CN112567031A - 新型crispr相关蛋白及其用途 - Google Patents

Abstract

本发明涉及一种新型CRISPR相关蛋白及其用途。根据本发明，由SEQ ID NO：1或SEQ ID NO：3所示氨基酸序列的蛋白具有核酸内切酶的活性，其识别并切割与向导RNA连接的细胞内核酸序列。因此，在CRISPR‑Cas系统中，本发明的新型CRISPR相关蛋白可以用于基因组编辑的不同核酸酶。

Description

新型CRISPR相关蛋白及其用途

技术领域

本发明涉及一种新型CRISPR相关蛋白及其用途。

背景技术

基因组编辑是一种可以自由编辑生物体遗传信息的技术。生命科学领域的进步和基因组测序技术的发展使得了解广泛的遗传信息成为可能。例如，引起各种人类遗传疾病的基因突变以及生产生物燃料的动植物繁殖，疾病和生长相关基因已被了解；但是，为了改善生物体和治疗人类疾病，必须进一步发展技术以直接利用这种了解。

基因组编辑技术可用于改变动物的遗传信息，包括人、植物和微生物，因此可大大扩展其应用范围。遗传剪刀是为精确剪切所需遗传信息而设计和制造的分子工具，在基因组编辑技术中起着关键作用。与将基因测序领域推向新高度的下一代测序技术类似，使用基因剪刀已成为提高遗传信息利用速度和范围以及开辟新工业领域的关键技术。

迄今为止已经开发的遗传剪刀可以根据它们出现的顺序分为三代。第一代遗传剪刀是锌指核酸酶(ZFN)；第二代遗传剪刀是转录激活子样效应核酸酶(TALEN)；最近研究的，聚簇的规则间隔的短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)是第三代遗传剪刀。

CRISPR是包含多个短直接重复序列的基因座，在约40％的测序细菌和90％的测序古细菌的基因组中存在。当与称为CRISPR RNA(crRNA)和反式激活crRNA(tracrRNA)的两个RNA复合时，Cas9蛋白形成一个活性核酸内切酶，从而在入侵的噬菌体或质粒中切割外源遗传元件以保护宿主细胞。crRNA从过去被外来入侵者占据过的宿主基因组的CRISPR元件转录而来。

源自此CRISPR-Cas系统的RNA向导核酸酶提供了能够编辑基因组的工具。特别地，与使用单向导RNA(sgRNA)和Cas蛋白来编辑细胞和器官的基因组技术有关的研究已被积极开展。最近，Cpf1蛋白(源自普雷沃菌属(Prevotella)和弗朗西斯氏菌属1(Francisella1))被报道为CRISPR-Cas系统中的另一种核酸酶蛋白(B.Zetsche等人，2015)，这使得基因组编辑有了更多选择。

发明公开内容

技术问题

为了开发一种在基因组编辑方面比已知核酸酶更有效的蛋白，在不断努力下，本发明人发现了一种新型CRISPR相关的蛋白质，该蛋白质可识别和切割靶核酸序列，从而完成了本发明。

因此，本发明的目的是提供一种识别并切割靶核酸序列的新型CRISPR相关蛋白。

技术问题的解决方案

为了实现上述目的，本发明提供了具有SEQ ID NO：1所示氨基酸序列的Cas12a蛋白。

此外，本发明提供了具有SEQ ID NO：1所示氨基酸序列的Cas12a蛋白，其中925位的赖氨酸(Lys)被另一个氨基酸取代。

此外，本发明提供了具有SEQ ID NO：3所示氨基酸序列的Cas12a蛋白。

此外，本发明提供了具有SEQ ID NO：3所示氨基酸序列的Cas12a蛋白，，其中930位的赖氨酸(Lys)被另一个氨基酸取代。

此外，本发明提供了具有SEQ ID NO：1所示氨基酸序列的Cas12a蛋白，其中877位的天冬氨酸(Asp)被另一个氨基酸取代。

此外，本发明提供了具有SEQ ID NO：3所示氨基酸序列的Cas12a蛋白，其中873位的天冬氨酸(Asp)被另一个氨基酸取代。

此外，本发明提供了用于治疗癌症的药物组合物，其包含作为活性成分的：mgCas12a；以及crRNA，其靶向癌细胞中特异存在的核酸序列。

发明的有益效果

根据本发明，如SEQ ID NO：1或SEQ ID NO：3所示氨基酸序列的蛋白，具有识别和切割结合向导RNA的细胞内核酸序列的核酸内切酶活性。因此，本发明的新型CRISPR相关蛋白可以用作在CRISPR-Cas系统中进行基因组编辑的另一种核酸酶。

附图说明

图1示出了从宏基因组中发现Cas12a的过程的示意图。

图2A示出了发现的Cas12a的系统发生树。

图2B示出了新型Cas12a和AsCas12a的结构。

图3至图8示出了使用ESPript程序比对本发明的现有Cas12a和mgCas12a中的氨基酸序列。

图9A和9B示出了通过比较和总结本发明的Cas12a和mgCas12a的序列信息获得的表。

图10至12示出了根据pH确定本发明的mgCas12a的活性而获得的结果。另一方面，图10中的crRNA#1具有SEQ ID NO：25所示的核苷酸序列，图11中的crRNA#2具有SEQ ID NO：26所示的核苷酸序列。

图13示出了靶核酸序列和crRNA结合的位置被指出的图。

图14示出了在使用每个CCR5和DNMT1基因的crRNA的情况下，通过鉴定由各个蛋白质(对照蛋白，mgCas12a-1和mgCas12a-2)实现的基因编辑效率所获得的结果。

图15示出了在使用各自基因FucT14-1和FucT14-2的两个crRNA的情况下，通过鉴定各个蛋白质(FnCpf1、mgCas12a-1和mgCas12a-2)实现的基因编辑效率的结果。

图16A和16B示出了通过鉴定FnCas12a、WT mgCas12a-1或WT mgCas12a-2蛋白的DNA切割活性而获得的结果。

图17示出了通过鉴定现有Cas12a(AsCas12a、FnCas12a或LbCas12a)和新型Cas12a(WT mgCas12a-1、d_mgCas12a-1、WTmgCas12a-2或d_mgCas12a-2)的非特异性DNase功能获得的结果。

图18A和18B示出了通过鉴定在没有crRNA时，FnCas12a、WT mgCas12a-1或WTmgCas12a-2蛋白是否具有非特异性DNase功能的结果。

图19示出了通过使用现有Cas12a的5'端(柄端)(handle)鉴定mgCas12a是否可以进行DNA切割而获得的结果。

图20A和20B示出了FnCas12a、mgCas12a-1或mgCas12a-2蛋白在二价离子中的DNA切割活性。

发明实施的最佳方式

在本发明的一方面，提供了一种从宏基因组获得的新型Cas12a蛋白。

如本文所用，术语“Cas12a”是CRISPR相关蛋白，并且也可以称为Cpf1。另外，Cpf1是在V型CRISPR系统中发现的效应蛋白。作为单一效应蛋白的Cas12a与在II型CRISPR系统中发现的效应蛋白Cas9相似，因为它与crRNA结合以切割靶基因。但是，两者的工作方式有所不同。Cas12a蛋白可与单个crRNA协同工作。因此，对于Cas12a蛋白，无需像Cas9中那样，同时使用crRNA和反式激活crRNA(tracrRNA)或通过合成tracrRNA和crRNA的组合来构建单向导RNA(sgRNA)。

此外，与Cas9不同，Cas12a系统可识别靶序列5'端存在的PAM。此外，在Cas12a系统中，确定靶标的向导RNA的长度也比Cas9的短。此外，Cas12a的优势在于，它在靶DNA的切割位点产生5'突出端(粘性末端)，而不是平末端，因此能够进行更准确和多样化的基因编辑。

按照惯例，Cas12a蛋白可能来自暂定菌属(Candidatus genus)、毛螺菌属(Lachnospira genus)、丁酸弧菌属(Butyrivibrio genus)、异域菌属(Peregrinibacteriagenus)、氨基酸球菌属(Acidominococcus genus)、卟啉单胞菌属(Porphyromonas genus)、普雷沃氏菌属(Prevotella genus)、、弗朗西斯菌属(Francisella genus)、暂定甲烷支原体属(Candidatus Methanoplasma genus)、或优杆菌属(Eubacterium genus)。具体地，PbCas12a是源自螺旋体属GWC2011_GWC2_44_17的蛋白；PeCas12a是源自异域菌属GW2011_GWA_33_10的蛋白；AsCas12a是衍生自氨基酸球菌(Acidaminococcus sp.BVBLG)的蛋白质；PmCas12a是一种源自猕猴卟啉单胞菌(Porphyromonas macacae)的蛋白；LbCas12a是一种来自毛螺科菌(Lachnospiraceae bacterium)ND2006的蛋白；PcCas12a是一种来源于狗齿龈卟啉单胞菌(Porphyromonas crevioricanis)的蛋白；PdCas12a是一种源自解糖胨普雷沃氏菌(Prevotella disiens)的蛋白；FnCas12a是源自新凶手弗朗西丝氏菌(Francisellanovicida)U112的蛋白。然而，每种Cas12a蛋白都可能具有不同的活性，取决于其来源的微生物。

在本发明中，通过分析宏基因组中的基因已鉴定出新型Cas12a。在下文中，衍生自宏基因组的Cas12a可以被称为mgCas12a。像AsCas12a一样，本发明的mgCas12a包括WED、REC、PI、RuvC、BH和NUC结构域(图2)。此外，已经确定，与先前已知的Cas12a蛋白相似，本发明的mgCas12a蛋白可以通过包括crRNA和5'-端的gRNA进行基因切割。已确定mgCas12a使用有与FnCas12a具有相同序列的5'-端RNA。具体地，5'-端RNA可以具有AAUUUCUACUGUUGUGUAGAU(SEQ ID NO：12)的序列。然而，已确定mgCas12a甚至可以与AsCas12a和LbCas12a中的5'端RNA一起作用(图19)。

mgCas12a可另外包含用于分离和纯化的标签。标签可以结合至mgCas12a的N末端或C末端。另外，标签可以同时结合至mgCas12a的N末端和C末端。标签的一个特定示例可以是6XHis标签。

作为mgCas12a的一个具体实例，提供了一种具有SEQ ID No：1所示氨基酸序列的蛋白。此外，只要不改变mgCas12a的活性，可以在其中进行部分氨基酸的缺失或取代。具体而言，mgCas12a可以是具有SEQ ID NO：1所示氨基酸序列的蛋白质，其中925位的赖氨酸(Lys)被另一氨基酸取代。在此，其他氨基酸可以是选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任何一种。具体地，所述蛋白质可以具有SEQ ID NO：1所示的氨基酸序列，其中925位的赖氨酸被谷氨酰胺取代。即，该蛋白质可以具有SEQ ID NO：5所示的氨基酸序列。

此外，编码具有SEQ ID NO：1所示氨基酸序列的蛋白的基因可以是如SEQ ID NO：2所示的核苷酸序列的多核苷酸。此外，具有SEQ ID NO：1所示氨基酸序列的mgCas12a，根据本发明，在pH 7.0至pH 7.9下可具有最佳活性。

作为mgCpf1的另一个具体实例，提供了一种具有如SEQ ID NO：3所示氨基酸序列的蛋白。此外，只要不改变mgCpf1的活性，可以在其中进行部分氨基酸的缺失或取代。具体而言，mgCpf1可以是具有SEQ ID NO：3所示氨基酸序列的蛋白质，其中930位的赖氨酸(Lys)被另一氨基酸取代。在此，其他氨基酸可以是选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任一种。具体地，所述蛋白质可以具有SEQ ID NO：3所示的氨基酸序列，其中930位的赖氨酸被谷氨酰胺取代。即，该蛋白质可以具有SEQ ID NO：6所示的氨基酸序列。

编码如SEQ ID NO：3所示氨基酸序列的蛋白质的基因可以是如SEQ ID NO：4所示的核苷酸序列的多核苷酸。

此外，根据本发明，具有SEQ ID NO：3所示氨基酸序列的mgCas12a，在pH 7.0至pH7.9下可具有最佳活性。

在本发明的另一方面，提供了一种具有降低的核酸内切酶活性的mgCas12a蛋白。其一个具体实例可以是具有如SEQ ID NO：1所示氨基酸序列的mgCas12a，其中877位的天冬氨酸(Asp)被另一氨基酸取代。在此，其他氨基酸可以是选自下组的精氨酸(Arg)、组氨酸(His)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任一种。具体地，蛋白可以是通过用丙氨酸(Ala)取代天冬氨酸(Asp)而获得的蛋白。

mgCas12a蛋白的另一个具体实例可以是具有如SEQ ID NO：3所示氨基酸序列的mgCas12a，其中873位的天冬氨酸(Asp)被另一个氨基酸取代。在此，其他氨基酸可以是选自下组的精氨酸(Arg)、组氨酸(His)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任一种。具体地，蛋白可以是通过用丙氨酸(Ala)取代天冬氨酸(Asp)而获得的蛋白。在此，具有降低的核酸内切酶活性的mgCas12a可以被称为死亡mgCas12a或d_mgCas12a。d_mgCas12a可以具有如SEQ ID NO：13或SEQ ID NO：14所示的氨基酸序列。

