WO2018228183A1 - 基于碱基编辑的基因敲除方法及其应用 - Google Patents

基于碱基编辑的基因敲除方法及其应用 Download PDF

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WO2018228183A1
WO2018228183A1 PCT/CN2018/088925 CN2018088925W WO2018228183A1 WO 2018228183 A1 WO2018228183 A1 WO 2018228183A1 CN 2018088925 W CN2018088925 W CN 2018088925W WO 2018228183 A1 WO2018228183 A1 WO 2018228183A1
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王小平
许先进
刘慧莹
张锋
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苏州茂行生物科技有限公司
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Definitions

  • the present invention relates to gene knockout strategies and, more particularly, to gene editing methods based on base editing and applications thereof.
  • the terminally differentiated somatic cell clone is difficult to completely dedifferentiate into undifferentiated cells, affecting embryonic development, resulting in developmental defects; 2, all genetic material is only a maternal source; 3, low success rate.
  • Traditional gene targeting techniques limit gene knockout.
  • Programmable endonuclease technology includes zinc-finger nucleases (ZFNs) technology and transcription activator-like effectors nucleases (TALENs), as well as regular clustering intervals.
  • ZFNs zinc-finger nucleases
  • TALENs transcription activator-like effectors nucleases
  • CRISPR-associated, CRISPR/Cas9 Clustered regular interspaced short palindromic repeat
  • the invention and promotion of such technologies breaks the limitations of omnipotent embryonic stem cells, making genetic manipulation of different species possible.
  • the CRISPR/Cas9 system which is simple, efficient, and inexpensive, has swept the world immediately after its emergence. It has become the latest in the field of genetic editing, but the fastest growing and most widely used technology has triggered a revolution in the field of genetic editing.
  • the CRISPR/Cas9 system has now been successfully used for DNA knockout, DNA knock-in, DNA substitution, DNA modification, RNA modification, DNA labeling, gene transcription regulation, etc. [Hsu et al., 2014; Komor et al., 2017] . It has been successfully applied to gene editing of multiple species [Barrangou R&Doudna JA, 2016; Komor et al., 2017].
  • CRISPR/Cas9-mediated specific gene editing is the use of sgRNA (single guided RNA) to guide the Cas9 protein to cleave double-stranded DNA through target sequence complementation, resulting in double-strand breaks (DSB) in the absence of template conditions.
  • sgRNA single guided RNA
  • DSB double-strand breaks
  • NHEJ non-homologous end joining
  • a frameshift mutation resulting in knockout
  • HDR homologous recombination
  • HDR homologous recombination
  • One of the objects of the present invention is to provide an efficient and accurate gene knockout strategy.
  • Cas9 fusion protein based on CRISPR/Cas9 technology can be used as a "base editor (BE)".
  • BE base editor
  • These fusion proteins contain the dCas9 or Cas9 nickase and the rat cytidine deaminase APOBEC1, which converts cytosine (C) to uracil by deamination without the need to cleave DNA. Thereafter, uracil is converted to thymine (T) by DNA replication or repair. Similarly, it can also convert a single base G to A.
  • BE3 consisting of Cas9 nicking enzyme and APOBEC1 can significantly improve base editing efficiency to 15-75%.
  • the inventors have skillfully designed a gene knockout strategy based on the above-described BE-mediated single-base mutations, especially the precision and specificity of BE3-mediated single-base editing: introduction of stop codons by CT mutations, such as CAA , CAG, CGA mutated to stop codon TAA, TAG, TGA, or TGG was mutated to stop codon TAA, TGA, TAG by GA mutation, and the translation of the coding gene was terminated, thereby achieving gene knockout.
  • CT mutations such as CAA , CAG, CGA mutated to stop codon TAA, TAG, TGA, or TGG was mutated to stop codon TAA, TGA, TAG by GA mutation, and the translation of the coding gene was terminated, thereby achieving gene knockout.
  • a gene knockout method comprising:
  • the 20 bp-NGG target sequence (PAM sequence) of the coding region (CDS region) of the gene to be knocked out is selected to contain the complete target codon CAA, CAG or CGA;
  • sgRNA sequences to map BE3 to a target sequence such that the target single base C in the target codon becomes T to introduce a stop codon TAA or TAG, TGA, respectively, to effect gene knockout,
  • the target single base C is located at positions 1-8 of the target sequence (left end), preferably at positions 4-8, and the target codon is 12-14 bp apart from NGG, preferably 14 bp, and the base of the target codon is immediately adjacent to the base.
