CN113136376B - 一种Cas12a变体及其在基因编辑中的应用 - Google Patents

一种Cas12a变体及其在基因编辑中的应用 Download PDF

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CN113136376B
CN113136376B CN202110578385.XA CN202110578385A CN113136376B CN 113136376 B CN113136376 B CN 113136376B CN 202110578385 A CN202110578385 A CN 202110578385A CN 113136376 B CN113136376 B CN 113136376B
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殷雷
周进
陈鹏
王宏建
刘欢
方嘉凌
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Abstract

本发明提供了一种Cas12a变体及其在基因编辑中的应用，所述Cas12a变体包括如下中的至少一种：Lb2Cas12a‑K518R，氨基酸序列分别如SED ID NO.1所示；LbCas12a‑K538R，氨基酸序列分别如SED ID NO.2所示；AsCas12a‑K548R，氨基酸序列分别如SED ID NO.3所示。本发明首次通过蛋白改造，获得Cas12a变体，使Cas12a的PAM识别相比较对应野生型更严格，从而降低脱靶效应；所述三种Cas12a变体能够在crRNA的介导下定点对真核生物基因组进行基因编辑且具有更低的脱靶效应，三种Cas12a变体的发现进一步扩大了基因编辑工具的种类。

Description

一种Cas12a变体及其在基因编辑中的应用
技术领域
本发明涉及基因编辑技术领域，特别涉及一种Cas12a变体及其在基因编辑中的应用。
背景技术
自2013年以来，基因编辑技术取得了突破性进展，此项技术已经在基础科学研究、医药、临床、生物技术等许多领域引起了新的变革。除了具有代表性的Cas9系统之外，Cas12，又名Cpf1，作为又一种被发现的具有基因编辑效应的CRISPR系统新成员，极大的扩大了基因编辑系统靶点的可编辑范围，相比于Cas9系统，Cas12a所具有的加工前提RNA的功能，为其介导多基因编辑提供了相比与Cas9系统更为便捷高效的编辑能力。除此之外，相比于Cas9的向导RNA，Cas12a的向导RNA组成更为简单，设计更为方便。
2015年，张峰团队首次发现了Cas9系统之外的另外一种具有基因编辑能力的新成员，Cas12a，又名Cpf1，将其划分到CRISPR系统2类V型中。相比于Cas9系统，Cas12a的编辑效率与Cas9的效率相当，在有些靶点低于Cas9。Cas12a的脱靶率极低，相比于Cas9脱靶率高的特性，Cas12a是一种安全的基因编辑工具。Cas12a在切割之后形成粘性末端，而Cas9形成平末端，已有研究表明，Cas12a切割之后的粘性末端相比于Cas9的平末端而言，更容易发生同源重组修复，这也为基因的定点插入和修复提供了更好的工具。在向导RNA的加工方面，Cas12a具有明显的优势，仅仅只需要Cas12a本身就能够完成对前提RNA的加工，而Cas9系统则需要RNaseIII的加工，这极大地促进Cas12a在多基因编辑上的应用。在PAM的识别上，Cas12a识别5’-TTTN-3’或5’-KYTV-3’，Cas9则识别5’-NGG-3’。
因此，Cas12a作为一种新型基因编辑工具，与Cas9系统一道，为科学研究和疾病的治疗提供了有力的工具。基于对目前已有的Cas12a的研究，为应对将来各种情况下的基因编辑事件，发现更多的具有一定特性的Cas12a变体是一件具有重要意义的事情。
发明内容
本发明目的是提供一种Cas12a变体及其在基因编辑中的应用，该Cas12a变体包括Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R，其在体外比对应野生型PAM识别更严格；且在体内脱靶效应更低。
在本发明的第一方面，提供了一种Cas12a变体，所述Cas12a变体包括如下中的至少一种：
Lb2Cas12a-K518R，氨基酸序列分别如SED ID NO.1所示；
LbCas12a-K538R，氨基酸序列分别如SED ID NO.2所示；
AsCas12a-K548R，氨基酸序列分别如SED ID NO.3所示。
在本发明的第二方面，提供了一种重组表达载体，所述重组载体表达所述的Cas12a变体。
进一步地，所述重组表达载体包括质粒载体、病毒载体和噬菌体载体中的一种。
在本发明的第三方面，提供了一种重组菌或重组细胞系或重组病毒，包含所述的重组表达载体。
在本发明的第四方面，提供了一种重组病毒，所述重组病毒采用所述的病毒载体包装获得，所述重组病毒包括表达所述的Cas12a变体的腺病毒或慢病毒。
在本发明的第五方面，提供了一种所述的Cas12a变体、所述的重组表达载体、所述的重组菌或重组细胞系和所述的重组病毒在基因编辑中的应用。
在本发明的第六方面，提供了一种包括所述Cas12a变体的CRISPR/Cas12a基因编辑系统。
在本发明的第七方面，提供了所述系统还包括：靶向目标基因的crRNA或crDNA。
在本发明的第八方面，提供了一种对受体中的目的基因进行基因编辑的方法，借助CRISPR/Cas12a系统对受体中的目的基因进行基因编辑，所述方法包括：
通过将所述的重组表达载体导入受体提供Cas12a变体；
通过靶向目标基因的crRNA或crDNA质粒导入受体提供crRNA；
所述Cas12a变体和所述crRNA在受体中形成复合物识别目的基因并完成编辑。
进一步地，所述受体包括293T细胞、ATDC5细胞和C6细胞中的一种。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
