CN108795902A - 一种安全高效的CRISPR/Cas9基因编辑技术 - Google Patents

一种安全高效的CRISPR/Cas9基因编辑技术 Download PDF

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CN108795902A
CN108795902A CN201810733106.0A CN201810733106A CN108795902A CN 108795902 A CN108795902 A CN 108795902A CN 201810733106 A CN201810733106 A CN 201810733106A CN 108795902 A CN108795902 A CN 108795902A
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姜舒
纪惜銮
张芸
郭明
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Shenzhen Three Zhi Medical Science And Technology Co Ltd
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Abstract

本发明针对现有CRISPR/Cas9基因编辑技术脱靶效应的技术缺陷，对Cas9蛋白的DNA序列结合的位点进行了氨基酸位点定点突变，获得了脱靶效率低的Cas9蛋白，从而实现靶向性更好更高效的基因编辑功能。同时，本发明还提供了一种采用此基因编辑方法进行HEK293T细胞基因编辑的方法。

Description

一种安全高效的CRISPR/Cas9基因编辑技术
技术领域
本发明涉及基因编辑技术领域，尤其涉及一种安全高效的CRISPR/Cas9基因编辑技术
背景技术
规律性重复短回文序列簇/CRISPR相关基因(Clustered regulatoryinterspaced short palindromic repeat/CRISPR-associated genes,CRISPR/Cas)系统是细菌对噬菌体的长期抗争过程中进化产生的一种获得性免疫防御系统，普遍存在于细菌与古生菌中。
CRISPR是一类独特的DNA串联重复序列，CRISPR基因座由一个前导区(Leader)、多个短而高度保守的重复序列区(Repeat)和多个间隔序列区(Spacer)组成。Cas基因一般位于CRISPR基因座附近。根据Cas基因核心元件序列的不同，CRISPR/Cas免疫系统被分为3种类型：Ⅰ型、Ⅱ型和Ⅲ型。Ⅰ型和Ⅲ型CRISPR/Cas免疫系统需要多个Cas蛋白形成复合体切割DNA双链，而Ⅱ型CRISPR/Cas免疫系统只需要一个Cas9蛋白来切割DNA双链。Ⅱ型系统是目前被改造得最为成功的人工核酸酶，其CRISPR/Cas基因座结构包括5'端的tracrRNA(trans-activating crRNA)基因，tracrRNA主要与CRISPR转录产物(CRISPR-derived RNA，crRNA)形成crRNA：tracrRNA复合体，帮助Cas9蛋白识别并与crRNA：tracrRNA复合体结合，实现靶点特异性识别；中间是一系列Cas蛋白编码基因(包括Cas9、Cas1、Cas2和Csn2)，主要发挥核酸酶切割作用；3'端是CRISPR基因座。
目前，CRISPR/Cas9系统已作为一种基因组编辑技术被广泛地研究和利用，该技术利用人工设计的向导RNA(single-guide RNA,sgRNA)介导外源表达的Cas9蛋白与基因组靶点特异性结合，以实现对基因组DNA的特异性切割，切割后的基因组DNA通过非同源末端连接或同源重组的方式进行修复，从而实现基因的敲除、敲入等。相比传统的基因打靶技术如锌指核酸酶(Zinc-fingernucleases，ZFN)和转录激活因子样效应因子核酸酶(transcription activator-likeeffector nucleases，TALEN)，CRISPR/Cas系统技术具有技术流程简易、基因编辑效率高、特异性更强、应用范围更广的优势。
然而，作为基因编辑技术，脱靶效应是不可避免的一个缺点，影响了其临床转化应用的进程。因此，如何在提高CRISPR/Cas技术基因编辑效率的同时提高其靶向性，是该技术目前面临的一大难题。
发明内容
为解决以上技术问题，本发明对pSpCas9载体进行了修饰，提高其靶向性。