  • (H) cannot be G;
  • the sgRNA sequence is a 20 bp sequence corresponding to a complementary target sequence.
  • the CCN-20 bp target sequence (PAM sequence) of the coding region of the gene to be knocked out may also be selected to include the entire target codon TGG, and the target codon is immediately adjacent to the base ( D) cannot be C; accordingly, the target single base G is located at positions 1-8 of the target sequence (right end), preferably at positions 4-8, and the target codon is spaced from the CCN by 12-14 bp, preferably 14 bp.
  • BE3 may be selected from the group consisting of: rAPOBEC1-SaCas9-NLS-UGI-NLS; 3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS; rAPOBEC1-SpCas9-NLS-UGI-NLS; 3xUGI-rAPOBEC1-SpCas9-NLS-UGI -NLS, preferably the latter two.
  • the method according to the present invention can be used to knock out the following eight target genes: human PD1, LAG3, TIGIT, VISTA, 2B4, CD160, and mouse TIM3 and LAG3, and their corresponding sgRNA sequences and target gene sequences shown in sequences one to eight, respectively. Complementary.
  • the use of the above method for knockout of human PD1, LAG3, TIGIT, VISTA, 2B4, CD160 genes in the cell line HEK293T is provided.
  • a third aspect of the present invention there is provided the use of the above method for human PD, LAG3, TIGIT, VISTA, 2B4, CD160 gene knockdown in human T cells.
  • an isolated T cell or cell line obtained according to the above application or a subculture thereof.
  • a kit for gene knockout comprising the above-described sgRNA, BE3 and a corresponding amplification reagent (corresponding to a gene to be knocked out) is provided.
  • the invention utilizes the base editing technology developed on the basis of CRISPR/Cas9 to create a stop codon by precise CT or GA single base mutation, thereby establishing a gene knocker which is more efficient, more precise and less off target than CRISPR/Cas9. In addition to the strategy.
  • FIG. 1 is a schematic diagram showing the knockdown of a target gene using a CT mutation according to the present invention
  • Figure 2-5 shows the structure of different BE3.
  • BE3s as shown in Figure 2-5.
  • the different Cas9nickase and cytidine deaminase (APOBEC1) were fused to form the following four types of BE3:
  • any of the above BE3s may be employed, preferably (3) or (4).
  • Base-point editing is the use of sgRNA to localize or target BE3 to specific target sites.
  • the selection and design of target gene-specific sgRNAs is a key aspect of the present invention.
  • the invention selects the design sgRNA as follows:
  • the 20 bp-NGG target sequence (PAM sequence) of the coding region of the gene to be knocked out is selected to contain the complete target codon CAA, CAG or CGA;
  • the target single base C is preferably located at positions 4-8 of the target sequence (left end), the target codon is preferably 14 bp apart from NGG, and the base immediately adjacent to the target codon (H) cannot be G;
  • a 20 bp sgRNA sequence corresponding to the complement of the target sequence was prepared.
  • the CCN-20bp target sequence (PAM sequence) of the coding region of the gene to be knocked out is selected to contain the entire target codon TGG, and the base immediately below the target codon (D) cannot be C Accordingly, the target single base G is preferably located at positions 4-8 of the target sequence (right end), and the target codon is preferably 14 bp apart from the CCN.
  • the present invention selects the following target gene sequences to design corresponding sgRNAs (bold underlined to indicate PAM; in italics)
  • the stroke indicates the candidate mutation coding):
  • Sg-1 CTA CAA CTGGGCTGGCGGCC AGG
  • Sg-2 CAG CAA CCAGACGGACAAGC TGG
  • Sg-1 AAAA CAG CTGAGACTTAAAA GGG
  • human PD1 (3), LAG3 (3), TIGIT, VISTA, 2B4, CD160 and murine TIM3, LAG3, construct corresponding sgRNA expression vectors, and introduce different sgRNA into pGL3- U6-sgRNA.
  • BE3 mediated base editing was performed on the cell line, and a stop codon was introduced to achieve gene knockout.
  • Gene knockout (by electroporation or lipofection) of cell lines is performed as usual, and liposome transfection is exemplified.