本发明提供的一种Cas12a变体及其在基因编辑中的应用，本发明首次通过蛋白改造，获得Cas12a变体包括Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R，使Cas12a的PAM识别相比较对应野生型更严格，从而降低脱靶效应；所述三种Cas12a变体能够在crRNA的介导下定点对真核生物基因组进行基因编辑且具有更低的脱靶效应，三种Cas12a变体的发现进一步扩大了基因编辑工具的种类，对基础科研和临床治疗具有十分重要的作用。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1为应用例1中Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R在基因编辑中的应用的结果；其中，A代表野生型Lb，B代表LbK538R，C代表野生型Lb2，D代表Lb2K518R，E代表野生型As，F代表AsK548R；
图2为应用例2中二代测序分析原理图；
图3为应用例2中Lb2Cas12a-K518R，LbCas12a-K538R在体外比对应野生型PAM识别更严格的结果；
图4为应用例3中Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R在体内脱靶效应更低的结果；
图5为应用例3中Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R在体内脱靶效应更低的结果。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
除非另有特别说明，本发明中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买获得或者可通过现有方法获得。
本发明技术方案的总体思路如下：
本申请发明人通过实验发现：
对野生型Cas12a进行如下改造：
Lb2Cas12a-K518R，氨基酸序列分别如SED ID NO.1所示；具体改造为：第518位氨基酸K突变为氨基酸R，
LbCas12a-K538R，氨基酸序列分别如SED ID NO.2所示；具体改造为：第538位氨基酸K突变为氨基酸R，
AsCas12a-K548R，氨基酸序列分别如SED ID NO.3所示；具体改造为：第548位氨基酸K突变为氨基酸R，
采用上述改造后，通过实验证实本发明实施例的Cas12a变体在体外比对应野生型PAM识别更严格，脱靶效应更低。
下面将结合实施例及实验数据对本申请的一种Cas12a变体及其在基因编辑中的应用进行详细说明。
实施例1重组表达载体的构建
1、Lb2Cas12a-K518R的表达载体的构建
(1)Lb2Cas12a-K518R的原核表达载体以NcoI和XhoI为双酶切位点构建到pET28a载体上，具体步骤为(下划线为酶切位点NcoI和XhoI)：
正向引物：5’-CATGCCATGGGCATGTACTATGAGTCCCTGACCAAGCAGTAC-3’(SED IDNO.49)
反向引物：5’-CCGCTCGAGTTTCAGCAGGTGTGTCTGGGCATACTCCAGC-3’(SED ID NO.50)
采用所述引物对对Lb2Cas12a-K518R真核表达载体pcDNA3.1-Lb2Cas12a(K518R)模板扩增，获得PCR产物，PCR纯化回收后用NcoI和XhoI酶切，再次PCR产物回收。
将pET28a载体进行NcoI和XhoI双酶切，胶回收后，将线性化的pET28a与上面酶切回收的片段进行酶连，获得pET28a-Lb2Cas12a(K518R)。
(2)真核表达载体直接在addgene上购买的Lb2Cas12a原始表达载体(编号：#69983)的基础上突变得到。具体突变步骤为(下划线表示精氨酸R对应的密码子)：
正向引物：5’-CTGAACGGCTGGGATCGGAATAGGGAGACAGACAAC-3’(SED ID NO.51)
反向引物：5’-GTTGTCTGTCTCCTTATTCCGATCCCAGCCGTTCAG-3’(SED ID NO.52)
采用所述引物对对pcDNA3.1-Lb2Cas12a进行转圈PCR，获得PCR产物后进行PCR产物回收，然后用DpnI酶切，酶切后的产物直接转化DH5α大肠杆菌，长出菌落提质粒测序分析即可得到pcDNA3.1-Lb2Cas12a(K518R)。
2、LbCas12a-K538R的表达载体的构建
(1)LbCas12a-K538R的原核表达载体以NcoI和XhoI为双酶切位点构建到pET28a载体上；具体步骤为(下划线为酶切位点NcoI和XhoI)：
正向引物：5’-CATGCCATGGGCATGAGCAAGCTGGAGAAGTTTACAAACTG-3’(SED IDNO.53)
反向引物：5’-CCGCTCGAGGTGCTTCACGCTGGTCTGGGCGTACTCCAG-3’(SED ID NO.54)
采用所述引物对对LbCas12a-K538R真核表达载体pcDNA3.1-LbCas12a(K538R)模板扩增，获得PCR产物，PCR纯化回收后用NcoI和XhoI酶切，再次PCR产物回收。
将pET28a载体进行NcoI和XhoI双酶切，胶回收后，将线性化的pET28a与上面酶切回收的片段进行酶连，获得pET28a-LbCas12a(K538R)。
(2)真核表达载体直接在addgene上购买的LbCas12a原始表达载体(编号：#69988)的基础上突变得到。具体突变步骤为(下划线表示精氨酸R对应的密码子)：
正向引物：5’-GGCGGCTGGGACAAGGATAGGGAGACAGACTATCGGGC-3’(SED ID NO.55)
反向引物：5’-GCCCGATAGTCTGTCTCCTTATCCTTGTCCCAGCCGCC-3’(SED ID NO.56)
采用所述引物对对pcDNA3.1-LbCas12a进行转圈PCR，获得PCR产物后进行PCR产物回收，然后用DpnI酶切，酶切后的产物直接转化DH5α大肠杆菌，长出菌落提质粒测序分析即可得到pcDNA3.1-LbCas12a(K538R)。
3、AsCas12a-K548R的表达载体的构建
(1)AsCas12a-K548R的原核表达载体以NcoI和XhoI为双酶切位点构建到pET28a载体上；具体步骤为(下划线为酶切位点NcoI和XhoI)：
正向引物：5’-CATGCCATGGGCATGACACAGTTCGAGGGCTTTACCAACC-3’(SED ID NO.57)
反向引物：5’-CCGCTCGAGGTTGCGCAGCTCCTGGATGTAGGCCAGCCAGTC-3’(SED IDNO.58)