本发明针对已发表文献中的Cas9蛋白的晶体结构(见图1)中DNA序列结合的位点进行氨基酸突变，获得了脱靶效率低的Cas9蛋白，其序列见序列表中序号1的Cas9蛋白序列，晶体形态见图1，其中突变位点有：848号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸；1003号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸；1060号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸。新得到的SpCas9我们命名为SpCas9-SZ。
相比现有的CRISPR-Cas9系统技术，本发明构建的CRISPR-Cas9系统具有以下优势：
1.优化设计的Cas9酶可提高基因编辑靶向性，减少脱靶效应，减少脱靶导致的基因突变。本技术方案使用的是pSpCas9载体。我们在传统的pSpCas9载体中引入了新的点突变，这些点突变位于Cas9蛋白与DNA结合的关键部位，使得改造后的Cas9蛋白能够更加精准地识别目标序列，从而提高基因编辑效率，降低脱靶效应。
2.通过各种软件设计、评估后的sgRNA靶向性强，同时，靶位点设计在基因的内含子区域，剪切该内含子后利用同源重组技术，将携带有目的基因的DNA模板重组到细胞的基因组中，可修复下游基因突变，从而达到特异性治疗的目的。该技术方法将基因靶点设计在基因的内含子区域，不用通过剪切外显子来进行基因编辑，同以往的基因编辑相比，降低了基因编辑技术脱靶效应导致的风险。
3.该技术方案中，我们利用了腺相关病毒载体(Adeno-associated Virus,AAV)进行Cas9蛋白和sgRNA的输送。该系统为目前最为安全的病毒载体系统，可以提高基因治疗的安全性。通过实验筛选，我们得到的新的血清型的AAV载体能够显著地提高对一些难转染的细胞如干细胞、神经元细胞的转染效率。
4.该技术中设计的Cas9蛋白和sgRNA也可以直接用电转染的方式输送到细胞内。该种方式操作简单，细胞转染效率高，是病毒转染技术的一个良好的补充。
附图说明
图1 Cas9蛋白晶体结构。
图2构建好的针对人类IX凝血因子(F9)基因的psgRNA-F9-SpCas9-SZ结构示意图。
图3psgRNA-F9-SpCas9-SZ转染HEK293细胞后的细胞观察图片。左侧为明视野下细胞图片；右侧为荧光下细胞图片。
图4 T7endonucleaseⅠ酶切法检测插入/缺失效率，以验证psgRNA-F9-SpCas9-SZ对人类血红蛋白基因切割效率。图中1为不含psgRNA-F9-SpCas9-SZ的对照组，2～6为psgRNA-F9-SpCas9-SZ编辑后基因酶切结果图。
图5 T7endonucleaseⅠ酶切法检测插入/缺失效率的统计结果，Data＝Mean±SE,n＝3。
图6 F9-sgRNA-1的靶向序列和人类基因组其他序列进行比对，从中挑选的3个和F9-sgRNA-4靶向序列最为接近的序列。
图7分析psgRNA-F9-SpCas9-SZ的脱靶效率测序结果。
图8重组后的细胞表达EGFP荧光图片。
图9同源重组后的F9基因的测序结果。图中有四个标注序列区域，其中1.1和1.2为F9基因同源臂，2为EF1启动子，3为EGFP基因序列。
具体实施方式
实施例1
以下根据血友病IX凝血因子(F9)基因突变位点进行HEK293T细胞的基因编辑作为具体基因编辑方案来详细说明本发明专利。
1.针对F9基因，设计合成5个sgRNA，其序列见下表。
其中sgRNA编号1-5的共10个DNA序列依上表顺序见生物序列表中编号2到编号11的序列，且其名称与上表中的名称相对应。
每对sgRNA包括两条序列，其中一条为正义链DNA(S)，另一条为反义链DNA(AS)。其中加下划线及斜体部分的序列为和F9基因互补的DNA序列。
2.首先配置sgRNA DNA正义链和反义链的悬液，使其终浓度为100μM，按以下反应体系进行退火：
组分 体积比
sgRNA S(100μM) 1
sgRNA-AS(100μM) 1
T4ligation buffer，10× 1
T4PNK 1
ddH2O 6
总共 10
37℃，30min；95℃，5min；以5℃·min-1的速度降至25℃。获得的产物用ddH2O按1:200稀释至总体积为200μl。
3.将SpCas9-SZ载体用BbSI进行酶切。按以下反应体系进行酶切和连接反应。
组分 体积比
pSpCas9-SZ，1μg/μl 1
10×NEB buffer 2
FastDigest BbsI 1
ddH2O 16
总共 20
37℃酶切反应1h，在上述反应体系基础上添加以下组分：
组分 体积比
步骤2的产物 1
10X T4ligase buffer 2.5
T7ligase 1
ddH2O 0.5