  • HEK293T cells are seeded and cultured in DMEM high glucose medium (HyClone, SH30022.01B) supplemented with 10% FBS, which contains penicillin ( 100 U/ml) and streptomycin (100 ⁇ g/ml).
  • DMEM high glucose medium HyClone, SH30022.01B
  • FBS penicillin
  • streptomycin 100 ⁇ g/ml
  • Some cells were collected and lysed with 100 ⁇ g/ml proteinase K in lysate (10 ⁇ M Tris-HCl, 0.4 M NaCl, 2 ⁇ M EDTA, 1% SDS), and then extracted with phenol-chloroform and dissolved in 50 ⁇ l of deionized water.
  • PCR amplification was performed using a pair of primers N-For and N-Rev.
  • the PCR product was purified by AxyPrep PCR cleanup, and 200 ng was diluted to 20 ⁇ l for denaturation and annealing.
  • the procedure was as follows: 95 ° C, 5 min; 95-85 °C at -2 ° C / s; 85 - 25 ° C at - 0.1 ° C / s; hold at 4 ° C.
  • Sg-2 CAGCAACCAGACGGACAAGC TGG
  • the genes such as PD1, LAG3, TIGIT, VISTA, 2B4 and CD160 were successfully knocked out.
  • BE3 mediated base editing was performed on primary cells, and a stop codon was introduced to achieve gene knockout.
  • A. Collect peripheral blood with an anticoagulant tube, and shake the peripheral blood to fully mix the peripheral blood with the anticoagulant;
  • peripheral blood cells and the lymphocyte separation solution are mixed in equal volume, centrifuged, and the cells of the white blood layer after centrifugation are aspirated;
  • the obtained white blood layer cells are mixed with PBS or serum-free cell culture medium 1640, and then centrifuged, and the precipitate is the PBMC cells.
  • PBMC cell concentration adjusted to 50x10 6 cell / ml.
  • the cells after electroporation were rapidly transferred to AIM-V medium supplemented with 10% FBS which was preheated in advance, and cultured for 2 hours in a 37-degree 5% carbon dioxide incubator.
  • the cells after electroporation were completely exchanged, and the cells were resuspended at a density of 1 ⁇ 10 6 /ml and cultured overnight.
  • the T cells were collected into a 15 ml centrifuge tube, and the centrifuge tube was placed in a magnetic stand, and the supernatant was slowly transferred to another clean 15 ml centrifuge tube, and this step was repeated once.
  • Some cells were collected and lysed with 100 ⁇ g/ml proteinase K in lysate (10 ⁇ M Tris-HCl, 0.4 M NaCl, 2 ⁇ M EDTA, 1% SDS), and then extracted with phenol-chloroform and dissolved in 50 ⁇ l of deionized water.
  • PCR amplification was performed using a pair of primers N-For and N-Rev.
  • the PCR product was purified by AxyPrep PCR cleanup, and 200 ng was diluted to 20 ⁇ l for denaturation and annealing.
  • the procedure was as follows: 95 ° C, 5 min; 95-85 °C at -2 ° C / s; 85 - 25 ° C at - 0.1 ° C / s; hold at 4 ° C.
  • Sg-2 CAGCAACCAGACGGACAAGC TGG
  • the genes such as PD1, LAG3, TIGIT, VISTA, 2B4 and CD160 were successfully knocked out.
  • mice Mouse embryo collection, microinjection, embryo culture, and embryo transfer were performed as usual. Knockout mice were constructed using the TIM3 and LAG3 genes as examples.