此外，在本发明的另一个方面，提供了一种用于治疗癌症的药物组合物，其包含作为活性成分的mgCas12a；和靶向癌细胞中特异存在的核酸序列的crRNA。在此，mgCas12a可以具有选自下组的任一种氨基酸序列：SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：5、以及SEQID NO：6。如本文所用，术语“在癌细胞中特异性存在的核酸序列”是指在正常细胞中不存在并且仅在癌细胞中存在的核酸序列。即，该术语是指与正常细胞中的序列不同的序列，并且两个序列可以相差至少一个核酸。此外，这种差异可能是由于部分基因的替换或缺失引起的。作为一个具体实例，癌细胞中特异性存在的核酸序列可以是癌细胞中存在的SNP。存在于癌细胞中的具有上述序列的靶DNA与具有与该靶DNA互补的序列的向导RNA特异性地结合。

特别地，关于癌细胞中特异性存在的核酸序列，可以通过各种癌症组织的基因组测序并使用它们来发现仅存在于癌细胞中的特定SNP来创建crRNA。这以表现出癌细胞特异性毒性的方式完成，因此使得开发患者特异性抗癌治疗药物成为可能。此外，与正常细胞不同，癌细胞中特异性存在的核酸序列可以是癌细胞中具有高拷贝数变异(CNV)的基因。

一个具体癌症的例子可以是选自以下癌症的任何一种：膀胱癌、骨癌、血液癌、乳腺癌、黑素瘤、甲状腺癌、甲状旁腺癌、骨髓癌、直肠癌、喉癌、喉部癌、肺癌、食道癌、胰腺癌、胃癌、舌癌、皮肤癌、脑瘤、子宫癌、头颈癌、胆囊癌、口腔癌、结肠癌、肛周癌、中枢神经系统肿瘤、肝癌和结直肠癌。特别地，癌症可以是胃癌、结直肠癌、肝癌、肺癌和乳腺癌，在韩国被称为五种主要癌症。

在此，靶向癌细胞中特异性存在的核酸序列的crRNA可以包括一个或多个gRNA序列。例如，crRNA可以使用能够同时靶向卵巢癌或乳腺癌中存在的BRCA1的外显子10和11的gRNA。此外，crRNA可以使用两个或多个靶向BRCA1外显子11的gRNA。因此，可以根据癌症治疗的目的和癌症的类型适当地选择gRNA的组合。即，可以选择和使用不同的gRNA。

发明方式

在下文中，将通过以下实施例更详细地描述本发明。但是，以下实施例仅用于说明目的，本发明的范围不限于此。

实施例1.来自宏基因组的Cas12a蛋白的发现

从NCBI基因银行(Genbank)BLAST数据库下载宏基因组核苷酸序列，并将其构建到本地BLASTp数据库中。另外，从Uniprot数据库下载了16个Cas12a和各种CRISPR相关蛋白(Cas1)的氨基酸序列。使用宏CRT(MetaCRT)程序在宏基因组中查找CRISPR重复序列和间隔区序列。然后，仅提取具有CRISPR序列的宏基因组序列，并使用Prodigal程序预测其基因。

在预测的基因中，提取CRISPR序列上游或下游10kb范围内的那些基因，并将Cas12a的氨基酸序列用于预测讨论中的相关基因之间的Cas12a同源物。Cas1基因用于预测Cas12a同源物上游或下游是否有Cas1同源物；选择了在Cas1附近大约800到1,500个氨基酸之间的Cas12a基因。对于这些基因中的每一个，都在NCBI基因银行(Genbank)非冗余数据库中使用了BLASTp来确定该基因是否已经报道或该基因是否与CRISPR完全无关。

去除不以蛋氨酸(Met)开头的Cas12a片段后，使用快速傅里叶变换(MAFFT)程序使用多重比对对这些基因进行比对。然后，使用MEGA7通过邻接法(100x引导程序)绘制系统树。选择与先前已知的Cas12a基因形成单系分类群的基因，并使用MEGA7，最大似然和1000x引导程序绘制其系统树以及现有Cas12a的氨基酸序列，以检查其进化关系。在此，从宏基因组中发现Cas12a的过程在图1中示出。此外，在图2A中示出了Cas12a的系统树。在此，新型蛋白质具有氨基酸序列如SEQ ID NO：1所示，被命名为WT mgCas12a-1。此外，新型蛋白质具有氨基酸序列如SEQ ID NO：3所示，被命名为WT mgCas12a-2。此外，AsCas12a、mgCas12a-1和mgCas12a-2的结构在图2B中示出。

实施例2.mgCas12a变体的生产

使用ESPript程序，基于AsCas12a和LbCas12a的结构比对候选Cas12a。对于WTmgCas12a-1和WT mgCas12a-2，进行部分氨基酸的取代以增加其核酸内切酶活性。其中第925个氨基酸Lys(K)被Glu(Q)取代的WT mgCas12a-1被命名为mgCas12a-1。此外，其中第930个氨基酸Lys(K)被Glu(Q)取代的WT mgCas12a-2被命名为mgCas12a-2。考虑人、拟南芥属和大肠杆菌中密码子的用法，对所得变体进行密码子优化，并对其基因合成提出了仿生学的要求。这里，人密码子优化的mgCas12a-1和mgCas12a-2的核苷酸序列分别示于SEQ ID NO：7和SEQ ID NO：8中。此外，现有Cas12a的氨基酸序列(AsCas12a(SEQ ID NO：9)、LbCas12a(SEQ ID NO：10)，和FnCas12a(SEQ ID NO：11))以及通过ESPript程序比对的候选Cas12a(mgCas12a-1和mgCas12a-2)，在图3至8中示出；通过比较和总结它们的序列信息获得的结果在图9A和9B中示出。

然后，将已经克隆到pUC57载体中的WT mgCas12a-1、WT mgCas12a-2、mgCas12a-1和mgCas12a-2基因的每个再次插入pET28a-KanR-6xHis-BPNLS载体中，然后进行克隆。将克隆的载体分别转化到大肠杆菌菌株DH5a和Rosetta中。从宏基因组CRISPR重复序列中提取了crRNA的5'端序列。提取的RNA被合成为DNA寡核苷酸。使用MEGAshortscript T7 RNA转录酶试剂盒进行DNA寡聚体的转录，并通过FLUOstar Omega检查转录的5'-端的浓度。

实施例3.蛋白质表达和纯化

将5ml培养过夜的大肠杆菌Rosetta(DE3)接种到500ml补充了100mg/ml卡那霉素抗生素的液体TB培养基中。将培养基在37℃的培养箱中培养，直至OD600达到0.6。为了进行蛋白质表达，用0.4uM的异丙基β-D-1-硫代半乳糖吡喃糖苷(IPTG)处理，然后在22℃进一步培养16至18小时。离心后，将获得的细胞与10ml裂解缓冲液(20mM HEPES pH 7.5、100mMKCl，20mM咪唑，10％甘油和不含EDTA的蛋白酶抑制剂混合物)混合，然后通过超声处理以裂解细胞。将该裂解物以6,000rpm离心3次，每次20分钟，然后通过0.22微米的过滤器过滤。

之后，使用镍柱(HisTrap FF，5ml)和300mM咪唑缓冲液进行洗涤和洗脱，并通过亲和色谱纯化蛋白质。通过SDS-PAGE电泳检查蛋白质大小，并针对透析缓冲液(20mM HEPESpH 7.5、100mM KCl、1mM DTT，10％甘油)进行过夜透析。然后，根据蛋白质的大小对蛋白质进行选择性过滤和浓缩(Amicon Ultra Centrifugal Filter 100,000MWCO)。对于蛋白质，使用Bradford定量方法测量其浓度。然后，将蛋白质储存在-80℃并使用。

实施例4.通过裂解分析鉴定适用于mgCas12a的pH范围

通过PCR扩增莴苣(Lactuca sativa)的木糖基转移酶以预测原间隔邻近基序(PAM)，并为此设计了向导RNA(gRNA)。对于mgCas12a-1和mgCas12a-2的核糖核蛋白(RNP)复合物，将每种mgCas12a蛋白与gRNA以1：1.25的分子比例在室温下混合20分钟，以生产每种RNP复合物。将纯化的木糖基转移酶PCR产物用各种浓度的RNP进行处理。然后，用NEBuffer1.1(1X缓冲液成分、10mM Bis-Tris-丙烷-HCl、10mM MgCl₂和100μg/ml BSA)，NEBuffer2.1(1X缓冲液成分，50mM NaCl、10mM Tris-HCl、10mM MgCl₂和100μg/ml BSA)和NEBuffer3.1(1X缓冲液成分、100mM NaCl、50mM Tris-HCl、10mM MgCl₂和100μg/ml BSA)进行浓度调节，并在37℃进行了体外裂解分析。在此，在25℃时，NEBuffer 1.1、NEBuffer 2.1和NEBuffer 3.1分别具有pH 7.0、pH 7.9和pH 7.9值。在完成每个反应之后，在65℃下温育10分钟来终止反应，并通过1.5％琼脂糖凝胶电泳检查完成的反应。结果在图10至图12中示出。在图10至图12中，mgCas12a-1和mgCas12a-2分别由hemgCas12a-1和hemgCas12a-2表示。另外，在图13中显示了木糖基转移酶中的靶核酸序列，以及crRNA结合的位置在图表中示出。

如图10至图12中示出，在用NEBuffer 1.1处理mgCas12a-1和crRNA复合物的情况下，切割靶标dsDNA。此外，在用NEBuffer 1.1处理mgCas12a-2和crRNA复合物的情况下，靶dsDNA被切割。从这些结果，发现mgCas12a-1和mgCas12a-2在pH 7.0下具有活性。

实施例5.mgCas12a在动物细胞中的基因编辑效率分析

实施例5.1.包含mgCas12a-1或mgCas12a-2的RNP的产生，用于CCR5和DNMT1的基因编辑

在37℃，HEK 293T细胞在5％CO₂培养箱中，在补充有10％胎牛血清(FBS)和青霉素-链霉素(P/S)的DMEM培养基中培养。在室温下将每100pmole的mgCas12a-1蛋白和mgCas12a-2蛋白以及200pmole的各靶向CCR5的crRNA和靶向DNMT1的crRNA孵育20分钟，以制备每个RNP。此处，CCR5和DNMT1的crRNA序列由集成DNA技术公司(IDT)合成，并在下表1中显示。

[表1]

将培养的2×10⁵的HEK293T细胞与20μl核转染试剂混合，然后与10μlRNP复合物混合。随后，使用4D-核转染设备(Lonza)用于转染。转染后48和72小时，使用PureLink^TM基因组DNA迷你试剂盒(英杰公司)从细胞中提取基因组DNA。

实施例5.2.靶标处的测序分析

使用下表2中所示的CCR5或DNMT1的衔接子引物扩增实施例5.1中提取的基因组DNA。

[表2]

随后，按照Illumina的方案进行纯化和测序文库制备，然后使用MiniSeq设备在目标位点进行深度测序分析。由mgCas12a-1和mgCas12a-2蛋白实现的基因编辑效率在图14中示出,针对靶位点的测序分析结果示于下表3。如图14所示，mgCas12a-1和mgCas12a-2蛋白表现出比对照蛋白(mock)更高的基因编辑效率。

[表3]

实施例6.mgCas12a在植物细胞中的基因编辑效率分析

实施例6.1.植物原生质体分离

通过50％Clorox处理，灭菌烟草种子1分钟。将灭菌后的种子放在用于种子发芽的培养基上，培养1周。然后，将种子转移到用于培养的品红色盒中，生长3周。所用的光培养条件是光照下16小时和黑暗下8小时，并且种子在25℃至28℃的温度下生长。对于植物，使用生长4至6周的叶子。将叶片放在玻璃板上，并从其上切下叶片的顶端和叶柄，从而仅使用叶片的内部。在此，将叶子切成0.5mm或更小的片。将切下的叶片置于10mL的酶溶液中，在黑暗中于室温在定轨振荡器(50rpm)上孵育3至4个小时。

孵育后，添加10mL W5溶液并小心混合。使用细胞过滤器(70μm)过滤存在于酶溶液中的原生质体。将过滤的原生质体在100×g下离心6分钟。丢弃上清液，并通过添加MMG溶液小心地悬浮原生质体沉淀。然后，将悬浮液置于冰上10至30分钟。对于悬浮液的一部分，使用血细胞计数器(它是一种计数板)和显微镜计数原生质体的数量。随后，进一步添加MMG溶液进行稀释，以使原生质体浓度达到2×10⁶细胞/mL。下表4中示出了酶溶液、MMG溶液和PEG溶液各自的组成。

[表4]

