal expansion culture and plasmid extraction as in 2.1, the final plasmid product is STST-pX330, which can simultaneously express STST elements and Cas9 protein.

2.5 STSTST表达元件的质粒构建2.5 Plasmid construction of STSTST expression elements

表4Table 4

以合成的质粒STS-pUC57为模板，用以上四对引物分别进行PCR反应，BbsI酶切以及连接转化，克隆鉴定得到产物STSTST-pUC57。具体实验条件操作步骤参见2.1和2.3。根据2.4中单质粒构建方法，最终质粒产物记作STSTST-pX330。该构建可以表达Cas9蛋白和3个不同sgRNA。Using the synthetic plasmid STS-pUC57 as a template, the above four pairs of primers were used for PCR reaction, BbsI digestion and ligation transformation, and the product STSTST-pUC57 was cloned and identified. See Tables 2.1 and 2.3 for specific experimental conditions. According to the single plasmid construction method in 2.4, the final plasmid product was designated as STSTST-pX330. This construct can express the Cas9 protein and three different sgRNAs.

2.6对照质粒的构建2.6 Construction of control plasmid

表5table 5

引物退火：将引物稀释成100μM，按如下条件混合：引物F 1μl；引物R 1μl；10*T4连接酶缓冲液1μl；T4PNK(NEB，M0201S)0.5μl；水补至10μl。实验混合物放置37℃反应30分钟，在95℃3分钟，所得产物经试剂盒回收(QIAGEN 28706)最终产物溶于水并进行定量。Primer annealing: The primers were diluted to 100 μM and mixed under the following conditions: primer F 1 μl; primer R 1 μl; 10*T4 ligase buffer 1 μl; T4PNK (NEB, M0201S) 0.5 μl; water supplemented to 10 μl. The experimental mixture was allowed to react at 37 ° C for 30 minutes, and at 95 ° C for 3 minutes, the resulting product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.

酶切模板反应：模板SpCas9-pX330质粒(Addgene#71707)3μg；BbsI(NEB#R0539S)3μl；NEB Cutsmart缓冲液3μl；H ₂O补平30μl。酶切在37℃水浴中进行2小时。酶切产物经1％琼脂糖凝胶电泳分离，所得产物经试剂盒回收(QIAGEN 28706)最终产物溶于水并进行定量。 Digestion template reaction: template SpCas9-pX330 plasmid (Addgene #71707) 3 μg; BbsI (NEB #R0539S) 3 μl; NEB Cutsmart buffer 3 μl; H ₂ O to fill 30 μl. The digestion was carried out in a 37 ° C water bath for 2 hours. The digested product was separated by 1% agarose gel electrophoresis, and the obtained product was recovered by a kit (QIAGEN 28706) and the final product was dissolved in water and quantified.

设置连接反应：酶切回收后模板100ng；退火后引物2μl；T4连接酶(NEB#M0202L)：0.6μl；T4连接酶缓冲液1μl；H ₂O补平10μl，连接在室温下进行2小时。连接产物进行细菌转化，单克隆测序鉴定如上。最终质粒产物分别记作S1-pX330，S2-pX330和S3-pX330，均可以表达Cas9蛋白和对应的sgRNA。 The ligation reaction was set: 100 ng of template after enzymatic cleavage; 2 μl of primer after annealing; T4 ligase (NEB #M0202L): 0.6 μl; 1 μl of T4 ligase buffer; 10 μl of H ₂ O, and ligated at room temperature for 2 hours. The ligation product was subjected to bacterial transformation, and monoclonal sequencing was identified as above. The final plasmid products, designated S1-pX330, S2-pX330 and S3-pX330, respectively, can express the Cas9 protein and the corresponding sgRNA.

3.细胞内编辑效率测定3. Determination of intracellular editing efficiency

3.1细胞转染：3.1 cell transfection:

细胞计数：293T细胞复苏后，培养至第三代或以上，于转染前一天进行细胞传代，分细胞于24孔板，每个孔约5万个细胞。Cell count: After 293T cells were resuscitated, cultured to the third generation or above, cells were passaged one day before transfection, and the cells were divided into 24-well plates with about 50,000 cells per well.

转染时的组合及条件：细胞生长至约占培养板底面积40％-60％时，用Lipo2000(Life 11668-027)作为转染试剂，每个孔转染总量为500ng的质粒。每个实验条件都重复三次。Combinations and conditions at the time of transfection: When cells were grown to about 40% to 60% of the bottom area of the plate, Lipo2000 (Life 11668-027) was used as a transfection reagent, and a total of 500 ng of plasmid was transfected per well. Each experimental condition was repeated three times.

3.2编辑效率测定3.2 Editing efficiency measurement

收集细胞：细胞转染后48小时，将细胞反复吹打，形成细胞悬液，收集在EP管里。Collecting cells: 48 hours after cell transfection, the cells were repeatedly beaten to form a cell suspension, which was collected in an EP tube.