  • genomic DNA was extracted from the tail of conventional mice, and the coding region was amplified by PCR. Sanger sequencing was performed. The sequencing results were as follows (bold under the head indicates PAM; italic indicates the mutation code; and italic underline indicates the mutated base. ):

Abstract

提供了一种碱基编辑的基因敲除方法：选定待敲除基因的编码区的20bp–NGG目标序列，使其包含完整的目标密码子CAA、CAG或CGA；利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，其中目标单碱基C优选位于目标序列的第4-8位，目标密码子与NGG间隔12-14bp,并且目标密码子的上游紧邻碱基(H)不能为G；该sgRNA序列为与目标序列互补对应的20bp序列。

Description

基于碱基编辑的基因敲除方法及其应用 技术领域
本发明涉及基因敲除策略，更具体来说，涉及基于碱基编辑的基因敲除方法及其应用。
背景技术
传统的真核生物靶向基因操作是通过全能胚胎干细胞的同源重组和囊胚注射实现的。由于受到建立全能胚胎干细胞这一限制，主要是小鼠(也有大鼠的报道)可以通过全能胚胎干细胞的同源重组完成基因靶向改造[Capecchi,2005]。进行基因靶向操作的另一种途径是克隆，即体细胞的基因改造和核移植。不过，克隆技术存在一些缺陷[Carter et al.,2002；Zhu et al.,2004]。比如，1、终末分化的体细胞克隆后很难完全去分化成未分化细胞，影响胚胎发育，造成发育缺陷；2、所有的遗传物质仅为母源；3、成功率低等。传统的基因靶向操作技术制约了基因敲除。
可编程核酸内切酶技术包括锌指核酸酶(zinc-finger nucleases,ZFNs)技术和转录激活因子样效应物核酸酶(transcription activator-like effectors nucleases,TALENs)技术，以及规律成簇间隔短回文重复系统(clustered regularly interspaced short palindromic repeat；CRISPR-associated，CRISPR/Cas9)[Kim and Kim,2014]。这类技术的发明和推广打破了全能胚胎干细胞的限制，使得不同物种的基因操作变得可能。特别是CRISPR/Cas9系统，由于简便、高效、价廉，出现之后立即席卷全球，成为了基因编辑领域最新，但发展最快、应用最广的技术，引发了基因编辑领域的革命。现在CRISPR/Cas9系统已经被成功地用于DNA敲除、DNA敲入、DNA替代、DNA修饰、RNA修饰、DNA标记、基因转录调节等[Hsu et al.,2014；Komor et al.,2017]。并已经成功应用于多个物种的基因编辑[Barrangou R&Doudna JA,2016；Komor et al.,2017]。
CRISPR/Cas9介导特异性基因编辑是利用sgRNA(single guided RNA)通过靶序列互补引导Cas9蛋白定位剪切双链DNA，造成双链DNA断裂(double-strand breaks,DSB)，在没有模板的条件下，发生非同源末端连接(non-homologous end joining,NHEJ)修复，造成移码突变(frameshift mutation)，导致基因敲除(knockout)；在有模板的条件下，通过同源重组进行修复(homology-directed repair,HDR)，实现基因敲入(knockin)[Hsu et al.,2014；Kim and Kim,2014；Komor et al.,2017]。由于HDR效率低(整合很少发生)，而且非同源性末端接合机制容易产生随机插入和删除(indel)，使得在断裂点附近可能随机引入新的碱基，从而导致不精确的基因编辑。此外，CRISPR/Cas9介导的基因编辑总有一些脱靶效应[Gorski et al.,2017]。
发明内容
本发明的目的之一是提供一种高效精准的基因敲除策略。
最新研究表明，基于CRISPR/Cas9技术所构建的Cas9融合蛋白可作为“碱基编辑器(Base editor，BE)”。这些融合蛋白包含dCas9或Cas9切口酶以及大鼠胞苷脱氨酶APOBEC1，它通过脱氨基作用将胞嘧啶(C)转化为尿嘧啶，而无需切割DNA。之后，通过DNA复制或修复，尿嘧啶被转化成胸腺嘧啶(T)。类似地，其也能将单碱基G转化成A。特别是Cas9切口酶与APOBEC1组成的BE3，其能够将碱基编辑效率显著提高到15～75％。由于不需切割DNA造成DSB，形成的indel低于1％，实现的基因编辑更精确[Komor et al.,2016]；而且，这种方式将脱靶效率降低到低于自然背景的10倍，实现的基因编辑更安全[Nishida et al.,2017]。BE3已经被成功地用于在体内碱基编辑，实现了小鼠的CT突变，效率达到44～57％[Kim et al.,2017]。