采用所述引物对对AsCas12a-K548R真核表达载体pcDNA3.1-AsCas12a(K548R)模板扩增，获得PCR产物，PCR纯化回收后用NcoI和XhoI酶切，再次PCR产物回收。
将pET28a载体进行NcoI和XhoI双酶切，胶回收后，将线性化的pET28a与上面酶切回收的片段进行酶连，pET28a-AsCas12a(K548R)。
(2)真核表达载体直接在addgene上购买的AsCas12a原始表达载体(编号：#69982)的基础上突变得到；具体突变步骤为(下划线表示精氨酸R对应的密码子)：
正向引物：5’-CCTCTGGCTGGGACGTGAATAGGGAGAAGAACAATGGCGC-3’(SED ID NO.59)
反向引物：5’-GCGCCATTGTTCTTCTCCCTATTCACGTCCCAGCCAGAGG-3’(SED ID NO.60)
采用所述引物对对pcDNA3.1-AsCas12a进行转圈PCR，获得PCR产物后进行PCR产物回收，然后用DpnI酶切，酶切后的产物直接转化DH5α大肠杆菌，长出菌落提质粒测序分析即可得到pcDNA3.1-AsCas12a(K548R)。
实施例2 Cas12a变体的CRISPR/Cas12a基因编辑系统和方法
1、获得crRNA质粒
本发明实施例选择了DNMT1，PD1，B2M，CTLA4，CFTR 5个293T细胞内源基因靶点；
对于以上5个靶点对应的crRNA质粒构建过程如下：
crRNA包括直接重复序列(AsCas12a，LbCas12a，Lb2Cas12a的直接重复序列有所不同)和靶点对应的spacer序列，将二者序列连接起来并加上NotI和XbaI酶切位点合成一对互为反补的引物，退火获得双链DNA；将pU6载体进行NotI和XbaI双酶切后与所述退火获得的双链DNA进行酶连获得。所述涉及到的引物对如表1。
表1
Figure BDA0003085308210000061
Figure BDA0003085308210000071
Figure BDA0003085308210000081
5个靶点对应的crRNA的序列(其中下划线表示直接重复序列)分别如表2所示；
表2
Figure BDA0003085308210000082
2、对受体中的目的基因进行基因编辑的方法
将实施例1所述的重组表达载体分别导入受体提供Cas12a变体；
将靶向目标基因的crRNA质粒导入受体提供crRNA；
所述Cas12a变体和所述crRNA在受体中形成复合物识别目的基因并完成编辑。
所述编辑为使所述靶点序列或其邻近序列发生碱基插入和/或缺失；
所述crRNA靶向靶点序列如表3：
表3
靶点名称 PAM Guide sequence(5'to 3')
DNMT1 TTTG GCTCAGCAGGCACCTGCCTCAGC
PD1 TTTA GCACGAAGCTCTCCGATGTGTTG
B2M TTTA CTCACGTCATCCAGCAGAGAATG
CTLA4 TTTC AGCGGCACAAGGCTCAGCTGAAC
CFTR TTTC GTATAGAGTTGATTGGATTGAGA
应用例1、基因编辑效率测定
我们选择了DNMT1，PD1，B2M，CTLA4，CFTR 5个293T细胞内源基因靶点，分别同时转染Cas12a及其变体和crRNA质粒，48h收细胞，做3次重复的T7E1实验。Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R与对应野生型的平均编辑效率如图1所示。
由图1可知，Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R在基因编辑中与对应野生型效率相当。
应用例2、基因识别情况测定
1、获得含有256种PAM底物的混合物，
该混合底物是通过PCR的方法先从pcDNA-EGFP上P出PAM处4个碱基随机排列，在长度为150bp大小的256种不同PAM底物(总共进行256次PCR)，将256种PAM底物等质量混合为PAM文库。其中PCR引物为：
正向引物：5’-TAGAGAACCCACTGCTTACTGGC-3’
反向引物：5’-TGAACTTGTGGCCGNNNNCGTCGCCGTCCAGCTCGACC-3’(N为A,G,C,T中的一种，共256种)
2、将所述含有256种PAM底物的文库与LbCas12a-crRNA，Lb2Cas12a-crRNA，LbCas12a(K538R)-crRNA，Lb2Cas12a(K518R)-crRNA分别孵育0min，1min，2min，3min，4min，5min，6min，7min，8min，10min，15min，25min，采用PCR扩增未被切割的残留底物，二代测序分析，原理如图2。
其中256种随机PAM为5′-NNNN-3′，从5′到3′分别为PAM的4，3，2，1号核苷酸。分析各个时间点切割产物，结果如图3所示；
由图3可知，颜色越深表示切割能力越强，LbCas12a，Lb2Cas12a，Lb-K538R，LbCas12a-K518R对256种不同PAM的底物切割能力各不相同。带有颜色的格子数越多，表示能识别的PAM越多。Lb2Cas12a-K518R，LbCas12a-K538R在体外比对应野生型PAM识别更严格。其中Lb2Cas12a-K518R相较于Lb2Cas12a PAM由5′-YYN-3′(Y＝T/C，N＝A/T/C/G)变为5′-TYN-3′；LbCas12a-K538R相较于LbCas12a来说，几乎不再识别CTTV,TCTV,and TTCV(V＝A/G/C)等含C的非典型PAM。
应用例3、体内脱靶效应测定
在全基因组水平上探究LbCas12a与LbCas12a-K538R，Lb2Cas12a与Lb2Cas12a-K518R，AsCas12a与AsCas12a-K548R在基因编辑中的保真度，对比Cas12a及其相应变体在全基因组水平的脱靶效应。选取HEK293 site 1，PRKCH，POLQ target 1，POLQ target 2,B2M5个293T细胞内源基因靶点，采用GUIDE-seq方法分析全基因组水平的脱靶效应，GUIDE-seq为评估脱靶效应的利器，GUIDE-seq的原理是利用一种短双链寡核苷酸标签来标记CRISPR-Cas诱导的断裂，然后对标签所在的基因区域进行高通量测序，最后利用生物信息学分析确定脱靶突变的位置以及突变效率。某个脱靶突变的出现频率低至0.1％，通过GUIDE-seq也能检测得到。具体操作为：