实施例6.2.靶标处的测序分析及其编辑效率的确定

将crRNA，mgCas12a蛋白和NEB缓冲液1.1添加到2mL e-管中至最终体积为20μL，然后使反应在室温下进行10分钟。将实施例6.1中获得的200μL(5×10⁵细胞)原生质体和反应后的crRNA和mgCas12蛋白(20μL体积)添加到e-管(2mL)中，充分混合，然后在干净的工作台中培养10分钟。随后，向其中添加与孵育体积等体积的220μL PEG溶液并仔细混合。将混合物在室温下培养15分钟。然后，向其中加入840μL的W5溶液并充分混合。以100×g离心2分钟后，弃去上清液。然后，在W5溶液中培养两天。然后，收获细胞并从中提取DNA。

使用提取的DNA对靶标部分进行PCR，然后通过下一代测序(NGS)鉴定靶标基因编辑效率。结果示于下表5。如表5所示，通过mgCas12a-1蛋白实现的基因编辑效率比FnCpf1高1.8倍。

[表5]

此外，使用两个crRNA鉴定了每种蛋白对烟草FucT14基因的基因编辑效率。结果在图15中示出。如图15所示，mgCas12a-1蛋白的基因编辑效率比FnCpf1高2倍。在此，靶基因NbFucT14_1和NbFucT14_2的crRNA和引物序列如下表6和7中所示。

[表6]

[表7]

实施例7.FnCas12a和mgCas12a基因编辑效率的比较

为了形成由FnCas12a、WT mgCas12a-1或WT mgCas12a-2蛋白和crRNA组成的每个核糖核蛋白(RNP)复合物，将6pmol FnCas12a、WT mgCas12a-1或WT mgCas12a-2蛋白和7.5pmol crRNA用NEB1.1缓冲液和1X蒸馏水，室温下混合30分钟。为了使用crRNA依赖的Cas12a(FnCas12a、WT mgCas12a-1或WT mgCas12a-2)鉴定dsDNA切割活性，向其中加入0.3pmol的靶dsDNA(线性或环状)，在37℃下反应2小时。此处，HsCCR5、HsDNMT1和HsEMX1被用作DNA。此外，实验中使用的线性DNA(SEQ ID NO：27至SEQ ID NO：29)为PCR纯化的产物，以及环状DNA(SEQ ID NO：30至SEQ ID NO：32)是纯化的质粒。向其中加入SDS和EDTA(凝胶负载染料，NEB)，然后将混合物在-20℃下保存10分钟以终止反应。将每个DNA加载到1％琼脂糖凝胶上，然后电泳，以检查由FnCas12a、WT mgCas12a-1或WT mgCas12a-2引起的DNA切割活性。结果在16A(线性DNA)和16B(环形DNA)中示出。在图16A和16B中，S表示底物，并且在凝胶底部指示的每个数字表示底物DNA带的暗度。

实施例8.鉴定mgCas12a的非特异性DNase活性

为了识别Cas12a(AsCas12a，FnCas12a或LbCas12a)和mgCas12a(WT mgCas12a-1、d_mgCas12a-1、WTmgCas12a-2或d_mgCas12a-2)的随机DNase功能，以与实施例7中相同的方式进行了实验。此处，d-mgCas12a-1和d_mgCas12a-2是指分别从WT mgCas12a-1和WTmgCas12a-2获得的蛋白，通过取代Asp(在WT mgCas12a-1的877位或WT mgCas12a-2的873位)为Ala。

具体地，为了形成由7种类型的Cas12a和crRNA组成的每种核糖核蛋白(RNP)复合物，让6pmol的每种Cas12a蛋白和7.5pmol的crRNA在NEB1.1缓冲液和1X蒸馏水存在下，于室温下反应30分钟。随后，向其中加入0.3pmol的靶标dsDNA，然后在37℃下进行12小时或24小时的反应。此处，HsCCR5、HsDNMT1和HsEMX1被用作DNA。向其中加入SDS和EDTA(凝胶负载染料，NEB)，然后将混合物在-20℃下保存10分钟以终止反应。将每个DNA加载到1％琼脂糖凝胶上，然后电泳，以检查由7种类型的Cas12a引起的DNA切割活性。结果在图17中示出。在图17中，S表示底物，并且在凝胶底部指示的每个数字表示底物DNA带的暗度。

如图17所示，由WT mgCas12a-1、d_mgCas12a-1、WTmgCas12a-2或d_mgCas12a-2(新型Cas12a)和crRNA组成的各个核糖核蛋白复合物的非特异性DNase功能比由AsCas12a、FnCas12a或LbCas12a(已存在的Cas12a)和crRNA组成的核糖核蛋白复合物的非特异性DNase功能弱。此外，总体而言，可以推测Cas12a RNP与DNA的反应会导致非特异性DNase功能。

实施例9.不含crRNA的条件下Cas12a非特异性DNase功能的鉴定

为了鉴定在没有crRNA时，Cas12a是否也具有随机DNase功能，对于FnCas12a、WTmgCas12a-1或WTmgCas12a-2蛋白，以与实施例7相同的方式、进行了不同时间的实验，除了使用没有crRNA的条件。结果在图18A和18B中示出。如图18A和18B中所示，FnCas12a、WTmgCas12a-1或WT mgCas12a-2蛋白即使在没有crRNA的情况下也具有随机DNase功能，其中首先出现了FnCas12a蛋白的随机DNase功能。

实施例10.使用现有Cas12a的柄端(handle)鉴定mgCas12a的DNA切割功能

为了确定新型Cas12a(d_mgCas12a或WT mgCas12a)是否可以使用位于现有Cas12a(AsCas12a、FnCas12a或LbCas12a)序列5'端的柄端进行DNA切割，以与实施例7相同的方式、不同的反应时间进行了实验，除了使用各AsCas12a、FnCas12a或LbCas12a的柄端。结果在图19中示出。

如图19所示，使用AsCas12a、FnCas12a或LbCas12a的柄端对d_mgCas12a或WTmgCas12a蛋白进行DNA切割，使用三种类型的柄端的所有d_mgCas12a或WT mgCas12a蛋白均具有DNA切割功能，尽管效率根据各自的柄端略有不同。从这些结果发现，对于DNA切割，mgCas12a可以使用AsCas12a、FnCas12a或LbCas12a的柄端。

实施例11.鉴定FnCas12a或mgCas12a在二价离子中的活性

此外，为了确定FnCas12a、mgCas12a-1或mgCas12a-2蛋白在二价离子(CaCl₂、CoCl₂、CuSO₄、FeCl₂、MnSO₄、NiSO₄或ZnSO₄)中的DNA切割活性，采用与实施例4中相同的方法进行实验。除了使用预定量的二价离子代替NEBuffer 1.1外，其余均与实施例4相同。结果在图20A和20B中示出。如图20A和20B中所示，在相同的二价离子中，FnCas12a、mgCas12a-1或mgCas12a-2蛋白显示出相似的DNA裂解活性。

<110> G+FLAS生命科学有限公司

首尔大学校产学协力团

<120> 新型CRISPR相关蛋白及其用途

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<151> 2018-08-09

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<213> 人工序列（Artificial Sequence）

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Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser

1 5 10 15

Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln

20 25 30

Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly

35 40 45

Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly

50 55 60

Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser

65 70 75 80

Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp

85 90 95

Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr Lys Lys

100 105 110

Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile

115 120 125

Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala

130 135 140

Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe

145 150 155 160

Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser

165 170 175

Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn Asp Asn

180 185 190

Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys

195 200 205

Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile Lys Asp

210 215 220

Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr

225 230 235 240

Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp Ile Cys

245 250 255

Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn Lys Glu

260 265 270

Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile Leu Cys

275 280 285

Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu

290 295 300

Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys

305 310 315 320

His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr

325 330 335

Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser

340 345 350

Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile

355 360 365

His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys

370 375 380

Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile

385 390 395 400

Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn Ile Lys

405 410 415

Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu

420 425 430

Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu

435 440 445

Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala

450 455 460

Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp

465 470 475 480

Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Thr

485 490 495

Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro

500 505 510

Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala

515 520 525

Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu

530 535 540

Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys

545 550 555 560

Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp

565 570 575

Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile

580 585 590

Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro

595 600 605

Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu Lys Ser

610 615 620

Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe

625 630 635 640

Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp

645 650 655

Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu

660 665 670

Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys

675 680 685

Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His

705 710 715 720

Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile

725 730 735

Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser

740 745 750

Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg

755 760 765

Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val

770 775 780

Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe

785 790 795 800

Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys

805 810 815

Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr

820 825 830

Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn

835 840 845

Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu Gln Tyr

850 855 860

Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg Gly Glu

865 870 875 880

Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val

885 890 895

Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys

900 905 910

Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu Trp Lys

915 920 925

Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val

930 935 940

Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala

945 950 955 960

Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys Val Glu

965 970 975

Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys Leu Asn

980 985 990

Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu

995 1000 1005

Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val Gly

1010 1015 1020

His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys

1025 1030 1035 1040

Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys Asp Leu

1045 1050 1055

Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser Ile Arg

1060 1065 1070

Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr Asn Asn

1075 1080 1085

Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser Val Tyr

1090 1095 1100

Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg Phe Ser

1105 1110 1115 1120

Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys Thr Leu

1125 1130 1135

Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg Gln Asp

1140 1145 1150

Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe Arg Leu

1155 1160 1165

Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr

1170 1175 1180

Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp

1185 1190 1195 1200

Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly

1205 1210 1215

Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr

1220 1225 1230

Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile

1235 1240 1245

Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu

1250 1255 1260

<210> 2

<211> 3792

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> mgCas12a-1

<400> 2

atgaataacg gaacaaataa ctttcagaac tttatcggaa tttcttcttt gcagaagact 60

cttaggaatg ctctcattcc aacagaaaca acacagcaat ttattgttaa aaatggaata 120

attaaagaag atgaactcag aggagaaaat cgtcagatac ttaaagatat catggatgat 180

tattacagag gtttcatttc agaaacttta tcgtcaattg atgatattga ctggacctct 240

ttatttgaga aaatggaaat tcagttaaaa aatggagata ataaagacac tcttataaaa 300

gaacaggctg aaaaacgtaa ggcaatctat aaaaaatttg cagatgatga tagatttaaa 360

aatatgttca gtgcaaaatt aatctcagat attcttcctg aatttgtcat tcataacaat 420

aattattctg catcagaaaa ggaagaaaaa acacaggtaa ttaaattatt ttccagattt 480

gcaacatcat tcaaggacta ttttaaaaac agggctaatt gtttttctgc tgatgatata 540

tcttcttctt cttgtcatag aatagttaat gataatgcag aaatattttt tagtaatgca 600

ttggtgtata ggagaattgt aaaaaatctt tcaaatgatg atataaataa aatatccgga 660

gatattaagg attcattaaa ggaaatgtct ctggaggaaa tttattctta tgaaaaatat 720

ggggaattta ttacacagga aggtatatct ttttataatg atatatgcgg taaagtaaat 780

tcatttatga atttatattg ccagaaaaat aaagaaaaca aaaatctcta taagctgcga 840

aagcttcata aacagatact gtgcatagca gatacttctt atgaggtgcc gtataaattt 900

gaatcagatg aagaggttta tcaatcagtg aatggatttt tggacaatat tagttcaaaa 960

catatcgttg aaagattgcg taagattgga gacaactata acggctacaa tcttgataag 1020

atttatattg ttagtaaatt ctatgaatca gtttcacaaa agacatatag agattgggaa 1080

acaataaata ctgcattaga aattcattac aacaatatat tacccggaaa tggtaaatct 1140

aaagctgaca aggtaaaaaa agcggtaaag aatgatctgc aaaaaagcat tactgaaatc 1200

aatgagcttg ttagcaatta taaattatgt ccggatgata atattaaagc agagacatat 1260

atacatgaaa tatcacatat tttgaataat tttgaagcac aggagcttaa gtataatcct 1320

gaaattcatc tggtggaaag tgaattgaaa gcatctgaat taaaaaatgt tctcgatgta 1380

ataatgaatg cttttcattg gtgttcggtt ttcatgacag aggagctggt agataaagat 1440

aataattttt atgcggagtt agaagagata tatgacgaaa tatatacggt aatttcattg 1500

tataatcttg tgcgtaatta tgtaacgcag aagccatata gtacaaaaaa aattaaattg 1560

aattttggta ttcctacact agcggatgga tggagtaaaa gtaaagaata tagtaataat 1620

gcaattattc tcatgcgtga taatttgtac tatttaggaa tatttaatgc aaaaaataag 1680

cctgacaaaa agataattga aggtaataca tcagaaaata aaggggatta taagaagatg 1740

atttataatc ttctgccagg accaaataaa atgatcccca aggtattcct ctcttcaaaa 1800

accggagtgg aaacatataa gccgtctgcc tatatattgg agggctataa acaaaacaag 1860

catcttaaat cctctaagga ttttgatata acgttttgtc acgatttgat tgattatttt 1920

aagaactgta tagcaataca tcctgaatgg aagaattttg gctttgattt ttctgacacc 1980

tccacatatg aagatatcag cggattttac agagaagtcg aattgcaagg ttataaaatt 2040

gactggacat atatcagcga aaaggatatt gatttgttgc aggaaaaagg acagttatat 2100

ttatttcaaa tatataacaa agatttttcc aagaaaagta ccggaaatga taatcttcat 2160

actatgtatt tgaagaattt gtttagcgaa gagaatttaa aggatattgt actgaaatta 2220

aacggtgagg cggaaatctt ctttagaaaa tcaagcataa agaatccaat aattcataaa 2280

aaaggctcta ttcttgttaa tagaacatat gaagcagagg aaaaagatca atttggaaat 2340

atccagatag tcagaaaaac cataccggaa aatatatatc aggagcttta taaatatttc 2400

aatgataaaa gtgataaaga actttcggat gaagcagcta agcttaagaa tgtagtaggt 2460

catcatgagg ctgctacaaa catagtaaaa gattatagat atacatatga taaatatttt 2520

cttcatatgc ctattacaat caattttaaa gccaataaga caagctttat taatgacaga 2580

atattacaat atattgctaa agaaaagaat ttgcatgtaa taggcattga tcgtggtgaa 2640

agaaacctga tatatgtttc agtaattgat acttgtggaa atattgttga acaaaaatcg 2700

tttaacattg ttaatggata tgattatcag attaagctca agcagcagga gggggcgcga 2760

caaatcgcac gaaaagaatg gaaagaaatc ggcaaaataa aagaaattaa agaaggctat 2820

ttatctcttg taattcatga aatttcaaag atggttatta aatataatgc cataattgca 2880

atggaggatt taagctacgg atttaaaaaa ggtcgtttca aggttgagcg acaggtttac 2940

cagaagtttg agacaatgct tatcaacaaa ctcaactatc tggtatttaa agatatatcc 3000

ataactgaaa acggtggtct tctaaaggga tatcagctta catatattcc agataaactg 3060

aaaaatgtgg gtcatcaatg tggttgtata ttttacgtac ctgctgccta tacatcaaaa 3120

atagatccta caaccggatt tgtaaatata ttcaaattta aagatttaac agttgatgca 3180

aagagagaat ttataaaaaa atttgacagt atcagatatg attcagaaaa aaaactgttt 3240

tgttttacat ttgattataa taactttatt acgcaaaata ctgttatgtc aaagtcaagc 3300

tggagtgtat atacgtacgg agttaggata aaaagaagat ttgtcaatgg caggttctca 3360

aatgaatcgg atacaattga tataacaaaa gatatggaaa aaaccctcga aatgacagat 3420

ataaattgga gagatggtca tgatctgagg caggatatta ttgattatga aatcgtacaa 3480

cacatatttg agatttttag attgactgta caaatgagaa acagtttaag tgaattagaa 3540

gacagggatt atgaccgttt gatttctccg gtgctcaatg aaaataatat attttatgat 3600

tcagctaaag caggagatgc gttacctaaa gacgcagatg ctaatggtgc atattgtata 3660

gctctaaaag gcttgtatga aatcaaacaa attacagaga attggaaaga agacggtaag 3720

ttttcaagag ataaacttaa aatttccaat aaggactggt ttgactttat tcaaaataaa 3780

aggtatttat aa 3792

<210> 3

<211> 1275

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> mgCas12a-2

<400> 3

Met Gly Lys Asn Gln Asn Phe Gln Glu Phe Ile Gly Val Ser Pro Leu

1 5 10 15

Gln Lys Thr Leu Arg Asn Glu Leu Ile Pro Thr Glu Thr Thr Lys Lys

20 25 30

Asn Ile Thr Gln Leu Asp Leu Leu Thr Glu Asp Glu Ile Arg Ala Gln

35 40 45

Asn Arg Glu Lys Leu Lys Glu Met Met Asp Asp Tyr Tyr Arg Asn Val

50 55 60

Ile Asp Ser Thr Leu His Val Gly Ile Ala Val Asp Trp Ser Tyr Leu

65 70 75 80

Phe Ser Cys Met Arg Asn His Leu Arg Glu Asn Ser Lys Glu Ser Lys

85 90 95

Arg Glu Leu Glu Arg Thr Gln Asp Ser Ile Arg Ser Gln Ile His Asn

100 105 110

Lys Phe Ala Glu Arg Ala Asp Phe Lys Asp Met Phe Gly Ala Ser Ile

115 120 125

Ile Thr Lys Leu Leu Pro Thr Tyr Ile Lys Gln Asn Ser Glu Tyr Ser

130 135 140

Glu Arg Tyr Asp Glu Ser Met Glu Ile Leu Lys Leu Tyr Gly Lys Phe

145 150 155 160

Thr Thr Ser Leu Thr Asp Tyr Phe Glu Thr Arg Lys Asn Ile Phe Ser

165 170 175

Lys Glu Lys Ile Ser Ser Ala Val Gly Tyr Arg Ile Val Glu Glu Asn

180 185 190

Ala Glu Ile Phe Leu Gln Asn Gln Asn Ala Tyr Asp Arg Ile Cys Lys

195 200 205

Ile Ala Gly Leu Asp Leu His Gly Leu Asp Asn Glu Ile Thr Ala Tyr

210 215 220

Val Asp Gly Lys Thr Leu Lys Glu Val Cys Ser Asp Glu Gly Phe Ala

225 230 235 240

Lys Ala Ile Thr Gln Glu Gly Ile Asp Arg Tyr Asn Glu Ala Ile Gly

245 250 255

Ala Val Asn Gln Tyr Met Asn Leu Leu Cys Gln Lys Asn Lys Ala Leu

260 265 270

Lys Pro Gly Gln Phe Lys Met Lys Arg Leu His Lys Gln Ile Leu Cys

275 280 285

Lys Gly Thr Thr Ser Phe Asp Ile Pro Lys Lys Phe Glu Asn Asp Lys

290 295 300

Gln Val Tyr Asp Ala Val Asn Ser Phe Thr Glu Ile Val Thr Lys Asn

305 310 315 320

Asn Asp Leu Lys Arg Leu Leu Asn Ile Thr Gln Asn Ala Asn Asp Tyr

325 330 335

Asp Met Asn Lys Ile Tyr Val Val Ala Asp Ala Tyr Ser Met Ile Ser

340 345 350

Gln Phe Ile Ser Lys Lys Trp Asn Leu Ile Glu Glu Cys Leu Leu Asp

355 360 365

Tyr Tyr Ser Asp Asn Leu Pro Gly Lys Gly Asn Ala Lys Glu Asn Lys

370 375 380

Val Lys Lys Ala Val Lys Glu Glu Thr Tyr Arg Ser Val Ser Gln Leu

385 390 395 400

Asn Glu Val Ile Glu Lys Tyr Tyr Val Glu Lys Thr Gly Gln Ser Val

405 410 415

Trp Lys Val Glu Ser Tyr Ile Ser Ser Leu Ala Glu Met Ile Lys Leu

420 425 430

Glu Leu Cys His Glu Ile Asp Asn Asp Glu Lys His Asn Leu Ile Glu

435 440 445

Asp Asp Glu Lys Ile Ser Glu Ile Lys Glu Leu Leu Asp Met Tyr Met

450 455 460

Asp Val Phe His Ile Ile Lys Val Phe Arg Val Asn Glu Val Leu Asn

465 470 475 480

Phe Asp Glu Thr Phe Tyr Ser Glu Met Asp Glu Ile Tyr Gln Asp Met

485 490 495

Gln Glu Ile Val Pro Leu Tyr Asn His Val Arg Asn Tyr Val Thr Gln

500 505 510

Lys Pro Tyr Lys Gln Glu Lys Tyr Arg Leu Tyr Phe His Thr Pro Thr

515 520 525

Leu Ala Asn Gly Trp Ser Lys Ser Lys Glu Tyr Asp Asn Asn Ala Ile

530 535 540

Ile Leu Val Arg Glu Asp Lys Tyr Tyr Leu Gly Ile Leu Asn Ala Lys

545 550 555 560

Lys Lys Pro Ser Lys Glu Ile Met Ala Gly Lys Glu Asp Cys Ser Glu

565 570 575

His Ala Tyr Ala Lys Met Asn Tyr Tyr Leu Leu Pro Gly Ala Asn Lys

580 585 590

Met Leu Pro Lys Val Phe Leu Ser Lys Lys Gly Ile Gln Asp Tyr His

595 600 605

Pro Ser Ser Tyr Ile Val Glu Gly Tyr Asn Glu Lys Lys His Ile Lys

610 615 620

Gly Ser Lys Asn Phe Asp Ile Arg Phe Cys Arg Asp Leu Ile Asp Tyr

625 630 635 640

Phe Lys Glu Cys Ile Lys Lys His Pro Asp Trp Asn Lys Phe Asn Phe

645 650 655

Glu Phe Ser Ala Thr Glu Thr Tyr Glu Asp Ile Ser Val Phe Tyr Arg