发明人基于上述BE介导单碱基突变，尤其是BE3介导的单碱基编辑的精确性和特异性，巧妙设计了一种基因敲除策略：通过CT突变引入终止密码子，例如将CAA、CAG、CGA突变成终止密码子TAA、TAG、TGA，或通过GA突变将TGG突变成终止密码子TAA、TGA、TAG，终止编码基因的翻译，从而实现基因敲除。
根据本发明的第一方面，提供了一种基因敲除方法，其包括：
选定待敲除基因的编码区(CDS区)的20bp–NGG目标序列(PAM序列)，使其包含完整的目标密码子CAA、CAG或CGA；
利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，
其中目标单碱基C位于目标序列的(左端)第1-8位，优选为第4-8位，目标密码子与NGG间隔12-14bp,优选为14bp，并且目标密码子的上游紧邻碱基(H)不能为G；
所述sgRNA序列为与目标序列互补对应的20bp序列。
可替代地，在上述方法中，也可选定待敲除基因的编码区的CCN‐20bp目标序列(PAM序列)，使其包含完整的目标密码子TGG，目标密码子的下游紧邻碱基(D)不能为C；相应地，目标单碱基G位于目标序列的(右端)第1-8位，优选为第4-8位，目标密码子与CCN间隔12-14bp，优选为14bp。
根据本发明，BE3可以选自：rAPOBEC1-SaCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS；rAPOBEC1-SpCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS，优选采用后两种。
根据本发明的方法可以用于敲除如下八个靶基因：人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，与其相应的sgRNA序列分别与序列一至八所示目标基因序列互补。
根据本发明的第二方面，提供了上述方法在细胞系HEK293T进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
根据本发明的第三方面，提供了上述方法在人T细胞进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
根据本发明的第四方面，提供了根据上述应用而获得的分离的T细胞或细胞系或它们的次代培养物。
根据本发明的第五方面，提供了一种用于基因敲除的试剂盒，包括(与待敲除基因相应的)上述sgRNA、BE3以及相应的扩增试剂。
本发明利用CRISPR/Cas9基础上发展的碱基编辑技术，通过精准的CT或GA单碱基突变创造终止密码子，从而建立了比CRISPR/Cas9更高效、更精确以及更少脱靶效应的基因敲除策略。
附图说明
图1为根据本发明的利用CT突变实现目标基因被敲除的示意图；
图2-5为不同BE3的结构示意图。
具体实施方式
首先，构建不同的BE3，如图2-5所示，将不同的Cas9nickase与胞苷脱氨酶(APOBEC1)融合后形成如下四种BE3：
(1)rAPOBEC1-SaCas9-NLS-UGI-NLS，图2，序列1；
(2)3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS，图3，序列2；
(3)rAPOBEC1-SpCas9-NLS-UGI-NLS，图4，序列3；
(4)3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS，图5，序列4。
在下面进行基因敲除时，可以采用上述任一种BE3，优选为(3)或(4)。
接下来进行sgRNA的设计。碱基定点编辑是利用sgRNA将BE3定位到或靶向特异靶位点，靶基因特异性sgRNA的选择和设计是本发明的关键之处。本发明如下选择设计sgRNA：
选定待敲除基因的编码区的20bp–NGG目标序列(PAM序列)，使其包含完整的目标密码子CAA、CAG或CGA；
目标单碱基C优选位于目标序列的(左端)第4-8位，目标密码子与NGG优选间隔14bp,并且目标密码子的上游紧邻碱基(H)不能为G；
制备与目标序列互补对应的20bp sgRNA序列。
在替代方案中，则选定待敲除基因的编码区的CCN-20bp目标序列(PAM序列)，使其包含完整的目标密码子TGG，目标密码子的下游紧邻碱基(D)不能为C；相应地，目标单碱基G优选位于目标序列的(右端)第4-8位，目标密码子优选与CCN间隔14bp。