基因组提取：将细胞悬液离心，弃上清。加入80μl的QE(Lucigen:QE09050)，混匀细胞。98℃加热2分钟，再置于65℃6分钟。Genomic extraction: Centrifuge the cell suspension and discard the supernatant. 80 μl of QE (Lucigen: QE09050) was added and the cells were mixed. Heat at 98 ° C for 2 minutes and then at 65 ° C for 6 minutes.

目标片段扩增，其引物信息如下Target fragment amplification, the primer information is as follows

表6Table 6

PCR设置：模板：1μl基因组；正向引物：1μl；反向引物：1μl；2X Amplitag(PCR预混液，Thermo,4398790)：10μl；H ₂O：7μl。程序设置：(95℃,7分钟；((95℃，30秒；58℃,30秒；72℃,30秒)34循环)；72℃,7分钟) PCR settings: template: 1 μl genome; forward primer: 1 μl; reverse primer: 1 μl; 2X Amplitag (PCR premix, Thermo, 4398790): 10 μl; H ₂ O: 7 μl. Program setting: (95 ° C, 7 minutes; ((95 ° C, 30 seconds; 58 ° C, 30 seconds; 72 ° C, 30 seconds) 34 cycles); 72 ° C, 7 minutes)

编辑效率分析：对于上述表6的PCR产物进行测序(GeneWiz公司)，对于TRAC基因片段提供TRAC-F引物，对于B2M基因片段提供B2M-R引物，对于EGFR基因片段提供EGFR-F引物。对于测序的结果进行TIDE分析 https://tide-calculator.nki.nl/。编辑效率结果如图(图2和图3)汇总。 Edit efficiency analysis: The PCR products of Table 6 above were sequenced (GeneWiz), TRAC-F primers were provided for the TRAC gene fragment, B2M-R primers were provided for the B2M gene fragment, and EGFR-F primers were provided for the EGFR gene fragment. For the results of the sequencing, a TIDE analysis was performed https://tide-calculator.nki.nl/ . The editing efficiency results are summarized in the figure (Figure 2 and Figure 3).

结果和讨论(图2和图3)Results and discussion (Figures 2 and 3)

我们在哺乳动物细胞体内试验了以上这些构建，并且同时比较使用了sgRNA-tRNA为基本单位的构建方法。确定通用型多sgRNA构建载体的形式。We tested these constructs in mammalian cells and compared construction methods using sgRNA-tRNA as the basic unit. The form of the universal multi-sgRNA construction vector is determined.

首先我们确认由tRNA连接gRNA可以有效释放并介导下游的基因编辑。对于选定两个靶点的编辑时，跟STS这个基础元件相比，其两端的tRNA影响较小，在我们这个测试细胞系中，除了TSTS这个组合略低之外，其余构建没有明显区别。出于对构建的简化，STST这个构建是我们选择的对象First we confirmed that tRNA-ligated gRNA can efficiently release and mediate downstream gene editing. For the editing of the selected two targets, the tRNA at both ends has less influence than the basic element of STS. In our test cell line, except for the combination of TSTS which is slightly lower, there is no significant difference in the other constructions. For the simplification of the build, the STST build is the object of our choice.

我们比较了不同sgRNA在STST构建中的位置对其编辑效率的影响。实验证明 sgRNA对下游编辑效率的影响取决于它本身的效率，而与其在STST构建中的位置无关，这也从侧面验证这个构建***在体内的加工释放sgRNA的高效性。We compared the effect of the location of different sgRNAs in the STST construction on their editing efficiency. Experiments have shown that the effect of sgRNA on downstream editing efficiency depends on its own efficiency, and it is independent of its position in the STST construction, which also verifies the efficiency of the sgRNA released by the construction system in vivo.

我们同时也测试了单质粒***的有效性，即把STST这个表达元件亚克隆到含有Cas9表达元件的质粒中。这个单质粒***同样维持了对各个靶点的高水平的编辑效率。We also tested the validity of the single plasmid system by subcloning the STST expression element into a plasmid containing the Cas9 expression element. This single plasmid system also maintains a high level of editing efficiency for each target.

我们比较(ST)n这个构建与对应的n个sgRNA表达载体共转的效率差别。实验证明这个新的构建可以如设想释放多个不同成熟的sgRNA并完成下游的编辑。在n＝2或3时，效率上没有明显区别。We compared the efficiency of (ST)n construction with the corresponding n sgRNA expression vectors. Experiments have shown that this new construction can be as envisioned to release multiple different mature sgRNAs and complete downstream editing. At n=2 or 3, there is no significant difference in efficiency.

实施例2.一步法构建自切割引导RNA表达载体Example 2. One-step construction of a self-cleavage guide RNA expression vector

方法及材料Method and material

1.模板合成制备1. Template synthesis preparation

通用单链(即sgRNA骨架序列)：(SEQ ID NO:43)Universal single strand (ie sgRNA backbone sequence): (SEQ ID NO: 43)

可变tRNA单链：(sgRNA SC B20-XXXXXX-tRNA)RCVariable tRNA single chain: (sgRNA SC B20-XXXXXX-tRNA) RC

注：SC表示sgRNA骨架，SC B20表示sgRNA骨架3’端的20个碱基Note: SC indicates the sgRNA backbone and SC B20 indicates 20 bases at the 3' end of the sgRNA backbone.