660 665 670

Glu Val Glu Lys Gln Gly Tyr Arg Val Glu Trp Thr Tyr Ile Asn Ser

675 680 685

Glu Asp Ile Gln Lys Leu Glu Glu Asp Gly Gln Leu Phe Leu Phe Gln

690 695 700

Ile Tyr Asn Lys Asp Phe Ala Val Gly Ser Thr Gly Lys Pro Asn Leu

705 710 715 720

His Thr Leu Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Arg Asp

725 730 735

Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser

740 745 750

Ser Val Gln Lys Pro Val Ile His Lys Cys Gly Ser Ile Leu Val Asn

755 760 765

Arg Thr Tyr Glu Ile Thr Glu Ser Gly Thr Thr Arg Val Gln Ser Ile

770 775 780

Pro Glu Ser Glu Tyr Met Glu Leu Tyr Arg Tyr Phe Asn Ser Glu Lys

785 790 795 800

Gln Ile Glu Leu Ser Asp Glu Ala Lys Lys Tyr Leu Asp Lys Val Gln

805 810 815

Cys Asn Lys Ala Lys Thr Asp Ile Val Lys Asp Tyr Arg Tyr Thr Met

820 825 830

Asp Lys Phe Phe Ile His Leu Pro Ile Thr Ile Asn Phe Lys Val Asp

835 840 845

Lys Gly Asn Asn Val Asn Ala Ile Ala Gln Gln Tyr Ile Ala Gly Arg

850 855 860

Lys Asp Leu His Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile

865 870 875 880

Tyr Val Ser Val Ile Asp Met Tyr Gly Arg Ile Leu Glu Gln Lys Ser

885 890 895

Phe Asn Leu Val Glu Gln Val Ser Ser Gln Gly Thr Lys Arg Tyr Tyr

900 905 910

Asp Tyr Lys Glu Lys Leu Gln Asn Arg Glu Glu Glu Arg Asp Lys Ala

915 920 925

Arg Lys Ser Trp Lys Thr Ile Gly Lys Ile Lys Glu Leu Lys Glu Gly

930 935 940

Tyr Leu Ser Ser Val Ile His Glu Ile Ala Gln Met Val Val Lys Tyr

945 950 955 960

Asn Ala Ile Ile Ala Met Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly

965 970 975

Arg Phe Lys Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu

980 985 990

Ile Ser Lys Leu Asn Tyr Leu Ala Asp Lys Ser Gln Ala Val Asp Glu

995 1000 1005

Pro Gly Gly Ile Leu Arg Gly Tyr Gln Met Thr Tyr Val Pro Asp Asn

1010 1015 1020

Ile Lys Asn Val Gly Arg Gln Cys Gly Ile Ile Phe Tyr Val Pro Ala

1025 1030 1035 1040

Ala Tyr Thr Ser Lys Ile Asp Pro Thr Thr Gly Phe Ile Asn Ala Phe

1045 1050 1055

Lys Arg Asp Val Val Ser Thr Asn Asp Ala Lys Glu Asn Phe Leu Met

1060 1065 1070

Lys Phe Asp Ser Ile Gln Tyr Asp Ile Glu Lys Gly Leu Phe Lys Phe

1075 1080 1085

Ser Phe Asp Tyr Lys Asn Phe Ala Thr His Lys Leu Thr Leu Ala Lys

1090 1095 1100

Thr Lys Trp Asp Val Tyr Thr Asn Gly Thr Arg Ile Gln Asn Met Lys

1105 1110 1115 1120

Val Glu Gly His Trp Leu Ser Met Glu Val Glu Leu Thr Thr Lys Met

1125 1130 1135

Lys Glu Leu Leu Asp Asp Ser His Ile Pro Tyr Glu Glu Gly Gln Asn

1140 1145 1150

Ile Leu Asp Asp Leu Arg Glu Met Lys Asp Ile Thr Thr Ile Val Asn

1155 1160 1165

Gly Ile Leu Glu Ile Phe Trp Leu Thr Val Gln Leu Arg Asn Ser Arg

1170 1175 1180

Ile Asp Asn Pro Asp Tyr Asp Arg Ile Ile Ser Pro Val Leu Asn Lys

1185 1190 1195 1200

Asn Gly Glu Phe Phe Asp Ser Asp Glu Tyr Asn Ser Tyr Ile Asp Ala

1205 1210 1215

Gln Lys Ala Pro Leu Pro Ile Asp Ala Asp Ala Asn Gly Ala Phe Cys

1220 1225 1230

Ile Ala Leu Lys Gly Met Tyr Thr Ala Asn Gln Ile Lys Glu Asn Trp

1235 1240 1245

Val Glu Gly Glu Lys Leu Pro Ala Asp Cys Leu Lys Ile Glu His Ala

1250 1255 1260

Ser Trp Leu Ala Phe Met Gln Gly Glu Arg Gly

1265 1270 1275

<210> 4

<211> 3828

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> mgCas12a-2

<400> 4

atgggtaaaa atcaaaattt tcaggaattt attggggtat caccacttca aaagacttta 60

agaaacgaat taatcccaac agaaacaaca aaaaagaata ttactcagct tgatcttttg 120

actgaggatg aaatccgcgc gcaaaatcga gagaagctga aagagatgat ggatgactac 180

taccggaatg tgattgatag cactttgcat gtgggtatag ctgttgattg gagctattta 240

ttttcgtgta tgcgaaatca tctaagggag aattccaaag agtcaaagcg ggaattggaa 300

cgaacacagg attctattcg ttcacaaatc cataataagt ttgctgaacg agcggatttt 360

aaggatatgt ttggagcatc gataataaca aaattacttc cgacatatat aaaacagaat 420

tcagaatatt ccgagcggta tgacgagagc atggaaattt tgaaactgta tggaaaattc 480

acaacatcgt tgaccgatta ctttgagaca agaaagaata tcttttctaa agagaaaata 540

tcttctgccg ttggatatcg aatcgtagag gaaaatgctg agatcttctt gcagaatcag 600

aatgcttacg acagaatctg taagatagcg ggactggatt tacatggatt ggataatgaa 660

ataacagcat atgttgatgg aaaaacatta aaagaagtat gttcggatga aggatttgca 720

aaggctatta cacaagaagg gattgatcgc tacaacgagg caatcggtgc agtaaatcaa 780

tatatgaatc tgttatgcca gaagaataag gcattaaaac cgggacaatt taagatgaag 840

cggctacata aacagattct ttgcaaagga acaacctctt tcgatattcc aaagaagttt 900

gaaaatgata aacaggtgta tgacgcagtt aattctttta cagagatagt aacgaagaat 960

aatgatttga agcgactgtt aaatattaca cagaatgcaa atgattatga catgaataaa 1020

atctatgtag tagccgatgc atatagtatg atttcacagt ttatcagtaa aaaatggaat 1080

ctgattgaag aatgcttgct ggattattat agcgataatt tgccgggaaa aggaaatgcg 1140

aaagaaaaca aagttaaaaa ggcggtaaag gaagaaacgt atcgcagtgt ttcacagttg 1200

aatgaagtta ttgagaaata ttatgtggaa aagaccggac agtcagtatg gaaagtggaa 1260

agttatattt ctagtctggc agaaatgatt aagctggaat tgtgccacga gatagataac 1320

gatgagaagc ataatctgat tgaagatgat gagaagatat ccgagattaa ggaactgttg 1380

gatatgtaca tggatgtatt tcatattata aaagtgttcc gggtgaatga agtattgaat 1440

ttcgatgaaa ccttttattc ggagatggat gagatctatc aggatatgca ggaaatcgtt 1500

ccattataca atcatgttcg aaactatgtt acacagaaac catataagca ggagaaatat 1560

cgtttatatt tccacactcc aacattggca aatggctggt ccaagagtaa ggaatatgac 1620

aacaacgcaa ttatattggt gcgagaagat aaatattatt taggtattct gaatgcgaaa 1680

aagaaaccat cgaaagaaat tatggcgggc aaagaggatt gttcagaaca tgcatatgca 1740

aagatgaatt attatttgtt gccgggcgcg aacaagatgc ttccaaaagt atttttatct 1800

aagaaaggaa tacaggacta tcacccatca tcatatattg ttgaaggata taatgaaaag 1860

aaacatatta aaggttccaa gaattttgat atccggtttt gtagggattt gattgactac 1920

ttcaaggaat gcattaaaaa acatccggat tggaataagt ttaactttga attttctgcg 1980

acagaaacat atgaggatat cagtgtcttt tatcgcgaag ttgaaaagca aggatatcgc 2040

gtagagtgga cgtatatcaa tagtgaagat attcagaaac tggaagaaga tggacagttg 2100

tttttatttc agatatataa caaagatttt gctgtgggaa gtacaggtaa accaaatctt 2160

catacattgt atctgaaaaa tctgttcagc gaagaaaatt tgcgggacat tgtattaaaa 2220

ctaaatgggg aagcagaaat attcttccgt aaatcaagtg ttcaaaaacc ggtgattcat 2280

aagtgcggca gtattttagt caatcgtacc tatgagatta ccgagagtgg aacaacacgg 2340

gtacaatcaa ttccggaaag tgaatacatg gaattatatc gctactttaa tagtgaaaag 2400

cagatagaat tatcagatga ggcaaaaaaa tatttggaca aggtgcaatg taataaggca 2460

aagacagata ttgtgaaaga ctaccgatac accatggaca agttttttat tcatcttccg 2520

attacgatta attttaaggt tgataagggt aacaatgtta atgccattgc acagcaatat 2580

attgcagggc ggaaagattt acatgtgata ggaattgatc gaggagaacg gaatctgatt 2640

tacgtttctg taattgacat gtatggtaga attttagagc agaaatcctt taaccttgtg 2700

gaacaggtat cgtcgcaggg aacgaagcga tattacgatt acaaagaaaa attacagaac 2760

cgggaagagg aacgggataa agcaagaaag agttggaaga caatcggcaa gattaaggaa 2820

ttaaaagagg ggtatctgtc gtcagtaatt catgagattg cacagatggt cgtaaagtat 2880

aacgcaatca ttgcaatgga agatttgaat tatggattta agcggggaag attcaaagta 2940

gagcgccagg tatatcagaa atttgaaacg atgttgatca gtaagttgaa ttatctggca 3000

gataaatctc aggctgtgga tgaaccggga ggtatattac ggggatatca gatgacttat 3060

gtgccggata atattaagaa tgttggaaga caatgtggaa taatctttta tgtgccggca 3120

gcatatacct ccaagattga tccgacaacc ggatttatca atgcatttaa gcgggatgtg 3180

gtatcaacaa atgatgcaaa agagaatttc ctgatgaagt ttgattctat tcagtacgat 3240

atagaaaaag gcttatttaa gttttcattt gattacaaaa attttgccac acataaactt 3300

acacttgcga agacaaaatg ggacgtatat acaaatggaa ctcgaataca aaacatgaaa 3360

gttgaaggac attggctttc aatggaagtt gaacttacaa cgaaaatgaa agagttgctg 3420

gatgactcgc atattccata tgaagaagga cagaatatat tggatgattt gcgggagatg 3480

aaagatataa caaccattgt gaatggtata ttggaaatct tctggttgac agtccagctt 3540

cggaatagca ggatagataa tccggattac gatagaatta tctcaccggt attgaataaa 3600

aatggagaat tttttgattc tgatgaatat aattcatata ttgatgcgca aaaggcaccg 3660

ttaccgatag atgccgatgc aaatggcgca ttttgcattg cattaaaagg aatgtatact 3720

gccaatcaga tcaaagaaaa ctgggttgaa ggggagaaac ttccggcgga ttgcttgaag 3780

atcgaacatg cgagttggtt agcatttatg caaggagaaa ggggatag 3828

<210> 5

<211> 1263

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> 改造的mgCas12a-1(K925Q)

<400> 5

Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser

1 5 10 15

Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln

20 25 30

Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly

35 40 45

Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly

50 55 60

Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser

65 70 75 80

Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp

85 90 95

Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr Lys Lys

100 105 110

Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile

115 120 125

Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala

130 135 140

Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe

145 150 155 160

Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser

165 170 175

Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn Asp Asn

180 185 190

Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys

195 200 205

Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile Lys Asp

210 215 220

Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr

225 230 235 240

Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp Ile Cys

245 250 255

Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn Lys Glu

260 265 270

Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile Leu Cys

275 280 285

Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu

290 295 300

Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys

305 310 315 320

His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr

325 330 335

Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser

340 345 350

Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile

355 360 365

His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys

370 375 380

Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile

385 390 395 400

Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn Ile Lys

405 410 415

Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu

420 425 430

Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu

435 440 445

Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala

450 455 460

Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp

465 470 475 480

Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Thr

485 490 495

Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro

500 505 510

Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala

515 520 525

Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu

530 535 540

Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys

545 550 555 560

Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp

565 570 575

Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile

580 585 590

Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro

595 600 605

Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu Lys Ser

610 615 620

Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe

625 630 635 640

Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp

645 650 655

Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu

660 665 670

Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys

675 680 685

Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His

705 710 715 720

Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile

725 730 735

Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser

740 745 750

Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg

755 760 765

Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val

770 775 780

Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe

785 790 795 800

Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys

805 810 815

Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr

820 825 830

Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn

835 840 845

Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu Gln Tyr

850 855 860

Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Asp Arg Gly Glu

865 870 875 880

Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val

885 890 895

Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys

900 905 910

Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Gln Glu Trp Lys

915 920 925

Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val

930 935 940

Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala

945 950 955 960

Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys Val Glu

965 970 975

Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys Leu Asn

980 985 990

Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu

995 1000 1005

Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val Gly

1010 1015 1020

His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys

1025 1030 1035 1040

Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys Asp Leu

1045 1050 1055

Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser Ile Arg

1060 1065 1070

Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr Asn Asn

1075 1080 1085

Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser Val Tyr

1090 1095 1100

Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg Phe Ser

1105 1110 1115 1120

Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys Thr Leu

1125 1130 1135

Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg Gln Asp

1140 1145 1150

Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe Arg Leu

1155 1160 1165

Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr

1170 1175 1180

Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp

1185 1190 1195 1200

Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly

1205 1210 1215

Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr

1220 1225 1230

Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile

1235 1240 1245

Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu

1250 1255 1260

<210> 6

<211> 1275

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> 改造的mgCas12a-2(K930Q)