针对八个靶基因——人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，本发明选定下述目标基因序列来设计相应的sgRNA(粗体下划表示PAM；斜体下划表示候选突变编码子)：
一.hPD-1
Sg-1:CTA CAACTGGGCTGGCGGCC AGG
Sg-2:CAG CAACCAGACGGACAAGC TGG
Sg-3:CGGC CAGTTCCAAACCCTGG TGG
二.hLAG3
Sg-1: CCAGACCATAGGAGAGATG TGGG
Sg-2: CCATAGGAGAGATG TGGGAGGCT
Sg-3: CCGGCGGCGCCCTCCTCC TGGGG
三.hTIGIT
Sg-1:GAT CGAGTGGCCCCAGGTCC CGG
四.hVISTA
Sg-1: CCTTCTACAAGACG TGGTACCGC
五.2B4
Sg-1:GCAGCT CAGCAGCAGGACAG TGG
六.hCD160
Sg-1:AAAA CAGCTGAGACTTAAAA GGG
七.mTIM3
Sg-1: CCTCGTGCCCGTCTGC TGGGGCA
八.mLAG3
Sg-1: CCAGACCATAGGAGAGATG TGG
针对上述选定的目标基因序列，人PD1(3条)、LAG3(3条)、TIGIT、VISTA、2B4、CD160以及鼠TIM3、LAG3，构建相应的sgRNA表达载体，将不同的sgRNA分别导入pGL3-U6-sgRNA。
实施例1
在细胞株上进行BE3介导的碱基编辑，引入终止密码子，实现基因敲除。按常规操作，进行细胞株的基因敲除(通过电转或脂质体转染)，以脂质体转染为例。
(1)以HEK293T细胞为例，本发明进行真核生物细胞的培养与转染：HEK293T细胞接种培养于添加10％FBS的DMEM高糖培养液中(HyClone,SH30022.01B),其中含penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前分至6孔板中，待密度达到70％-80％时进行转染。
(3)转染以脂质体转染为例。按照Lipofectamine TM2000Transfection Reagent(Invitrogen,11668-019)的操作手册，以SpCas9nickase为例，将2μg BE3质粒与2μg pGL3-U6-sgRNA质粒混匀,共转染至每孔细胞中，6-8小时后换液，72小时后收取细胞。
(4)基因型分析
A、收取部分细胞在裂解液(10μM Tris-HCl,0.4M NaCl,2μM EDTA,1％SDS)中用100μg/ml蛋白酶K裂解消化后,酚-氯仿抽提后溶解到50μl去离子水中。
B、使用一对引物N-For和N-Rev进行PCR扩增，用AxyPrep PCR cleanup纯化获得PCR回收产物，取200ng统一稀释到20μl进行变性、退火，程序如：95℃，5min；95-85℃ at-2℃/s；85-25℃ at-0.1℃/s；hold at 4℃。
C、获得的PCR回收产物用rTaq进行加A反应。加A反应体系为：
700-800ng PCR回收产物
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
补水至50μl体系。
37℃温育30分钟后，取1μl产物与pMD19-T vector(TAKARA,3271)连接并转化DH5感受态细胞(TransGen,CD201)。
D、挑取单克隆，用通用引物M13-F测序各靶基因突变，测序结果如下(粗体下划表示PAM；斜体表示突变编码子；斜体下划表示突变碱基)：
1.hPD-1
Sg-1:CTACAACTGGGCTGGCGGCC AGG
Mut:CTA TAACTGGGCTGGCGGCC AGG
Sg-2:CAGCAACCAGACGGACAAGC TGG
Mut:CAG TAACCAGACGGACAAGC TGG
Sg-3:CGGCCAGTTCCAAACCCTGG TGG
Mut:CGGC TAGTTCCAAACCCTGG TGG
2.hLAG3
Sg-1: CCAGACCATAGGAGAGATGTGGG
Mut: CCAGACCATAGGAGAGATGTG AG
Sg-2: CCATAGGAGAGATGTGGGAGGCT
Mut: CCATAGGAGAGATGTG AGAGGCT
Sg-3: CCGGCGGCGCCCTCCTCCTGGGG
Mut: CCGGCGGCGCCCTCCTCCTG AGG
3.hTIGIT
Sg-1:GATCGAGTGGCCCCAGGTCC CGG
Mut:GAT TGAGTGGCCCCAGGTCC CGG
4.hVISTA
Sg-1: CCTTCTACAAGACGTGGTACCGC
Mut: CCTTCTACAAGACGTG ATACCGC
5.2B4
Sg-1:GCAGCTCAGCAGCAGGACAG TGG