XXXXXX表示在基因组序列中紧连tRNA5’端的6个碱基，针对不同的tRNA其上下游序列可以由网站中http://gtrnadb.ucsc.edu/查询；RC表示对前括号里的序列取反向互补序列。XXXXXX indicates that 6 bases of the tRNA 5' end are closely linked in the genomic sequence. The upstream and downstream sequences of different tRNAs can be searched by http://gtrnadb.ucsc.edu/ on the website; RC indicates the reverse of the sequence in the parentheses. To the complementary sequence.

反应条件：将以上两条合成的单链分别用水溶解，终浓度均为100μM。设置PCR体系：(通用单链：2.5μl；可变tRNA单链：2.5μl；2X Q5(PCR预混液，NEB,MO494S)：25μl；H ₂O：20μl)；设置PCR反应条件：98℃，30秒；(95℃，10秒；55℃，20秒；72℃，20秒)35个循环；72℃：2分钟。反应结束后，样品放置4℃。样品经过PCR纯化试剂盒(QIAquick 154045041)纯化后，可以存放在-20℃，作为一步反应中所用到的模板。 Reaction conditions: The above two synthetic single chains were each dissolved in water to a final concentration of 100 μM. Set the PCR system: (general single strand: 2.5 μl; variable tRNA single strand: 2.5 μl; 2X Q5 (PCR premix, NEB, MO494S): 25 μl; H ₂ O: 20 μl); set PCR reaction conditions: 98 ° C, 30 seconds; (95 ° C, 10 seconds; 55 ° C, 20 seconds; 72 ° C, 20 seconds) 35 cycles; 72 ° C: 2 minutes. After the reaction was over, the sample was placed at 4 °C. After purification of the sample by PCR purification kit (QIAquick 154045041), the sample can be stored at -20 ° C as a template for use in a one-step reaction.

2.引物设计2. Primer design

表7：引物设计指导原则Table 7: Primer Design Guidelines

注解：U6TR 20表示在质粒上元件***位点后20个碱基，一般包括U6终止子(tttttt)以及其后14个碱基，需要根据具体质粒来确定。Note: U6TR 20 indicates 20 bases after the element insertion site on the plasmid, generally including the U6 terminator (tttttt) and the subsequent 14 bases, which are determined according to the specific plasmid.

(U6PRO B20)RC表示在质粒上，U6启动子最后20个碱基的反向互补序列S1，S2，S3……表示按顺序排列的任何单链引导RNA，F19表示5’端前19个碱基，RC表示取反向互补的序列。SC表示sgRNA骨架，SCN20表示sgRNA骨架5’端的20个碱基，B20表示3’端20个碱基，(tRNA B20)RC表示tRNA3’端20个碱基的反向互补序列。(U6PRO B20) RC indicates that the reverse complement of the last 20 bases of the U6 promoter, S1, S2, S3, ... indicates any single-stranded guide RNA arranged in sequence, and F19 indicates the first 19 bases at the 5' end. Base, RC represents a sequence that is reverse complementary. SC denotes the sgRNA backbone, SCN20 denotes 20 bases at the 5' end of the sgRNA backbone, B20 denotes 20 bases at the 3' end, and (tRNA B20) RC denotes a reverse complement of 20 bases at the 3' end of the tRNA.

自切割元件扩增：每对正反向引物经(Genewiz)合成后，分别用水溶解，终浓度为10μM。设置PCR体系：(正向引物：2.5μl；反向引物：2.5μl；步骤1得到的模板：10μg；2X Q5(PCR预混液，NEB,MO494S)：25μl；H ₂O：20μl)；设置PCR反应条件：98℃，30秒；(95℃，10秒；55℃，20秒；72℃，30秒)35个循环；72℃：2分钟。反应结束后，样品保存在4℃。将样品进行2％的琼脂糖凝胶电泳，切下目的条带进行回收纯化。纯化后，可以存放在-20℃。 Self-cleaving element amplification: Each pair of forward and reverse primers was synthesized by Genewiz and dissolved in water to a final concentration of 10 μM. Set the PCR system: (forward primer: 2.5 μl; reverse primer: 2.5 μl; template obtained in step 1: 10 μg; 2X Q5 (PCR premix, NEB, MO494S): 25 μl; H ₂ O: 20 μl); set PCR Reaction conditions: 98 ° C, 30 seconds; (95 ° C, 10 seconds; 55 ° C, 20 seconds; 72 ° C, 30 seconds) 35 cycles; 72 ° C: 2 minutes. After the reaction was over, the sample was stored at 4 °C. The sample was subjected to 2% agarose gel electrophoresis, and the target band was excised for recovery and purification. After purification, it can be stored at -20 °C.