25℃连接反应30min。
4.取上一步骤获得的连接产物2μL，热转化至Stbl3感受态细胞中，涂布平板，37℃过夜培养。挑取单克隆，37℃摇菌过夜，对抽提的质粒进行测序鉴定。正确的克隆命名为psgRNA-F9-SpCas9-SZ。以下是质粒测序结果，序列中斜体标记部分为正确插入的sgRNA的序列。
5.psgRNA-F9-SpCas9-SZ载体转染HEK293T细胞
按1.5×105个细胞/孔将HEK293T细胞接种于24孔板中，细胞悬液总体积为500ml。种板24h后进行细胞转染：A.用无血清培养基将2mg psgRNA-SpCas9-SZ和1mg EGFP载体稀释至250ml，温和混匀；B.用培养基将脂质体稀释至250ml，室温孵育5min；C.将上述A步骤和B步骤获取的混合物混匀，室温孵育20min；D.吸去HEK293T细胞中的培养液，将步骤C获得的混合物加入细胞，细胞置于细胞恒温培养箱37℃孵育6h后换成含10％FBS的DMEM培养基，转染48h后观察转染效率，效果图片见图3，其中左侧为明视野下细胞图片；右侧为荧光下细胞图片。
6.T7endonucleaseⅠ酶切法检测插入/缺失效率，以验证psgRNA-F9-SpCas9-SZ对人类血红蛋白基因切割效率。
6.1收集转染后的细胞，400g离心5分钟；弃去上清液，用1×PBS重悬细胞。
6.2利用细胞基因组DNA提取试剂盒提取其细胞基因组DNA。
6.3以基因组DNA为模板，利用F9基因特异性引物按如下程序进行PCR扩增。
针对F9靶序列的特异性引物：
正向引物：5＇－TTGGAGCCTGGGAAATAATG－3＇
反向引物：5＇－TGAGTCTTTGCACGAGTGATCC－3＇
PCR扩增程序：
程序 时间
①95℃ 3min
②95℃ 30s
③66℃ 30s
④72℃ 30s
⑤goto step② 26cycles
⑥72℃ 5min
⑦4℃ Hold
6.4扩增产物于1.5％琼脂糖凝胶中进行电泳。
6.5利用胶回收试剂盒回收目的DNA片段。
6.6取上一步所得纯化DNA 17ml共300ng，加入2ml T7endonucleaseⅠ缓冲液，按如下程序进行变性退火处理：
6.7于上述退火产物中加入1ml T7endonucleaseⅠ，混匀后37℃孵育酶切30min。
酶切产物于2％琼脂糖凝胶中电泳。比较被T7endonucleaseⅠ酶切及未被酶切的片段的比例即可检测出psgRNA-F9-SpCas9-SZ的切割效率。我们的实验结果表明(见图4，其中1为不含psgRNA-F9-SpCas9-SZ的对照组，2～6为psgRNA-F9-SpCas9-SZ编辑后基因酶切结果图)，所设计的5个sgRNA均有较好的编辑效率。图5为T7endonuclease Ⅰ酶切法检测插入/缺失效率的统计结果(Data＝Mean±SE,n＝3)，可知其中以F9-sgRNA-4编辑效率最高。
实施例2
分析psgRNA-F9-SpCas9-SZ的脱靶效率。
我们将F9-sgRNA-4靶向序列和人类基因组其他序列进行比对，从中挑选了和F9-sgRNA-4靶向序列最为接近的序列(见图6所示序列)，针对这些序列设计特异性的引物，以基因组DNA为模板进行PCR扩增，然后对扩增产物进行测序，测序结果(见图7)表明，我们的psgRNA-F9-SpCas9-SZ在这些位点上都无剪切。
实施例3
利用psgRNA-F9-SpCas9-SZ和AAV打靶载体DNA将目的基因重组于F9基因中，并分析重组效率。
构建pAAV-sgRNA-F9-EF1mini-SpCas9-SZ腺相关病毒载体和包含用于修复F9基因的供体基因序列的载体(pAAV-F9-Donor载体)，并进行病毒包装，感染HEK293T细胞。重组后的细胞表达EGFP的荧光图片见图8。
以流式细胞仪分选EGFP阳性的细胞，提取细胞基因组DNA，然后以特异性引物扩增同源臂之间的序列，将PCR产物送测序。测序结果见图9。结果显示，F9基因成功重组。
序列表
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<120> 一种安全高效的CRISPR/Cas9基因编辑技术
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Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Ala
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Ala Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Ala Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 1