Mut:GCAGCT TAGCAGCAGGACAG TGG
6.hCD160
Sg-1:AAAACAGCTGAGACTTAAAA GGG
Mut:AAAA TAGCTGAGACTTAAAA GGG
结果表明：靶基因发生了sgRNA靶向的碱基突变，引入了终止密码子，PD1、LAG3、TIGIT、VISTA、2B4、CD160等基因敲除成功。
实施例2
在原代细胞上进行BE3介导的碱基编辑，引入终止密码子，实现基因敲除。
按常规操作，以人的T细胞进原代细胞的基因敲除(通过电转或脂质体转染)，以电转为例。
(1)PBMC细胞的分离纯化：
A、用抗凝管采集外周血，边采集边摇晃使外周血与抗凝剂充分混合；
B、外周血细胞与淋巴细胞分离液等体积混合，离心，吸取离心后的白膜层细胞；
C、将得到的白膜层细胞与PBS或者无血清细胞培养基1640混合后离心，沉淀即为所述PBMC细胞。
重复三遍。
(2)CD3阳性细胞的富集
A、调整PBMC细胞浓度至50x10 6cell/ml。
B、按每1ml加入CD3+enriched antibodies cocktail 50μl，混匀后室温静置5分钟。
C、按每1ml加入magnet 150μl，混匀后室温静置10分钟，
D、将离心管置于磁力架上静置5分钟，吸取上层细胞悬液至新的15ml离心管中。
E、重复该操作一次。
F、室温离心300*g，10分钟，收集细胞。
G、细胞计数。
(3)CD3阳性细胞的电转
A、配置电转体系
向1.5ml离心管中分别加入8μg BE3质粒与8μg pGL3-U6-sgRNA质粒，并按照Lonza Amaxa电转试剂盒说明书要求，加入82μl电转缓冲液和18μl supplement1,混匀。
B、收取20X10 6个细胞到15ml离心管中，300g离心10分钟，弃掉上清。
C、以A中配好的质粒电转缓冲液混合物重悬细胞，并转移至电转杯中。
D、使用仪器Lonza 2B,U-014程序进行电转。
E、电转后的细胞迅速转移至提前预热的添加有10％FBS的AIM-V培养基中，37度5％二氧化碳培养箱中培养2小时。
F、电转后的细胞全换液，以1X10 6个/ml的密度重悬细胞，培养过夜。
(4)T细胞的激活培养
A、电转培养24小时后，向培养基中加入100U/ml IL-2，并按照1:1的比例加入CD3/CD28dynabeads，激活T细胞。
B、每两天对细胞半换液，或者是补加IL-2，细胞密度始终维持在1X10 6个/ml。
C、激活5天后，将T细胞收集到15ml离心管中，并将离心管置于磁力架中，慢慢将上清转移到另外一个干净的15ml离心管中，重复此步骤一次。
D、室温离心300*g，10分钟，弃上清，使用10％FBS,300U/ml IL-2AIM-V培养基重悬细胞，密度控制在1X10 6个/ml。
E、每两天对细胞半换液，或者是补加IL-2，并计数，细胞密度始终维持在1X10 6个/ml。
(5)基因型分析
A、收取部分细胞在裂解液(10μM Tris-HCl,0.4M NaCl,2μM EDTA,1％SDS)中用100μg/ml蛋白酶K裂解消化后,酚-氯仿抽提后溶解到50μl去离子水中。
B、使用一对引物N-For和N-Rev进行PCR扩增，用AxyPrep PCR cleanup纯化获得PCR回收产物，取200ng统一稀释到20μl进行变性、退火，程序如：95℃，5min；95-85℃ at-2℃/s；85-25℃ at-0.1℃/s；hold at 4℃。
C、获得的PCR回收产物用rTaq进行加A反应。加A反应体系为：
700-800ng PCR回收产物
5μl 10X Buffer(Mg2+PLUS)
4μl dNTP
0.5μl rTaq(TAKARA,R001AM)
补水至50μl体系。
37℃温育30分钟后，取1μl产物与pMD19-T vector(TAKARA,3271)连接并转化DH5感受态细胞(TransGen,CD201)。
D、挑取单克隆，用通用引物M13-F测序T细胞各靶基因突变，测序结果如下(粗体下划表示PAM；斜体表示候选突变编码子；斜体下划表示突变碱基)：
1.hPD-1
Sg-1:CTACAACTGGGCTGGCGGCC AGG
Mut:CTA TAACTGGGCTGGCGGCC AGG
Sg-2:CAGCAACCAGACGGACAAGC TGG
Mut:CAG TAACCAGACGGACAAGC TGG
Sg-3:CGGCCAGTTCCAAACCCTGG TGG
Mut:CGGC TAGTTCCAAACCCTGG TGG
2.hLAG3
Sg-1: CCAGACCATAGGAGAGATGTGGG
Mut: CCAGACCATAGGAGAGATGTG AG
Sg-2: CCATAGGAGAGATGTGGGAGGCT
Mut: CCATAGGAGAGATGTG AGAGGCT
Sg-3: CCGGCGGCGCCCTCCTCCTGGGG