<400> 6

Met Gly Lys Asn Gln Asn Phe Gln Glu Phe Ile Gly Val Ser Pro Leu

1 5 10 15

Gln Lys Thr Leu Arg Asn Glu Leu Ile Pro Thr Glu Thr Thr Lys Lys

20 25 30

Asn Ile Thr Gln Leu Asp Leu Leu Thr Glu Asp Glu Ile Arg Ala Gln

35 40 45

Asn Arg Glu Lys Leu Lys Glu Met Met Asp Asp Tyr Tyr Arg Asn Val

50 55 60

Ile Asp Ser Thr Leu His Val Gly Ile Ala Val Asp Trp Ser Tyr Leu

65 70 75 80

Phe Ser Cys Met Arg Asn His Leu Arg Glu Asn Ser Lys Glu Ser Lys

85 90 95

Arg Glu Leu Glu Arg Thr Gln Asp Ser Ile Arg Ser Gln Ile His Asn

100 105 110

Lys Phe Ala Glu Arg Ala Asp Phe Lys Asp Met Phe Gly Ala Ser Ile

115 120 125

Ile Thr Lys Leu Leu Pro Thr Tyr Ile Lys Gln Asn Ser Glu Tyr Ser

130 135 140

Glu Arg Tyr Asp Glu Ser Met Glu Ile Leu Lys Leu Tyr Gly Lys Phe

145 150 155 160

Thr Thr Ser Leu Thr Asp Tyr Phe Glu Thr Arg Lys Asn Ile Phe Ser

165 170 175

Lys Glu Lys Ile Ser Ser Ala Val Gly Tyr Arg Ile Val Glu Glu Asn

180 185 190

Ala Glu Ile Phe Leu Gln Asn Gln Asn Ala Tyr Asp Arg Ile Cys Lys

195 200 205

Ile Ala Gly Leu Asp Leu His Gly Leu Asp Asn Glu Ile Thr Ala Tyr

210 215 220

Val Asp Gly Lys Thr Leu Lys Glu Val Cys Ser Asp Glu Gly Phe Ala

225 230 235 240

Lys Ala Ile Thr Gln Glu Gly Ile Asp Arg Tyr Asn Glu Ala Ile Gly

245 250 255

Ala Val Asn Gln Tyr Met Asn Leu Leu Cys Gln Lys Asn Lys Ala Leu

260 265 270

Lys Pro Gly Gln Phe Lys Met Lys Arg Leu His Lys Gln Ile Leu Cys

275 280 285

Lys Gly Thr Thr Ser Phe Asp Ile Pro Lys Lys Phe Glu Asn Asp Lys

290 295 300

Gln Val Tyr Asp Ala Val Asn Ser Phe Thr Glu Ile Val Thr Lys Asn

305 310 315 320

Asn Asp Leu Lys Arg Leu Leu Asn Ile Thr Gln Asn Ala Asn Asp Tyr

325 330 335

Asp Met Asn Lys Ile Tyr Val Val Ala Asp Ala Tyr Ser Met Ile Ser

340 345 350

Gln Phe Ile Ser Lys Lys Trp Asn Leu Ile Glu Glu Cys Leu Leu Asp

355 360 365

Tyr Tyr Ser Asp Asn Leu Pro Gly Lys Gly Asn Ala Lys Glu Asn Lys

370 375 380

Val Lys Lys Ala Val Lys Glu Glu Thr Tyr Arg Ser Val Ser Gln Leu

385 390 395 400

Asn Glu Val Ile Glu Lys Tyr Tyr Val Glu Lys Thr Gly Gln Ser Val

405 410 415

Trp Lys Val Glu Ser Tyr Ile Ser Ser Leu Ala Glu Met Ile Lys Leu

420 425 430

Glu Leu Cys His Glu Ile Asp Asn Asp Glu Lys His Asn Leu Ile Glu

435 440 445

Asp Asp Glu Lys Ile Ser Glu Ile Lys Glu Leu Leu Asp Met Tyr Met

450 455 460

Asp Val Phe His Ile Ile Lys Val Phe Arg Val Asn Glu Val Leu Asn

465 470 475 480

Phe Asp Glu Thr Phe Tyr Ser Glu Met Asp Glu Ile Tyr Gln Asp Met

485 490 495

Gln Glu Ile Val Pro Leu Tyr Asn His Val Arg Asn Tyr Val Thr Gln

500 505 510

Lys Pro Tyr Lys Gln Glu Lys Tyr Arg Leu Tyr Phe His Thr Pro Thr

515 520 525

Leu Ala Asn Gly Trp Ser Lys Ser Lys Glu Tyr Asp Asn Asn Ala Ile

530 535 540

Ile Leu Val Arg Glu Asp Lys Tyr Tyr Leu Gly Ile Leu Asn Ala Lys

545 550 555 560

Lys Lys Pro Ser Lys Glu Ile Met Ala Gly Lys Glu Asp Cys Ser Glu

565 570 575

His Ala Tyr Ala Lys Met Asn Tyr Tyr Leu Leu Pro Gly Ala Asn Lys

580 585 590

Met Leu Pro Lys Val Phe Leu Ser Lys Lys Gly Ile Gln Asp Tyr His

595 600 605

Pro Ser Ser Tyr Ile Val Glu Gly Tyr Asn Glu Lys Lys His Ile Lys

610 615 620

Gly Ser Lys Asn Phe Asp Ile Arg Phe Cys Arg Asp Leu Ile Asp Tyr

625 630 635 640

Phe Lys Glu Cys Ile Lys Lys His Pro Asp Trp Asn Lys Phe Asn Phe

645 650 655

Glu Phe Ser Ala Thr Glu Thr Tyr Glu Asp Ile Ser Val Phe Tyr Arg

660 665 670

Glu Val Glu Lys Gln Gly Tyr Arg Val Glu Trp Thr Tyr Ile Asn Ser

675 680 685

Glu Asp Ile Gln Lys Leu Glu Glu Asp Gly Gln Leu Phe Leu Phe Gln

690 695 700

Ile Tyr Asn Lys Asp Phe Ala Val Gly Ser Thr Gly Lys Pro Asn Leu

705 710 715 720

His Thr Leu Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Arg Asp

725 730 735

Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser

740 745 750

Ser Val Gln Lys Pro Val Ile His Lys Cys Gly Ser Ile Leu Val Asn

755 760 765

Arg Thr Tyr Glu Ile Thr Glu Ser Gly Thr Thr Arg Val Gln Ser Ile

770 775 780

Pro Glu Ser Glu Tyr Met Glu Leu Tyr Arg Tyr Phe Asn Ser Glu Lys

785 790 795 800

Gln Ile Glu Leu Ser Asp Glu Ala Lys Lys Tyr Leu Asp Lys Val Gln

805 810 815

Cys Asn Lys Ala Lys Thr Asp Ile Val Lys Asp Tyr Arg Tyr Thr Met

820 825 830

Asp Lys Phe Phe Ile His Leu Pro Ile Thr Ile Asn Phe Lys Val Asp

835 840 845

Lys Gly Asn Asn Val Asn Ala Ile Ala Gln Gln Tyr Ile Ala Gly Arg

850 855 860

Lys Asp Leu His Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile

865 870 875 880

Tyr Val Ser Val Ile Asp Met Tyr Gly Arg Ile Leu Glu Gln Lys Ser

885 890 895

Phe Asn Leu Val Glu Gln Val Ser Ser Gln Gly Thr Lys Arg Tyr Tyr

900 905 910

Asp Tyr Lys Glu Lys Leu Gln Asn Arg Glu Glu Glu Arg Asp Lys Ala

915 920 925

Arg Gln Ser Trp Lys Thr Ile Gly Lys Ile Lys Glu Leu Lys Glu Gly

930 935 940

Tyr Leu Ser Ser Val Ile His Glu Ile Ala Gln Met Val Val Lys Tyr

945 950 955 960

Asn Ala Ile Ile Ala Met Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly

965 970 975

Arg Phe Lys Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu

980 985 990

Ile Ser Lys Leu Asn Tyr Leu Ala Asp Lys Ser Gln Ala Val Asp Glu

995 1000 1005

Pro Gly Gly Ile Leu Arg Gly Tyr Gln Met Thr Tyr Val Pro Asp Asn

1010 1015 1020

Ile Lys Asn Val Gly Arg Gln Cys Gly Ile Ile Phe Tyr Val Pro Ala

1025 1030 1035 1040

Ala Tyr Thr Ser Lys Ile Asp Pro Thr Thr Gly Phe Ile Asn Ala Phe

1045 1050 1055

Lys Arg Asp Val Val Ser Thr Asn Asp Ala Lys Glu Asn Phe Leu Met

1060 1065 1070

Lys Phe Asp Ser Ile Gln Tyr Asp Ile Glu Lys Gly Leu Phe Lys Phe

1075 1080 1085

Ser Phe Asp Tyr Lys Asn Phe Ala Thr His Lys Leu Thr Leu Ala Lys

1090 1095 1100

Thr Lys Trp Asp Val Tyr Thr Asn Gly Thr Arg Ile Gln Asn Met Lys

1105 1110 1115 1120

Val Glu Gly His Trp Leu Ser Met Glu Val Glu Leu Thr Thr Lys Met

1125 1130 1135

Lys Glu Leu Leu Asp Asp Ser His Ile Pro Tyr Glu Glu Gly Gln Asn

1140 1145 1150

Ile Leu Asp Asp Leu Arg Glu Met Lys Asp Ile Thr Thr Ile Val Asn

1155 1160 1165

Gly Ile Leu Glu Ile Phe Trp Leu Thr Val Gln Leu Arg Asn Ser Arg

1170 1175 1180

Ile Asp Asn Pro Asp Tyr Asp Arg Ile Ile Ser Pro Val Leu Asn Lys

1185 1190 1195 1200

Asn Gly Glu Phe Phe Asp Ser Asp Glu Tyr Asn Ser Tyr Ile Asp Ala

1205 1210 1215

Gln Lys Ala Pro Leu Pro Ile Asp Ala Asp Ala Asn Gly Ala Phe Cys

1220 1225 1230

Ile Ala Leu Lys Gly Met Tyr Thr Ala Asn Gln Ile Lys Glu Asn Trp

1235 1240 1245

Val Glu Gly Glu Lys Leu Pro Ala Asp Cys Leu Lys Ile Glu His Ala

1250 1255 1260

Ser Trp Leu Ala Phe Met Gln Gly Glu Arg Gly

1265 1270 1275

<210> 7

<211> 3789

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 人密码子优化改造的mgCas12a-1

<400> 7

atgaacaatg gcaccaacaa tttccagaac tttatcggaa ttagcagtct gcaaaagact 60

ctccggaatg cccttatacc caccgagaca acccagcagt tcatcgtgaa aaacgggatt 120

atcaaggaag acgagctgcg cggcgaaaat cggcaaattt tgaaagatat aatggacgat 180

tattaccgcg gttttatctc tgagactctg agctccattg acgatatcga ctggacctca 240

ctcttcgaaa agatggagat tcagcttaaa aacggcgata ataaggacac actgataaaa 300

gaacaggctg agaagcggaa agccatctat aagaaatttg cagatgacga tcgcttcaag 360

aacatgttta gcgccaaatt gattagtgac atcctgccgg aattcgttat tcacaataac 420

aattactctg ctagcgagaa ggaagagaaa acccaagtca taaagctctt ttcccggttc 480

gccacttcat ttaaagatta tttcaagaac cgcgcaaatt gctttagcgc cgacgatatc 540

agttctagct cctgtcatcg gattgtgaac gacaatgctg aaatcttctt ttcaaacgcc 600

cttgtatacc gccggattgt gaaaaatctg agcaacgatg acataaataa gatcagtgga 660

gatattaaag actctttgaa ggagatgagc ctggaagaga tctattccta cgaaaaatat 720

ggggagttca ttacccagga aggcatatca ttttacaacg atatctgcgg taaggttaat 780

agcttcatga acctctattg tcagaaaaat aaggagaaca aaaatcttta caagctgcgc 840

aaattgcaca agcaaattct gtgcatcgca gacacaagtt atgaagtccc ttacaaattt 900

gagtctgatg aagaggtgta tcagagcgta aacggcttcc tcgacaatat ttcctcaaag 960

catatagtgg aacggcttcg caaaatcgga gataactaca atgggtataa cctggacaag 1020

atttacatcg ttagcaaatt ttatgagagt gtctctcaga agacctaccg ggattgggaa 1080

actattaata ccgccttgga gatacactat aacaatatcc tgcccggcaa cggtaaaagc 1140

aaggctgaca aagtgaagaa agccgtaaag aatgatctcc aaaaatccat tacagaaatc 1200

aacgagcttg tgtcaaatta caagctgtgt ccggacgata acattaaagc agaaacctat 1260