<211> 25
<212> DNA
<213> F9-gRNA1-S(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
caccgtaggt attattgcaa cagtt 25
<210> 2
<211> 25
<212> DNA
<213> F9-gRNA1-As(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
aaacaactgt tgcaataata cctac 25
<210> 3
<211> 25
<212> DNA
<213> F9-gRNA2-S(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
caccgacatt actgagttac aacta 25
<210> 4
<211> 25
<212> DNA
<213> F9-gRNA2-As(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
aaactagttg taactcagta atgtc 25
<210> 5
<211> 25
<212> DNA
<213> F9-gRNA3-S(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
caccgggtac tgtgtcaggg tacta 25
<210> 6
<211> 25
<212> DNA
<213> F9-gRNA3-As(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
aaactagtac cctgacacag taccc 25
<210> 7
<211> 25
<212> DNA
<213> F9-gRNA4-S(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
caccggtact gtgtcagggt actag 25
<210> 8
<211> 25
<212> DNA
<213> F9-gRNA4-As(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
aaacctagta ccctgacaca gtacc 25
<210> 9
<211> 25
<212> DNA
<213> F9-gRNA5-S(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
caccgtgtca gggtactagg ggtat 25
<210> 10
<211> 25
<212> DNA
<213> F9-gRNA5-As(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
aaacataccc ctagtaccct gacac 25

Claims (9)

1.一种用于CRISPR/Cas9基因编辑系统的Cas蛋白突变体，其序列为序列表中序列1所示序列。
2.一种用于CRISPR/Cas9基因编辑系统的Cas蛋白突变体，其中突变增强位点有：848号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸；1003号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸；1060号氨基酸位点碱基序列为GCG，对应的氨基酸为丙氨酸。
3.利用如权利要求1或2所述Cas蛋白进行CRISPR/Cas9系统基因编辑的方法。
4.包括权利要求1或2所述蛋白的CRISPR/Cas9基因编辑系统。
5.权利要求3所述基因编辑方法在在肝脏细胞基因编辑领域的用途。
6.权利要求3所述基因编辑方法在血友病IX凝血因子(F9)基因突变位点编辑领域的用途。
7.权利要求6所述基因编辑方法在血友病IX凝血因子(F9)基因突变位点编辑领域的用途，其中所编辑细胞系为HEK293T细胞。
8.一组RNA序列，其序列如序列表中序号2至序号11所示，其特征在于可与权利要求3所述系统进行血友病IX凝血因子(F9)基因突变位点编辑。
9.如权利要求8所示的RNA序列，其特征在于其序列为序列表中序号8和序号9所示序列。
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Application publication date: 20181113