载体线性化：每对正反向引物经(Genewiz)合成后，分别用水溶解，终浓度为10μM。同上设置PCR体系。反应条件做如下改动：98℃，30秒；(95℃，10秒；55℃，20秒；72℃，载体长度(nt)/1000*30秒)35个循环(参见图4)；72℃：5分钟。反应结束后，样品保存在4℃。将样品进行1％的琼脂糖凝胶电泳，切下目的条带进行回收纯化。纯化后，可以存放在-20℃。Vector linearization: Each pair of forward and reverse primers was synthesized by Genewiz and dissolved in water to a final concentration of 10 μM. The PCR system is set up as above. The reaction conditions were modified as follows: 98 ° C, 30 seconds; (95 ° C, 10 seconds; 55 ° C, 20 seconds; 72 ° C, carrier length (nt) / 1000 * 30 seconds) 35 cycles (see Figure 4); 72 ° C :5 minutes. After the reaction was over, the sample was stored at 4 °C. The sample was subjected to 1% agarose gel electrophoresis, and the target band was excised for recovery and purification. After purification, it can be stored at -20 °C.

3.多片段组装：3. Multi-segment assembly:

线性化质粒和多个片段经胶回收后测量浓度。按照以下的方式混合：载体(300ng/μl)，目的片段1μg/条，2x Gibson预混液(NEB 2611S)：10μl，补水至20μl。反应混合物放置50℃，2小时。转化进感受态细胞TOP10(天根CB104)。挑取单克隆培养并测序。在片段组装的上下游200左右碱基的位置分别设计引物测序，测序对的克隆进行扩大培养，并通过去内毒素的质粒抽提试剂盒(QIAGEN 12362)进行提取质粒。The concentration of the linearized plasmid and the multiple fragments were recovered after gel recovery. The mixture was mixed in the following manner: carrier (300 ng/μl), target fragment 1 μg/strand, 2x Gibson master mix (NEB 2611S): 10 μl, hydrating to 20 μl. The reaction mixture was placed at 50 ° C for 2 hours. Transform into competent cell TOP10 (Tiangen CB104). Pick up the monoclonal culture and sequence it. Primer sequencing was designed at positions of about 200 bases upstream and downstream of the fragment assembly, and the cloned clones were expanded and cultured, and the plasmid was extracted by an endotoxin-free plasmid extraction kit (QIAGEN 12362).

4.单质粒构建4. Single plasmid construction

参考之前步骤2,3，更换载体及对应的引物。选用有Cas9表达元件的载体，比如SpCas9-pX330质粒(Addgene 71707)。在不影响其它元件功能的区域进行线性化，线性化引物设计参考方法2中针对载体的引物设计。Refer to previous steps 2, 3 to replace the carrier and the corresponding primer. A vector having a Cas9 expression element, such as the SpCas9-pX330 plasmid (Addgene 71707), is used. Linearization is performed in areas that do not affect the function of other elements, and linearized primer design refers to the primer design for the carrier in Method 2.

载体线性化：每对正反向引物经(Genewiz)合成后，分别用水溶解，终浓度为10μM。设置PCR体系：(正向引物：2.5μl；反向引物：2.5μl；模板(含有Cas9表达元件的质粒，如SpCas9-pX330质粒)：10μg；2X Q5(NEB M0494S))：25μl；H ₂O：20μl)；设置PCR反应条件：98℃，30秒；(95℃，10s；55℃，20s；72℃，(载体长度/1000*30)s)35个循环；72℃：2分钟。反应结束后，样品保存在4℃。将样品进行1％的琼脂糖凝胶电泳，切下目的条带进行回收纯化。纯化后，可以存放在-20℃。 Vector linearization: Each pair of forward and reverse primers was synthesized by Genewiz and dissolved in water to a final concentration of 10 μM. The PCR system was set up: (forward primer: 2.5 μl; reverse primer: 2.5 μl; template (plasmid containing Cas9 expression element, such as SpCas9-pX330 plasmid): 10 μg; 2X Q5 (NEB M0494S)): 25 μl; H ₂ O : 20 μl); setting PCR reaction conditions: 98 ° C, 30 seconds; (95 ° C, 10 s; 55 ° C, 20 s; 72 ° C, (carrier length / 1000 * 30) s) 35 cycles; 72 ° C: 2 minutes. After the reaction was over, the sample was stored at 4 °C. The sample was subjected to 1% agarose gel electrophoresis, and the target band was excised for recovery and purification. After purification, it can be stored at -20 °C.

线性化的载体与方法2中合成元件，运用方法3中组装方法，即得到可以同时表达Cas9蛋白和多个gRNA的单质粒。测序引物需要根据使用的质粒进行更改，一般在片段组装的上下游200左右碱基的位置分别设计引物。The linearized vector and the synthetic element in the method 2, using the assembly method in the method 3, obtain a single plasmid which can simultaneously express the Cas9 protein and a plurality of gRNAs. The sequencing primers need to be modified according to the plasmid used, and primers are generally designed at positions of about 200 bases upstream and downstream of the fragment assembly.