在293T细胞中同时转染pcDNA3.1-Cas12a质粒，pU6-crRNA质粒和GUIDE tag,GUIDE-tag为一段短的双链DNA，序列为：
5′-P-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3′
5′-P-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3′
其中，P表示磷酸化，*表示硫代磷酸修饰。48h收集细胞，提取基因组，构建DNA文库，测序结果整理后如图4，图5所示。
如图4和图5可知，Lb2Cas12a-K518R，LbCas12a-K538R，AsCas12a-K548R在全基因组上相较于对应野生型具有较低的脱靶效应。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
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1105 1110 1115 1120
Lys Ala Phe Tyr Ser Ser Phe Met Ala Leu Met Ser Leu Met Leu Gln
1125 1130 1135
Met Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile Ser
1140 1145 1150
Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu
1155 1160 1165
Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala
1170 1175 1180
Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys
1185 1190 1195 1200
Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys
1205 1210 1215
Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys
1220 1225
<210> 3
<211> 1307
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Arg Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn
1010 1015 1020
Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly Val Leu
1025 1030 1035 1040
Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala Lys Met Gly
1045 1050 1055
Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro Tyr Thr Ser Lys
1060 1065 1070
Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe Val Trp Lys Thr Ile
1075 1080 1085
Lys Asn His Glu Ser Arg Lys His Phe Leu Glu Gly Phe Asp Phe Leu
1090 1095 1100
His Tyr Asp Val Lys Thr Gly Asp Phe Ile Leu His Phe Lys Met Asn
1105 1110 1115 1120
Arg Asn Leu Ser Phe Gln Arg Gly Leu Pro Gly Phe Met Pro Ala Trp
1125 1130 1135
Asp Ile Val Phe Glu Lys Asn Glu Thr Gln Phe Asp Ala Lys Gly Thr
1140 1145 1150
Pro Phe Ile Ala Gly Lys Arg Ile Val Pro Val Ile Glu Asn His Arg
1155 1160 1165
Phe Thr Gly Arg Tyr Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala
1170 1175 1180
Leu Leu Glu Glu Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu
1185 1190 1195 1200
Pro Lys Leu Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val
1205 1210 1215
Ala Leu Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr
1220 1225 1230
Gly Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp Ala
1250 1255 1260
Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu Asn His
1265 1270 1275 1280
Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile Ser Asn Gln
1285 1290 1295
Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1300 1305
<210> 4
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
aauuucuacu cuuguagaug cucagcaggc accugccuca gc 42
<210> 5
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 5