atacatgaga tcagccacat tttgaataac ttcgaagccc aggagctgaa gtacaatcca 1320

gaaatccatc tcgttgagag tgaacttaaa gcttctgagc tgaagaacgt cttggacgtg 1380

attatgaatg cctttcactg gtgcagcgta ttcatgactg aagagctggt ggataaagac 1440

aacaattttt atgcagaact cgaggaaata tacgatgaga tctataccgt tatttccctt 1500

tacaacctgg tccgcaatta tgtgacacag aagccctact caaccaaaaa gatcaaattg 1560

aacttcggca ttccgactct ggccgacgga tggagcaaga gtaaagaata ttctaataac 1620

gctataatcc tcatgcggga taatctttac tatctgggga tttttaacgc caagaataaa 1680

cctgacaaga aaatcattga gggcaacacc agcgaaaata agggtgatta caaaaagatg 1740

atatataact tgctgcccgg cccgaataaa atgatcccaa aggtattcct ctcctcaaaa 1800

acaggagtgg agacctacaa gcccagcgca tatattcttg aagggtacaa acaaaacaag 1860

catctgaaaa gttctaagga ctttgatatc actttctgtc acgacttgat tgattatttt 1920

aaaaattgca tagccatcca tccggagtgg aagaacttcg gctttgactt cagcgatacc 1980

tccacatacg aagacatttc aggtttttat cgcgaggttg aactgcaggg ctacaaaatc 2040

gattggacct atattagcga gaaggacata gatctccttc aggaaaaagg acaactgtac 2100

ttgttccaga tctataataa ggactttagt aaaaagtcta ctgggaacga taatctgcac 2160

accatgtacc tcaaaaacct tttcagcgag gaaaatctga aggacattgt cttgaaactg 2220

aacggcgagg ctgaaatctt tttccggaag tcctcaatta aaaatcctat aatccataag 2280

aaaggtagca ttctcgtgaa ccgcacatat gaggccgaag agaaggatca gtttggcaat 2340

atccaaattg tacggaaaac catacccgaa aacatctacc aggagcttta taagtacttc 2400

aatgacaaaa gtgataagga actgtctgac gaggcagcca aattgaagaa cgtggttgga 2460

caccatgaag ctgccactaa tattgtcaaa gattatcgct acacctatga caagtacttt 2520

ctgcacatgc cgatcacaat taacttcaaa gcaaataaga ccagctttat aaacgatcgg 2580

attctccagt atattgccaa agagaagaat cttcatgtga tcgggattga ccgcggcgaa 2640

cggaacctga tatacgtatc cgtgatcgat acttgtggta atattgttga gcaaaaatca 2700

ttcaacatcg tcaatggcta tgactaccag attaagttga aacagcaaga aggagctcgc 2760

cagatagccc ggcaggagtg gaaggaaatc gggaaaatta aggagatcaa agaaggctat 2820

ctgagcctcg tgattcacga gataagtaag atggtaatca aatacaacgc aattatcgcc 2880

atggaagatc tttcttatgg ttttaagaaa ggccgcttca aggtggagcg gcaagtttac 2940

cagaaatttg aaaccatgct gattaataag ttgaactatc tggtcttcaa agacataagc 3000

atcacagaga atggagggct ccttaagggc taccagctga cctatattcc agataaattg 3060

aagaacgtgg gtcatcaatg cggctgtatc ttttacgtac ccgctgccta tacttccaaa 3120

attgacccga ccacaggatt cgtgaatata tttaagttca aagatctgac cgttgacgca 3180

aagcgcgaat ttatcaaaaa gttcgattca attcggtacg acagcgagaa aaagctcttt 3240

tgcttcactt ttgattataa caatttcatc acccagaaca cagtcatgag taaatctagc 3300

tggtccgtgt acacctatgg ggtacgcatt aagcggcgct ttgtgaatgg ccggttctca 3360

aacgaaagcg acactataga tatcaccaaa gacatggaga agacacttga aatgaccgat 3420

attaattggc gcgacggtca cgatctgcgg caggacatca ttgattacga gatagttcaa 3480

catatctttg aaattttccg cttgactgtc cagatgcgga acagtctgtc tgagctcgaa 3540

gaccgcgatt atgaccggct tatcagccct gtgctgaatg agaacaatat tttttacgat 3600

tccgccaaag ctggcgacgc cttgcccaag gatgcagacg ccaacggagc ttattgtata 3660

gccctgaaag ggctctacga aatcaagcag attaccgaga attggaaaga agatggcaag 3720

ttctcacgcg acaaacttaa gatcagcaac aaagattggt ttgacttcat tcaaaataag 3780

cggtatctg 3789

<210> 8

<211> 3825

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 人密码子优化改造的mgCas12a-2

<400> 8

atgggcaaaa accaaaattt ccaagaattt atcggagtga gccccctgca gaagaccctc 60

cggaacgagc ttattccgac tgagaccaca aagaaaaata taacccagct ggacttgctg 120

actgaagatg agatccgcgc ccagaaccgg gaaaagctca aagagatgat ggacgattat 180

taccgcaatg ttattgacag tacccttcac gtcgggatcg ctgtggattg gtcttatctg 240

ttcagctgca tgcggaacca tttgcgcgaa aattccaagg agtcaaaacg ggaactggag 300

cgcacacagg acagcattcg gagtcagata cacaacaagt ttgccgaacg cgcagatttc 360

aaagacatgt ttggcgcctc tatcattacc aagctccttc ctacttacat caaacaaaat 420

agcgagtatt ccgaacggta cgatgagtca atggaaattc tgaagttgta tggtaaattc 480

accacaagcc tgaccgacta ctttgagact cgcaagaaca tattcagtaa agaaaagatc 540

tctagcgctg taggctatcg gattgtggag gaaaatgccg agatctttct ccagaaccag 600

aatgcatacg atcgcatttg taaaatagcc ggacttgacc tgcatgggtt ggataacgaa 660

atcaccgctt atgttgacgg caagacactg aaagaggtct gctccgatga aggtttcgcc 720

aaggcaatta cccaagaggg catcgaccgg tacaatgaag ccattggagc tgtgaaccag 780

tatatgaatc tcctttgtca gaaaaacaag gccctgaaac ccgggcaatt taagatgaaa 840

cgcttgcaca agcagatact gtgcaaaggc actacctcat tcgatatccc gaagaaattt 900

gagaatgaca agcaggtata cgatgcagtg aacagcttca cagaaattgt taccaaaaat 960

aacgacctca agcggcttct gaatatcact caaaacgcca atgattatga catgaacaaa 1020

atttacgtcg tggctgatgc ctatagtatg atatctcagt ttatcagcaa gaaatggaat 1080

ttgattgagg aatgtctgct cgactactat tccgataacc ttccaggtaa gggcaatgca 1140

aaagagaaca aggtaaaaaa ggccgtgaaa gaagagacct accgctcagt tagccagctg 1200

aatgaagtca tcgagaagta ttacgtggaa aaaacaggac aaagtgtatg gaaggtggag 1260

tcttatatta gctccttggc tgaaatgata aaactggagc tctgccatga aatcgacaac 1320

gatgagaagc acaatcttat tgaagacgat gagaaaatct cagaaattaa ggagctgttg 1380

gacatgtaca tggatgtttt ccatataatc aaagtctttc gggtgaacga agtactgaat 1440

ttcgacgaga ccttttatag cgaaatggat gagatttacc aggacatgca ggaaatcgtg 1500

cccctctata accacgttcg caattacgtc actcaaaagc cgtataaaca ggagaagtac 1560

cggctttatt tccatacccc tacactggcc aacgggtgga gtaaatctaa ggaatacgat 1620

aataacgcaa ttatattggt gcgcgaggac aaatattacc tgggcatcct caatgccaag 1680

aaaaagccca gcaaagaaat tatggctggt aaggaggatt gttccgaaca cgcctatgca 1740

aaaatgaact actatcttct gccgggcgcc aataagatgt tgccaaaagt atttctgtca 1800

aagaaaggaa tccaggacta ccatcccagc agttatattg tggaggggta caacgaaaag 1860

aaacacataa agggctctaa aaatttcgat atccggtttt gccgcgacct cattgattat 1920

ttcaaggagt gtatcaaaaa gcatccggac tggaacaaat ttaatttcga atttagcgct 1980

accgagactt acgaagatat ttccgttttc tatcgggagg tcgaaaagca aggttaccgc 2040

gtggagtgga cctatataaa ctcagaagac atccagaaac ttgaggaaga tggccagctg 2100

tttttgttcc aaatttacaa taaggacttt gccgtaggaa gcacagggaa acctaacctg 2160

cacaccctct atcttaagaa tctgttcagt gaggaaaact tgcgggatat cgtgctgaaa 2220

ctcaatggcg aggcagaaat ttttttccgc aagtctagcg ttcagaaacc cgtcatacat 2280

aagtgcggtt ccatccttgt gaaccggact tacgagatta ccgaatcagg cacaacccgc 2340

gtacagagca tcccggagag tgaatatatg gagctgtacc ggtattttaa ttctgaaaaa 2400

caaattgagt tgagcgacga agccaagaaa tacctggata aggtgcagtg taacaaagct 2460

aagactgaca tagttaaaga ttatcgctac accatggaca agttctttat ccacctccca 2520

attacaatca atttcaaagt cgataaggga aacaatgtga acgccattgc acagcaatat 2580

atagccgggc ggaaagacct tcatgtaatc ggcattgatc gcggtgagcg gaatctgatc 2640

tacgtgtccg ttattgacat gtatggccgc atattggaac agaagtcatt taacctggtc 2700

gagcaggtga gcagtcaagg aaccaaacgg tactatgatt acaaggaaaa actccagaat 2760

cgcgaggaag agcgggacaa ggctcgccag tcttggaaaa ctatcgggaa gattaaagaa 2820

cttaaggagg gctatctgag ctccgtaatc cacgaaattg cccaaatggt ggttaaatac 2880

aacgcaataa tcgccatgga ggatttgaat tatggtttca agcggggccg ctttaaagtc 2940

gaacggcagg tgtaccagaa gttcgagacc atgctgattt caaaactcaa ctatcttgct 3000

gacaagagcc aagccgtaga tgaacccgga gggattctgc gcggctacca gatgacatat 3060

gtgccggaca atattaaaaa cgttggtcgg cagtgcggca taatctttta cgtccctgca 3120

gcctatacca gtaagattga tcccactacc ggattcatca atgcttttaa acgcgacgtg 3180

gtatctacaa acgatgccaa ggagaatttc ttgatgaaat ttgacagcat tcaatacgat 3240

atagaaaagg ggctgttcaa attttccttc gactataaga actttgcaac ccataaactc 3300

actcttgcca agaccaaatg ggatgtgtac acaaatggca cccggattca gaacatgaag 3360

gttgagggtc actggctgtc aatggaagtc gagttgacta ccaaaatgaa ggaactgctc 3420

gacgatagcc atattccgta tgaggaaggc cagaatatcc ttgacgatct gcgcgagatg 3480

aaagacatta caaccatagt gaacggaatc ttggaaattt tctggctgac tgtacaactc 3540

cggaatagtc gcatcgataa cccagactac gatcggatta tatctcccgt gcttaataag 3600

aacggggagt ttttcgacag cgatgaatat aattcctaca tcgacgctca gaaagccccg 3660

ctgcctattg atgcagacgc caacggcgct ttttgtatcg ccttgaaggg tatgtatacc 3720

gcaaatcaga ttaaagagaa ctgggttgaa ggcgagaagc tgcccgccga ttgcctcaaa 3780

atagaacacg cttcatggct tgccttcatg caaggagagc gcggg 3825

<210> 9

<211> 1307

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> AsCas12a

<400> 9

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 Lys 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> 10