结果与讨论Results and discussion

在本实施例中，主要概述了这个多引导RNA表达载体的通用构建方法，其中包括模板链的合成，特异性引物的合成引导原则，以及构建所需其他材料和完整流程(图4和图5)。In this example, the general construction method of this multi-guided RNA expression vector is mainly summarized, including the synthesis of template strands, the principle of synthesis of specific primers, and other materials and complete processes required for construction (Fig. 4 and Fig. 5). ).

由实施例2得到结论，以sgRNA-tRNA作为自切割元件，可以在体内释放出成熟的引导RNA，并介导多靶点的高效编辑。对于组装多个单元涉及到无缝克隆，因为单元之间不能有多余无关序列以影响体内的切割作用，进而影响sgRNA的正确加工释放。对比不同无缝克隆的方法，我们倾向于选择Gibson组装。因为这种方法不要求特异的识别位点，而且它所需要的重叠序列长度正好与可变区的长度相兼容，为后续实验提供很多便利。这样所有的单元产物与线性化的载体可以通过一步连接得到我们想要的质粒构建。如果原质粒中含有Cas9表达元件，则最终构建的质粒包含多位点靶向编辑的所有元件，单个质粒的导入即可实现体内多位点编辑效应。It was concluded from Example 2 that sgRNA-tRNA was used as a self-cleaving element to release mature guide RNA in vivo and to mediate efficient editing of multiple targets. For the assembly of multiple units involves seamless cloning, because there can be no extraneous sequences between the units to affect the cleavage in vivo, which in turn affects the correct processing release of sgRNA. Comparing the methods of different seamless clones, we tend to choose Gibson assembly. Because this method does not require a specific recognition site, and the length of the overlapping sequence it needs is exactly compatible with the length of the variable region, it provides a lot of convenience for subsequent experiments. Thus all of the unit products and the linearized vector can be constructed in one step to obtain the desired plasmid construction. If the original plasmid contains a Cas9 expression element, the final constructed plasmid contains all of the elements of the multi-site targeted editing, and the introduction of a single plasmid can achieve a multi-site editing effect in vivo.

而且它比之前采用的构建方法有几个优势：对于引导RNA的保守性结构来看，相当于延伸了它的骨架序列，使其有着在细胞内可以自行被加工并释放出成熟的引导RNA；该方法更为直接，从模板引物合成到完成最终构建可以在很短的时间里完成，主要只涉及PCR反应和无缝连接反应；它的构建中不涉及酶切位点，因此对几乎所有的sgRNA都适用，基本上可以应用于表达各种组合的sgRNA。并且充分结合Gibson组装的优势，让sgRNA中特异的序列(引导序列)成为桥接区，这样就保证了各元件可以高效有序地按我们的需求组装。Moreover, it has several advantages over the previously constructed construction method: for the conservative structure of the guide RNA, it is equivalent to extending its skeleton sequence, so that it can be processed in the cell and release the mature guide RNA; The method is more direct, from template primer synthesis to completion of the final construction can be completed in a short time, mainly involving PCR reactions and seamless ligation reactions; its construction does not involve enzyme cleavage sites, so for almost all Both sgRNAs are suitable and can be applied to sgRNAs expressing various combinations. And fully combined with the advantages of Gibson assembly, the specific sequence (guide sequence) in the sgRNA becomes the bridge region, thus ensuring that the components can be assembled efficiently and orderly according to our needs.

基于该模块化设计，我们简化所有必需的材料，将恒定区(sgRNA的骨架部分以及选定的tRNA序列)和可变区(不同sgRNA的引导序列)分开，既能保证实施的简便通用性，又能考虑到不同实验环境里对不同基因组合编辑的需求。在这里恒定区是个相对的定义，对于更换种属的Cas蛋白，其使用的sgRNA的骨架序列可以根据具体使用蛋白而改变，其他设计和操作不变；同时，对于连接不同sgRNA之间的元件也可以作为一个筛选平台，可以通过改变模板链中的一条来达到对不同连接元件的构建和效果评估。Based on this modular design, we simplify all necessary materials, separating the constant region (the skeletal portion of the sgRNA and the selected tRNA sequence) and the variable region (the guide sequence for the different sgRNAs), ensuring easy and versatile implementation. The need to edit different combinations of genes in different experimental environments can also be considered. Here, the constant region is a relative definition. For the replacement of the Cas protein of the species, the skeleton sequence of the sgRNA used can be changed according to the specific use of the protein, and other designs and operations are unchanged; at the same time, for the components connecting different sgRNAs, It can be used as a screening platform to change the construction and effect evaluation of different connected components by changing one of the template chains.

通过这个方法平台，可以测试不同sgRNA的编辑效应，还可以测不同tRNA作为桥接区的有效性。还可以延伸到tRNA替代元件(凡是有两端可精确加工的短链RNA)的功能。实施例3就是以这个方法平台去测试不同tRNA释放引导RNA的能力。Through this method platform, the editing effects of different sgRNAs can be tested, and the effectiveness of different tRNAs as bridging regions can also be measured. It can also be extended to the function of tRNA replacement elements (when there are short-chain RNAs that can be precisely processed at both ends). Example 3 is the ability to test the ability of different tRNAs to release guide RNA using this method platform.