aauuucuacu cuuguagaug cacgaagcuc uccgaugugu ug 42
<210> 6
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 6
aauuucuacu cuuguagauc ucacgucauc cagcagagaa ug 42
<210> 7
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 7
aauuucuacu cuuguagaua gcggcacaag gcucagcuga ac 42
<210> 8
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 8
aauuucuacu cuuguagaug uauagaguug auuggauuga ga 42
<210> 9
<211> 43
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 9
aaucucuacu aaguguagau gcucagcagg caccugccuc agc 43
<210> 10
<211> 43
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaucucuacu aaguguagau gcacgaagcu cuccgaugug uug 43
<210> 11
<211> 43
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaucucuacu aaguguagau cucacgucau ccagcagaga aug 43
<210> 12
<211> 43
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 12
aaucucuacu aaguguagau agcggcacaa ggcucagcug aac 43
<210> 13
<211> 43
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaucucuacu aaguguagau guauagaguu gauuggauug aga 43
<210> 14
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 14
aauuucuacu auuguagaug cucagcaggc accugccuca gc 42
<210> 15
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 15
aauuucuacu auuguagaug cacgaagcuc uccgaugugu ug 42
<210> 16
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 16
aauuucuacu auuguagauc ucacgucauc cagcagagaa ug 42
<210> 17
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 17
aauuucuacu auuguagaua gcggcacaag gcucagcuga ac 42
<210> 18
<211> 42
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 18
aauuucuacu auuguagaug uauagaguug auuggauuga ga 42
<210> 19
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aaggaaaaaa gcggccgcaa tttctactct tgtagatgct cagcaggcac ctgcctcagc 60
tctagactag 70
<210> 20
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ctagtctaga gctgaggcag gtgcctgctg agcatctaca agagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 21
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aaggaaaaaa gcggccgcaa tttctactct tgtagatgca cgaagctctc cgatgtgttg 60
tctagactag 70
<210> 22
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctagtctaga caacacatcg gagagcttcg tgcatctaca agagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 23
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
aaggaaaaaa gcggccgcaa tttctactct tgtagatctc acgtcatcca gcagagaatg 60
tctagactag 70
<210> 24
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ctagtctaga cattctctgc tggatgacgt gagatctaca agagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 25
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
aaggaaaaaa gcggccgcaa tttctactct tgtagatagc ggcacaaggc tcagctgaac 60
tctagactag 70
<210> 26