Mut: CCGGCGGCGCCCTCCTCCTG AGG
3.hTIGIT
Sg-1:GATCGAGTGGCCCCAGGTCC CGG
Mut:GAT TGAGTGGCCCCAGGTCC CGG
4.hVISTA
Sg-1: CCTTCTACAAGACGTGGTACCGC
Mut: CCTTCTACAAGACGT AGTACCGC
5.2B4
Sg-1:GCAGCTCAGCAGCAGGACAG TGG
Mut:GCAGCT TAGCAGCAGGACAG TGG
6.hCD160
Sg-1:AAAACAGCTGAGACTTAAAA GGG
Mut:AAAA TAGCTGAGACTTAAAA GGG
结果表明：靶基因发生了sgRNA靶向的碱基突变，引入了终止密码子，PD1、LAG3、TIGIT、VISTA、2B4、CD160等基因敲除成功。
实施例3
构建BE3介导的基因敲除小鼠
按常规操作进行小鼠的胚胎收集、显微注射、胚胎培养和胚胎移植等。以TIM3和LAG3基因为例构建敲除小鼠。
(1)显微注射：受精卵分别注射BE3mRNA和TIM3特异性sgRNA(对应于上述序列七)，或BE3mRNA和LAG3特异性sgRNA(对应于上述序列八)。常规进行胚胎移植；
(2)基因型分析：常规小鼠剪尾提取基因组DNA，分别PCR扩增编码区域，Sanger测序，测序结果如下(粗体下划表示PAM；斜体表示突变编码子；斜体下划表示突变碱基)：
7.mTIM3
Sg-1: CCTCGTGCCCGTCTGCTGGGGCA
Mut: CCTCGTGCCCGTCTGCT AGGGCA
8.mLAG3
Sg-1: CCAGACCATAGGAGAGATGTGG
Mut: CCAGACCATAGGAGAGATGTG A
上述结果确证TIM3和LAG3的CT突变和终止密码子的引入。构建TIM3和LAG3敲除小鼠成功。
Figure PCTCN2018088925-appb-000001
Figure PCTCN2018088925-appb-000002
Figure PCTCN2018088925-appb-000003
Figure PCTCN2018088925-appb-000004
Figure PCTCN2018088925-appb-000005
Figure PCTCN2018088925-appb-000006
Figure PCTCN2018088925-appb-000007
Figure PCTCN2018088925-appb-000008
Figure PCTCN2018088925-appb-000009
Figure PCTCN2018088925-appb-000010
Figure PCTCN2018088925-appb-000011
Figure PCTCN2018088925-appb-000012
Figure PCTCN2018088925-appb-000013
Figure PCTCN2018088925-appb-000014

Claims (7)

  1. 一种基因敲除方法，包括：
    选定待敲除基因的编码区的20bp–NGG目标序列，使其包含完整的目标密码子CAA、CAG或CGA；
    利用sgRNA序列来将BE3定位到目标序列以使目标密码子中的目标单碱基C变成T从而相应引入终止密码子TAA或TAG、TGA以实现基因敲除，
    其中目标单碱基C位于目标序列的第1-8位，目标密码子与NGG间隔12-14bp,并且目标密码子的上游紧邻碱基不能为G；
    所述sgRNA序列为与目标序列互补对应的20bp序列。
  2. 根据权利要求1所述的方法，其中BE3选自：rAPOBEC1-SaCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SaCas9-NLS-UGI-NLS；rAPOBEC1-SpCas9-NLS-UGI-NLS；3xUGI-rAPOBEC1-SpCas9-NLS-UGI-NLS。
  3. 根据权利要求1所述的方法，用于敲除如下八个靶基因：人PD1、LAG3、TIGIT、VISTA、2B4、CD160以及小鼠TIM3和LAG3，与其相应的sgRNA序列分别与序列一至八所示目标基因序列互补。
  4. 权利要求1所述方法在细胞系HEK293T进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
  5. 权利要求1所述方法在人T细胞进行人PD1、LAG3、TIGIT、VISTA、2B4、CD160基因敲除的应用。
  6. 根据权利要求4或5的应用而获得的分离的T细胞或细胞系或它们的次代培养物。
  7. 一种用于基因敲除的试剂盒，包括权利要求1或2中所述的sgRNA、BE3以及扩增试剂。
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