实施例3.不同连接tRNA的效率比较Example 3. Comparison of the efficiency of different tRNA connections

方法及材料Method and material

所有序列(包括通用单链，可tRNA单链以及引物)均由Genewiz合成提供All sequences (including universal single strands, tRNA single strands and primers) are provided by Genewiz synthesis

1.模板合成Template synthesis

tRNA选择tRNA1(SEQ ID NO:44)，tRNA2(SEQ ID NO:45)或tRNA3(SEQ ID NO:46)，所用的所有tRNA二级结构示意图参见图6。除了完整的tRNA以外，模板设计中需要在其前端加上6个碱基的前导序列(通过tRNA数据库查询http://gtrnadb.ucsc.edu/)以方便后期tRNA加工。The tRNA selects tRNA1 (SEQ ID NO: 44), tRNA2 (SEQ ID NO: 45) or tRNA3 (SEQ ID NO: 46), and a schematic representation of the secondary structure of all tRNAs used is shown in Figure 6. In addition to the complete tRNA, the template design requires a 6-base leader sequence (query via the tRNA database http://gtrnadb.ucsc.edu/) to facilitate later tRNA processing.

方法参见图4，合成以下序列：Method Referring to Figure 4, the following sequence was synthesized:

通用正向引物：(SEQ ID NO:38)Universal forward primer: (SEQ ID NO: 38)

针对不同tRNA模板合成反向引物：tRNA1反向引物：(SEQ ID NO:47)；tRNA2反向引物：(SEQ ID NO:48)；tRNA3反向引物：(SEQ ID NO:49)。Reverse primers were synthesized for different tRNA templates: tRNA1 reverse primer: (SEQ ID NO: 47); tRNA2 reverse primer: (SEQ ID NO: 48); tRNA3 reverse primer: (SEQ ID NO: 49).

通过退火扩增合成模板方法参见实施例2的方法材料1，合成的产物为tRNA1模板，tRNA2模板，tRNA3模板。For the method of synthesizing the template by annealing, see Method 1 of Example 2, and the synthesized product is a tRNA1 template, a tRNA2 template, and a tRNA3 template.

表8：模板合成Table 8: Template Synthesis

2.元件合成及载体线性化2. Component synthesis and carrier linearization

参看图4、图5，通过PCR扩增的方法进行元件合成以及载体线性化：载体线性化的模板统一选用STS-pUC57，元件合成的模板分别选用1中生成的不同tRNA模板，其使用的引物如表9，反应条件参考实施例2。Referring to Fig. 4 and Fig. 5, element synthesis and vector linearization were carried out by means of PCR amplification: STS-pUC57 was selected as the template for vector linearization, and different tRNA templates generated in 1 were selected for the template synthesized by the element, and the primers used were used. As shown in Table 9, the reaction conditions are referred to in Example 2.

表9：以步骤1中tRNA1模板作为扩增模板，合成各元件Table 9: Synthesis of each component using the tRNA1 template in step 1 as an amplification template

表10：以步骤1中tRNA2模板作为扩增模板，合成各元件Table 10: Synthesis of each element using the tRNA2 template in step 1 as an amplification template

表11：以1中tRNA3模板作为扩增模板，合成各元件Table 11: Synthesis of each component using the tRNA3 template in 1 as an amplification template

3.质粒构建3. Plasmid construction

以2中得到的各套线性化载体和元件分别进行组装，其组装方法参见实施例2。这样最终获得由不同tRNA(tRNA1,tRNA2,tRNA3)连接sgRNA的能表达(ST)n的质粒，记作T1，T2，T3。The sets of linearized carriers and components obtained in 2 are assembled separately, and the assembly method is as described in Embodiment 2. Thus, a plasmid capable of expressing (ST)n, which is ligated to different sgRNAs by different tRNAs (tRNA1, tRNA2, tRNA3), was designated as T1, T2, T3.

4.编辑效率检测4. Editing efficiency detection

对于293T细胞的质粒转染和编辑效率分析参见实施例1中方法材料。对于K562细胞通过电转仪(Thermo MPK5000)导入质粒，每个反应体系细胞数为10 ⁵，细胞培养基为89％RPMI medium 1640(Gibco C11875500BT)，10％FBS(Corning 35010155)，1％Pen Step(Gibco 15140-122)。电转参数如下：1450伏特，10毫秒，3脉冲。电转后培养两天，收获细胞，分析基因编辑效率。其方法与293T细胞一致，参见实施例1中方法材料。 See Method Materials in Example 1 for plasmid transfection and editing efficiency analysis of 293T cells. For K562 cells, the plasmid was introduced by electroporation (Thermo MPK5000), the number of cells per reaction system was 10 ⁵ , the cell culture medium was 89% RPMI medium 1640 (Gibco C11875500BT), 10% FBS (Corning 35010155), 1% Pen Step ( Gibco 15140-122). The electrical parameters are as follows: 1450 volts, 10 milliseconds, 3 pulses. After electroporation, the cells were cultured for two days, and the cells were harvested for analysis of gene editing efficiency. The method is identical to the 293T cells, see the method materials in Example 1.