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ctagtctaga gttcagctga gccttgtgcc gctatctaca agagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 27
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
aaggaaaaaa gcggccgcaa tttctactct tgtagatgta tagagttgat tggattgaga 60
tctagactag 70
<210> 28
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
ctagtctaga tctcaatcca atcaactcta tacatctaca agagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 29
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
aaggaaaaaa gcggccgcaa tctctactaa gtgtagatgc tcagcaggca cctgcctcag 60
ctctagacta g 71
<210> 30
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ctagtctaga gctgaggcag gtgcctgctg agcatctaca cttagtagag attgcggccg 60
cttttttcct t 71
<210> 31
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
aaggaaaaaa gcggccgcaa tctctactaa gtgtagatgc acgaagctct ccgatgtgtt 60
gtctagacta g 71
<210> 32
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ctagtctaga caacacatcg gagagcttcg tgcatctaca cttagtagag attgcggccg 60
cttttttcct t 71
<210> 33
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
aaggaaaaaa gcggccgcaa tctctactaa gtgtagatct cacgtcatcc agcagagaat 60
gtctagacta g 71
<210> 34
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ctagtctaga cattctctgc tggatgacgt gagatctaca cttagtagag attgcggccg 60
cttttttcct t 71
<210> 35
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
aaggaaaaaa gcggccgcaa tctctactaa gtgtagatag cggcacaagg ctcagctgaa 60
ctctagacta g 71
<210> 36
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ctagtctaga gttcagctga gccttgtgcc gctatctaca cttagtagag attgcggccg 60
cttttttcct t 71
<210> 37
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
aaggaaaaaa gcggccgcaa tctctactaa gtgtagatgt atagagttga ttggattgag 60
atctagacta g 71
<210> 38
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ctagtctaga tctcaatcca atcaactcta tacatctaca cttagtagag attgcggccg 60
cttttttcct t 71
<210> 39
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
aaggaaaaaa gcggccgcaa tttctactat tgtagatgct cagcaggcac ctgcctcagc 60
tctagactag 70
<210> 40
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ctagtctaga gctgaggcag gtgcctgctg agcatctaca atagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 41
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
aaggaaaaaa gcggccgcaa tttctactat tgtagatgca cgaagctctc cgatgtgttg 60
tctagactag 70
<210> 42
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ctagtctaga caacacatcg gagagcttcg tgcatctaca atagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 43
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
aaggaaaaaa gcggccgcaa tttctactat tgtagatctc acgtcatcca gcagagaatg 60
tctagactag 70
<210> 44
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ctagtctaga cattctctgc tggatgacgt gagatctaca atagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 45
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
aaggaaaaaa gcggccgcaa tttctactat tgtagatagc ggcacaaggc tcagctgaac 60
tctagactag 70
<210> 46
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ctagtctaga gttcagctga gccttgtgcc gctatctaca atagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 47
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
aaggaaaaaa gcggccgcaa tttctactat tgtagatgta tagagttgat tggattgaga 60
tctagactag 70
<210> 48
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
ctagtctaga tctcaatcca atcaactcta tacatctaca atagtagaaa ttgcggccgc 60
ttttttcctt 70
<210> 49
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
catgccatgg gcatgtacta tgagtccctg accaagcagt ac 42
<210> 50
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ccgctcgagt ttcagcaggt gtgtctgggc atactccagc 40
<210> 51
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
ctgaacggct gggatcggaa tagggagaca gacaac 36
<210> 52
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gttgtctgtc tccttattcc gatcccagcc gttcag 36
<210> 53
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
catgccatgg gcatgagcaa gctggagaag tttacaaact g 41
<210> 54
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ccgctcgagg tgcttcacgc tggtctgggc gtactccag 39
<210> 55
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ggcggctggg acaaggatag ggagacagac tatcgggc 38
<210> 56
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gcccgatagt ctgtctcctt atccttgtcc cagccgcc 38
<210> 57
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
catgccatgg gcatgacaca gttcgagggc tttaccaacc 40
<210> 58
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ccgctcgagg ttgcgcagct cctggatgta ggccagccag tc 42
<210> 59
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
cctctggctg ggacgtgaat agggagaaga acaatggcgc 40
<210> 60
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
gcgccattgt tcttctccct attcacgtcc cagccagagg 40

Claims (10)

1.一种Cas12a变体，其特征在于，所述Cas12a变体为Lb2Cas12a-K518R，氨基酸序列如SED ID NO.1所示。
2.一种重组表达载体，其特征在于，所述重组表达载体表达权利要求1所述的Cas12a变体。
3.根据权利要求2所述的重组表达载体，其特征在于，所述重组表达载体包括质粒载体、病毒载体和噬菌体载体中的一种。
4.一种重组菌或重组细胞系或重组病毒，其特征在于，包含权利要求2所述的重组表达载体。
5.一种重组病毒，其特征在于，所述重组病毒采用权利要求3所述的病毒载体包装获得，所述重组病毒包括表达权利要求1所述的Cas12a变体的腺病毒或慢病毒。
6.一种权利要求1所述的Cas12a变体、权利要求2-3任一所述的重组表达载体、权利要求4所述的重组菌或重组细胞系和权利要求5所述的重组病毒在基因编辑中的应用。
7.一种包括权利要求1所述Cas12a变体的CRISPR/Cas12a基因编辑系统。
8.根据权利要求7所述的CRISPR/Cas基因编辑系统，其特征在于，所述系统还包括：靶向目标基因的crRNA或crDNA。
9.一种对受体中的目的基因进行基因编辑的方法，其特征在于，借助CRISPR/Cas12a系统对受体中的目的基因进行基因编辑，所述方法包括：
通过将权利要求2-3任一所述的重组表达载体导入受体提供Cas12a变体；
通过靶向目标基因的crRNA或crDNA质粒导入受体提供crRNA；
所述Cas12a变体和所述crRNA在受体中形成复合物识别目的基因并完成编辑。
10.根据权利要求9所述的一种对受体中的目的基因进行基因编辑的方法，其特征在于，所述受体包括293T细胞、ATDC5细胞和C6细胞中的一种。
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