结果与讨论Results and discussion

实施例2提供了多个引导RNA共表达元件构建方法，这个方法可以应用到连接引导RNA的元件的快速筛选。这个连接的元件主要起隔开不同sgRNA并且在体内能够释放出所需要的各种sgRNA，这样对连接元件需要满足以下几个要求：长度短，不会引入过多冗余的DNA片段；结构独立明确，不会对sgRNA的结构造成不良影响；两端可以被精确切割，多依赖于核酸内切酶活性让这个自切割元件可以释放两端，而不对sgRNA序列造成过多干扰；体内加工活性高，在不依赖于引入外源内切酶***的情况下，细胞内自带的内切酶需要对该元件有着准确识别和高效的切割活性，或者这个元件有着高效的不依赖其他工具的自切割活性。tRNA是个可以满足以上各条件的一个连接元件。这个构建平台可以测试不同tRNA作为连接元件的效果。而且根据平台化的设计与通用的流程，要测试连接元件可以使用几种固定sgRNA检测编辑效率，需要改变的只是一条模板单链。Example 2 provides a method for constructing multiple guide RNA co-expression elements that can be applied to rapid screening of elements that link the guide RNA. This connected component mainly separates different sgRNAs and releases the various sgRNAs required in the body, so that the connecting elements need to meet the following requirements: short length, no excessive redundant DNA fragments are introduced; Does not adversely affect the structure of sgRNA; both ends can be precisely cut, relying on endonuclease activity to allow this self-cleaving element to release both ends without causing excessive interference to the sgRNA sequence; Without relying on the introduction of an exogenous endonuclease system, the endonuclease that is present in the cell requires accurate recognition and efficient cleavage activity on the element, or the element has an efficient self-cleavage activity independent of other tools. The tRNA is a connecting element that satisfies the above conditions. This build platform can test the effects of different tRNAs as connecting elements. And according to the platform design and the general process, to test the connection components can use several fixed sgRNA to detect the editing efficiency, only one template single chain needs to be changed.

我们比较在293T细胞系里，来自不同物种，对应不同氨基酸的tRNA作为sgRNA之间的连接对基因组编辑效率的影响(图7)。其材料及实验方法参考实施例1,2中所述。结果在细胞系293T中发现不同的tRNA对编辑结果的影响并不大。这在于体内加工tRNA的酶对具体tRNA的序列包括剪切位点附近的序列保守型要求低，而只是对tRNA形成的三维结构有要求。因此凡是体内能形成稳定tRNA经典结构的序列在哺乳细胞里都可以被正常识别加工，进而在我们设计的***中释放出预期的sgRNA。但考虑到对细胞引入的外来物质的安全性，倾向于使用来源于模式生物种属的tRNA序列。We compared the effect of tRNAs from different species on different amino acids in different 293T cell lines as sgRNAs on genome editing efficiency (Figure 7). The materials and experimental methods are described in Examples 1 and 2. As a result, it was found that different tRNAs in the cell line 293T had little effect on the editing results. This is because the enzymes that process the tRNA in vivo have low requirements for the sequence of the specific tRNA including the sequence near the cleavage site, but only the three-dimensional structure of the tRNA formation. Therefore, any sequence that can form a stable tRNA classical structure in the body can be normally recognized and processed in the mammalian cells, and the expected sgRNA is released in the system we designed. However, in view of the safety of foreign substances introduced into cells, tRNA sequences derived from model organism species tend to be used.

我们比较不同tRNA作为连接的构建在不同细胞系里的编辑作用(图8)。发现T2(人 -chr1.tRNA34-GlyGCC)普遍好于其他两种选择，特别在编辑效率较低的细胞系K562中，这种差别更为明显。所以我们这个体系可以快速筛选高效的连接元件，以保证其两端的sgRNA可以被有效加工释放出，参与下游的多靶点编辑。We compared the editing effects of different tRNAs as a linker in different cell lines (Figure 8). T2 (human-chr1.tRNA34-GlyGCC) was found to be generally better than the other two options, especially in the less edited cell line K562. Therefore, our system can quickly screen high-efficiency connecting elements to ensure that the sgRNA at both ends can be efficiently processed and released, and participate in downstream multi-target editing.

Claims

一种在细胞内表达CRISPR-Cas体系的方法，包括：A method of expressing a CRISPR-Cas system in a cell, comprising:

(d)构建包含表达(sgRNA-tRNA)n的DNA片段，其中n为1-8之间的整数；(d) constructing a DNA fragment comprising the expression (sgRNA-tRNA)n, wherein n is an integer between 1 and 8;

(e)将步骤(a)的DNA片段转入细胞内；(e) transferring the DNA fragment of step (a) into the cell;

(f)表达在细胞内的sgRNA与细胞中的Cas蛋白结合，形成CRISPR-Cas体系。(f) The sgRNA expressed in the cell binds to the Cas protein in the cell to form a CRISPR-Cas system.
权利要求1所述的方法，其中n为1或2或3。The method of claim 1 wherein n is 1 or 2 or 3.
权利要求1或2所述的方法，其中步骤(a)中包含表达(sgRNA-tRNA)n的DNA片段为DNA质粒。The method of claim 1 or 2, wherein the DNA fragment expressing (sgRNA-tRNA)n in step (a) is a DNA plasmid.
权利要求3所述的方法，其中细胞中的Cas蛋白是通过电穿孔转染法进入细胞的；或者是通过构建能表达Cas蛋白的DNA质粒，通过电穿孔转染，脂质体转染或病毒侵染在细胞中表达Cas蛋白。The method of claim 3, wherein the Cas protein in the cell enters the cell by electroporation transfection; or by constructing a DNA plasmid capable of expressing the Cas protein, transfected by electroporation, liposome transfection or virus Infection affects the expression of Cas protein in cells.
权利要求4所述的方法，其中表达Cas蛋白的DNA质粒是与步骤(a)的DNA片段属于同一个质粒。The method of claim 4, wherein the DNA plasmid expressing the Cas protein belongs to the same plasmid as the DNA fragment of step (a).
权利要求4所述的方法，其中表达Cas蛋白的DNA质粒与步骤(a)的DNA片段不属于同一个质粒。The method of claim 4, wherein the DNA plasmid expressing the Cas protein does not belong to the same plasmid as the DNA fragment of the step (a).
前述任一项权利要求所述的方法，其中表达sgRNA的序列为5’-(X)m-恒定区骨架序列-3’，其中X选自A、U、C和G的任一个碱基，m为0-20任一整数，优选m＝17，18，19或20。A method according to any of the preceding claims, wherein the sequence expressing the sgRNA is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from any one of A, U, C and G, m is any integer from 0 to 20, preferably m = 17, 18, 19 or 20.
前述任一项权利要求所述的方法，其中sgRNA能特异性结合B2M基因、TRAC基因或EGFR基因。The method of any of the preceding claims, wherein the sgRNA is capable of specifically binding to a B2M gene, a TRAC gene or an EGFR gene.
前述任一项权利要求所述的方法，其中tRNA选自人-chr17.tRNA2-6-GlyGCC、玉米-chr9.trna85-GlyGCC、人-chr1.tRNA34-GlyGCC和人-chr17.tRNA41-SerCGA中的一种。A method according to any of the preceding claims, wherein the tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.trna85-GlyGCC, human-chr1.tRNA34-GlyGCC and human-chr17.tRNA41-SerCGA One.
前述任一项权利要求所述的方法，其中的细胞是哺乳动物细胞。A method according to any of the preceding claims wherein the cells are mammalian cells.
权利要求10所述的方法，其中的细胞是293T或K562细胞。The method of claim 10 wherein the cells are 293T or K562 cells.
前述任一项权利要求所述的方法，其中的Cas蛋白是Cas9蛋白。A method according to any of the preceding claims wherein the Cas protein is a Cas9 protein.
权利要求11所述的方法，其中的Cas9蛋白是来源于化脓链球菌或金黄色葡萄球菌的Cas9蛋白。The method of claim 11 wherein the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes or Staphylococcus aureus.
一种质粒，包含了表达(sgRNA-tRNA)n的DNA片段，其中n为1-8之间的整数。A plasmid comprising a DNA fragment expressing (sgRNA-tRNA)n, wherein n is an integer between 1-8.
权利要求14所述的质粒，其中n为1或2或3。The plasmid of claim 14 wherein n is 1 or 2 or 3.
权利要求14或15所述的质粒，还包含了表达Cas蛋白的DNA序列。The plasmid according to claim 14 or 15, which further comprises a DNA sequence expressing the Cas protein.
权利要求14-16任一项所述的质粒，其中表达sgRNA的序列为5’-(X)m-恒定区骨架序列-3’，其中X选自A、T、C和G的任一个碱基，m为0-20任一整数，优选m＝17，18，19或20。The plasmid according to any one of claims 14-16, wherein the sequence expressing the sgRNA is a 5'-(X)m-constant region backbone sequence-3', wherein X is selected from any one of A, T, C and G The base, m is any integer from 0 to 20, preferably m = 17, 18, 19 or 20.
权利要求14-17任一项所述的方法，其中tRNA选自人-chr17.tRNA2-6-GlyGCC、玉米-chr9.tRNA85-GlyGCC、人-chr1.tRNA34-GlyGCC和人-chr17.tRNA41-SerCGA中的一种。The method of any one of claims 14-17, wherein the tRNA is selected from the group consisting of human-chr17.tRNA2-6-GlyGCC, maize-chr9.tRNA85-GlyGCC, human-chr1.tRNA34-GlyGCC, and human-chr17.tRNA41-SerCGA One of them.
权利要求14-18任一项所述的质粒，其中的Cas蛋白是Cas9蛋白。The plasmid of any one of claims 14-18, wherein the Cas protein is a Cas9 protein.
权利要求19所述的方法，其中的Cas9蛋白是来源于化脓链球菌或金黄色葡萄球菌的Cas9蛋白。The method of claim 19, wherein the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes or Staphylococcus aureus.