<211> 1228

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> LbCas12a

<400> 10

Ala Ala Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys

1 5 10 15

Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile

20 25 30

Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr

35 40 45

Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn

50 55 60

Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser

65 70 75 80

Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu

85 90 95

Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly

100 105 110

Ala Ala Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile

115 120 125

Leu Pro Glu Ala Ala Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser

130 135 140

Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu

145 150 155 160

Asn Xaa Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys

165 170 175

Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Xaa Asp Ile Phe Glu

180 185 190

Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu

195 200 205

Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu

210 215 220

Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala

225 230 235 240

Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu

245 250 255

Asn Glu Tyr Ile Asn Leu Tyr Asn Ala Lys Thr Lys Gln Ala Leu Pro

260 265 270

Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu

275 280 285

Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val

290 295 300

Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys

305 310 315 320

Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly

325 330 335

Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile

340 345 350

Phe Gly Glu Trp Asn Leu Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp

355 360 365

Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp

370 375 380

Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln

385 390 395 400

Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys

405 410 415

Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser

420 425 430

Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys

435 440 445

Lys Asn Asp Ala Val Val Ala Ile Xaa Lys Asp Leu Leu Asp Ser Val

450 455 460

Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu

465 470 475 480

Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp

485 490 495

Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val

500 505 510

Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn

515 520 525

Pro Gln Phe Xaa Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg

530 535 540

Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Xaa Asp

545 550 555 560

Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn

565 570 575

Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys

580 585 590

Xaa Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Xaa Ala Tyr Tyr Asn

595 600 605

Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys

610 615 620

Gly Asp Xaa Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe

625 630 635 640

Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe

645 650 655

Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg

660 665 670

Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys

675 680 685

Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Xaa Phe Gln

690 695 700

Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu

705 710 715 720

His Thr Xaa Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln

725 730 735

Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Xaa Arg Arg Ala Ser Leu

740 745 750

Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn

755 760 765

Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val

770 775 780

Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro

785 790 795 800

Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu

805 810 815

Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile

820 825 830

Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys

835 840 845

Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe

850 855 860

Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys

865 870 875 880

Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn

885 890 895

Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile

900 905 910

Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu

915 920 925

Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr

930 935 940

Gln Lys Phe Glu Lys Xaa Leu Ile Asp Lys Leu Asn Tyr Xaa Val Asp

945 950 955 960

Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln

965 970 975

Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Xaa Ser Thr Gln Asn Gly

980 985 990

Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser

995 1000 1005

Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Xaa Tyr Val Pro Glu

1025 1030 1035 1040

Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg Thr

1045 1050 1055

Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly Asn Arg

1060 1065 1070

Ile Arg Ile Phe Ala Ala Ala Lys Lys Asn Asn Val Phe Ala Trp Glu

1075 1080 1085

Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe Asn Lys Tyr Gly

1090 1095 1100

Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu Leu Cys Glu Gln Ser

1105 1110 1115 1120

Asp Lys Ala Phe Tyr Ser Ser Phe Xaa Ala Leu Xaa Ser Leu Xaa Leu

1125 1130 1135

Gln Xaa Arg Asn Ser Ile Thr Gly Arg Thr Asp Val Asp Phe Leu Ile

1140 1145 1150

Ser Pro Val Lys Asn Ser Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr

1155 1160 1165

Glu Ala Gln Glu Asn Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly

1170 1175 1180

Ala Tyr Asn Ile Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys

1185 1190 1195 1200

Lys Ala Glu Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn

1205 1210 1215

Lys Glu Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys

1220 1225

<210> 11

<211> 1300

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> FnCas12a

<400> 11

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr

1010 1015 1020

Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe

1025 1030 1035 1040

Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln

1045 1050 1055

Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly

1060 1065 1070

Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val

1075 1080 1085

Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys

1090 1095 1100

Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp

1105 1110 1115 1120

Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys

1125 1130 1135

Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile

1140 1145 1150

Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val

1155 1160 1165

Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu

1170 1175 1180

Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp

1185 1190 1195 1200

Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln

1205 1210 1215

Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro

1220 1225 1230

Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu

1250 1255 1260

Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys

1265 1270 1275 1280

Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln

1285 1290 1295

Asn Arg Asn Asn

1300

<210> 12

<211> 19

<212> RNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 5'-柄端 RNA

<400> 12

aauuucuacu guuguagau 19

<210> 13

<211> 1263

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> d_mgCas12a-1

<400> 13

Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser

1 5 10 15

Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln

20 25 30

Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly

35 40 45

Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly

50 55 60

Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser

65 70 75 80

Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp

85 90 95

Thr Leu Ile Lys Glu Gln Ala Glu Lys Arg Lys Ala Ile Tyr Lys Lys

100 105 110

Phe Ala Asp Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile

115 120 125

Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala

130 135 140

Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe

145 150 155 160

Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser

165 170 175

Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn Asp Asn

180 185 190

Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys

195 200 205

Asn Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Ile Lys Asp

210 215 220

Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr

225 230 235 240

Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp Ile Cys

245 250 255

Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn Lys Glu

260 265 270

Asn Lys Asn Leu Tyr Lys Leu Arg Lys Leu His Lys Gln Ile Leu Cys

275 280 285

Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu

290 295 300

Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys

305 310 315 320

His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr

325 330 335

Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser

340 345 350

Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile

355 360 365

His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys

370 375 380

Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile

385 390 395 400

Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Pro Asp Asp Asn Ile Lys

405 410 415

Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu

420 425 430

Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu

435 440 445

Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala

450 455 460

Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp

465 470 475 480

Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Thr

485 490 495

Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro

500 505 510

Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala

515 520 525

Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu

530 535 540

Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys

545 550 555 560

Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp

565 570 575

Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile

580 585 590

Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro

595 600 605

Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Leu Lys Ser

610 615 620

Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe

625 630 635 640

Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp

645 650 655

Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu

660 665 670

Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys

675 680 685

Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His

705 710 715 720

Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile

725 730 735

Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser

740 745 750

Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg

755 760 765

Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val

770 775 780

Arg Lys Thr Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe

785 790 795 800

Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys

805 810 815

Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr

820 825 830

Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn

835 840 845

Phe Lys Ala Asn Lys Thr Ser Phe Ile Asn Asp Arg Ile Leu Gln Tyr

850 855 860

Ile Ala Lys Glu Lys Asn Leu His Val Ile Gly Ile Ala Arg Gly Glu

865 870 875 880

Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val

885 890 895

Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys

900 905 910

Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu Trp Lys

915 920 925

Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val

930 935 940

Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala

945 950 955 960

Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys Val Glu

965 970 975

Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys Leu Asn

980 985 990

Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu

995 1000 1005

Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val Gly

1010 1015 1020

His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys

1025 1030 1035 1040

Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe Lys Asp Leu

1045 1050 1055

Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe Asp Ser Ile Arg

1060 1065 1070

Tyr Asp Ser Glu Lys Lys Leu Phe Cys Phe Thr Phe Asp Tyr Asn Asn

1075 1080 1085

Phe Ile Thr Gln Asn Thr Val Met Ser Lys Ser Ser Trp Ser Val Tyr

1090 1095 1100

Thr Tyr Gly Val Arg Ile Lys Arg Arg Phe Val Asn Gly Arg Phe Ser

1105 1110 1115 1120

Asn Glu Ser Asp Thr Ile Asp Ile Thr Lys Asp Met Glu Lys Thr Leu

1125 1130 1135

Glu Met Thr Asp Ile Asn Trp Arg Asp Gly His Asp Leu Arg Gln Asp

1140 1145 1150

Ile Ile Asp Tyr Glu Ile Val Gln His Ile Phe Glu Ile Phe Arg Leu

1155 1160 1165

Thr Val Gln Met Arg Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr

1170 1175 1180

Asp Arg Leu Ile Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp

1185 1190 1195 1200

Ser Ala Lys Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly

1205 1210 1215

Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr

1220 1225 1230

Glu Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile

1235 1240 1245

Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu

1250 1255 1260

<210> 14

<211> 1275

<212> PRT

<213> 人工序列（Artificial Sequence）

<220>

<223> d_mgCas12a-2

<400> 14

Met Gly Lys Asn Gln Asn Phe Gln Glu Phe Ile Gly Val Ser Pro Leu

1 5 10 15

Gln Lys Thr Leu Arg Asn Glu Leu Ile Pro Thr Glu Thr Thr Lys Lys

20 25 30

Asn Ile Thr Gln Leu Asp Leu Leu Thr Glu Asp Glu Ile Arg Ala Gln

35 40 45

Asn Arg Glu Lys Leu Lys Glu Met Met Asp Asp Tyr Tyr Arg Asn Val

50 55 60

Ile Asp Ser Thr Leu His Val Gly Ile Ala Val Asp Trp Ser Tyr Leu

65 70 75 80

Phe Ser Cys Met Arg Asn His Leu Arg Glu Asn Ser Lys Glu Ser Lys

85 90 95

Arg Glu Leu Glu Arg Thr Gln Asp Ser Ile Arg Ser Gln Ile His Asn

100 105 110

Lys Phe Ala Glu Arg Ala Asp Phe Lys Asp Met Phe Gly Ala Ser Ile

115 120 125

Ile Thr Lys Leu Leu Pro Thr Tyr Ile Lys Gln Asn Ser Glu Tyr Ser

130 135 140

Glu Arg Tyr Asp Glu Ser Met Glu Ile Leu Lys Leu Tyr Gly Lys Phe

145 150 155 160

Thr Thr Ser Leu Thr Asp Tyr Phe Glu Thr Arg Lys Asn Ile Phe Ser

165 170 175

Lys Glu Lys Ile Ser Ser Ala Val Gly Tyr Arg Ile Val Glu Glu Asn

180 185 190

Ala Glu Ile Phe Leu Gln Asn Gln Asn Ala Tyr Asp Arg Ile Cys Lys

195 200 205

Ile Ala Gly Leu Asp Leu His Gly Leu Asp Asn Glu Ile Thr Ala Tyr

210 215 220

Val Asp Gly Lys Thr Leu Lys Glu Val Cys Ser Asp Glu Gly Phe Ala

225 230 235 240

Lys Ala Ile Thr Gln Glu Gly Ile Asp Arg Tyr Asn Glu Ala Ile Gly

245 250 255

Ala Val Asn Gln Tyr Met Asn Leu Leu Cys Gln Lys Asn Lys Ala Leu

260 265 270

Lys Pro Gly Gln Phe Lys Met Lys Arg Leu His Lys Gln Ile Leu Cys

275 280 285

Lys Gly Thr Thr Ser Phe Asp Ile Pro Lys Lys Phe Glu Asn Asp Lys

290 295 300

Gln Val Tyr Asp Ala Val Asn Ser Phe Thr Glu Ile Val Thr Lys Asn

305 310 315 320

Asn Asp Leu Lys Arg Leu Leu Asn Ile Thr Gln Asn Ala Asn Asp Tyr

325 330 335

Asp Met Asn Lys Ile Tyr Val Val Ala Asp Ala Tyr Ser Met Ile Ser

340 345 350

Gln Phe Ile Ser Lys Lys Trp Asn Leu Ile Glu Glu Cys Leu Leu Asp

355 360 365

Tyr Tyr Ser Asp Asn Leu Pro Gly Lys Gly Asn Ala Lys Glu Asn Lys

370 375 380

Val Lys Lys Ala Val Lys Glu Glu Thr Tyr Arg Ser Val Ser Gln Leu

385 390 395 400

Asn Glu Val Ile Glu Lys Tyr Tyr Val Glu Lys Thr Gly Gln Ser Val

405 410 415

Trp Lys Val Glu Ser Tyr Ile Ser Ser Leu Ala Glu Met Ile Lys Leu

420 425 430

Glu Leu Cys His Glu Ile Asp Asn Asp Glu Lys His Asn Leu Ile Glu

435 440 445

Asp Asp Glu Lys Ile Ser Glu Ile Lys Glu Leu Leu Asp Met Tyr Met

450 455 460

Asp Val Phe His Ile Ile Lys Val Phe Arg Val Asn Glu Val Leu Asn

465 470 475 480

Phe Asp Glu Thr Phe Tyr Ser Glu Met Asp Glu Ile Tyr Gln Asp Met

485 490 495

Gln Glu Ile Val Pro Leu Tyr Asn His Val Arg Asn Tyr Val Thr Gln

500 505 510

Lys Pro Tyr Lys Gln Glu Lys Tyr Arg Leu Tyr Phe His Thr Pro Thr

515 520 525

Leu Ala Asn Gly Trp Ser Lys Ser Lys Glu Tyr Asp Asn Asn Ala Ile

530 535 540

Ile Leu Val Arg Glu Asp Lys Tyr Tyr Leu Gly Ile Leu Asn Ala Lys

545 550 555 560

Lys Lys Pro Ser Lys Glu Ile Met Ala Gly Lys Glu Asp Cys Ser Glu

565 570 575

His Ala Tyr Ala Lys Met Asn Tyr Tyr Leu Leu Pro Gly Ala Asn Lys

580 585 590

Met Leu Pro Lys Val Phe Leu Ser Lys Lys Gly Ile Gln Asp Tyr His

595 600 605

Pro Ser Ser Tyr Ile Val Glu Gly Tyr Asn Glu Lys Lys His Ile Lys

610 615 620

Gly Ser Lys Asn Phe Asp Ile Arg Phe Cys Arg Asp Leu Ile Asp Tyr

625 630 635 640

Phe Lys Glu Cys Ile Lys Lys His Pro Asp Trp Asn Lys Phe Asn Phe

645 650 655

Glu Phe Ser Ala Thr Glu Thr Tyr Glu Asp Ile Ser Val Phe Tyr Arg

660 665 670

Glu Val Glu Lys Gln Gly Tyr Arg Val Glu Trp Thr Tyr Ile Asn Ser

675 680 685

Glu Asp Ile Gln Lys Leu Glu Glu Asp Gly Gln Leu Phe Leu Phe Gln

690 695 700

Ile Tyr Asn Lys Asp Phe Ala Val Gly Ser Thr Gly Lys Pro Asn Leu

705 710 715 720

His Thr Leu Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Arg Asp

725 730 735

Ile Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser

740 745 750

Ser Val Gln Lys Pro Val Ile His Lys Cys Gly Ser Ile Leu Val Asn

755 760 765

Arg Thr Tyr Glu Ile Thr Glu Ser Gly Thr Thr Arg Val Gln Ser Ile

770 775 780

Pro Glu Ser Glu Tyr Met Glu Leu Tyr Arg Tyr Phe Asn Ser Glu Lys

785 790 795 800

Gln Ile Glu Leu Ser Asp Glu Ala Lys Lys Tyr Leu Asp Lys Val Gln

805 810 815

Cys Asn Lys Ala Lys Thr Asp Ile Val Lys Asp Tyr Arg Tyr Thr Met

820 825 830

Asp Lys Phe Phe Ile His Leu Pro Ile Thr Ile Asn Phe Lys Val Asp

835 840 845

Lys Gly Asn Asn Val Asn Ala Ile Ala Gln Gln Tyr Ile Ala Gly Arg

850 855 860

Lys Asp Leu His Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Ile

865 870 875 880

Tyr Val Ser Val Ile Asp Met Tyr Gly Arg Ile Leu Glu Gln Lys Ser

885 890 895

Phe Asn Leu Val Glu Gln Val Ser Ser Gln Gly Thr Lys Arg Tyr Tyr

900 905 910

Asp Tyr Lys Glu Lys Leu Gln Asn Arg Glu Glu Glu Arg Asp Lys Ala

915 920 925

Arg Lys Ser Trp Lys Thr Ile Gly Lys Ile Lys Glu Leu Lys Glu Gly

930 935 940

Tyr Leu Ser Ser Val Ile His Glu Ile Ala Gln Met Val Val Lys Tyr

945 950 955 960

Asn Ala Ile Ile Ala Met Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly

965 970 975

Arg Phe Lys Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu

980 985 990

Ile Ser Lys Leu Asn Tyr Leu Ala Asp Lys Ser Gln Ala Val Asp Glu

995 1000 1005

Pro Gly Gly Ile Leu Arg Gly Tyr Gln Met Thr Tyr Val Pro Asp Asn

1010 1015 1020

Ile Lys Asn Val Gly Arg Gln Cys Gly Ile Ile Phe Tyr Val Pro Ala

1025 1030 1035 1040

Ala Tyr Thr Ser Lys Ile Asp Pro Thr Thr Gly Phe Ile Asn Ala Phe

1045 1050 1055

Lys Arg Asp Val Val Ser Thr Asn Asp Ala Lys Glu Asn Phe Leu Met

1060 1065 1070

Lys Phe Asp Ser Ile Gln Tyr Asp Ile Glu Lys Gly Leu Phe Lys Phe

1075 1080 1085

Ser Phe Asp Tyr Lys Asn Phe Ala Thr His Lys Leu Thr Leu Ala Lys

1090 1095 1100

Thr Lys Trp Asp Val Tyr Thr Asn Gly Thr Arg Ile Gln Asn Met Lys

1105 1110 1115 1120

Val Glu Gly His Trp Leu Ser Met Glu Val Glu Leu Thr Thr Lys Met

1125 1130 1135

Lys Glu Leu Leu Asp Asp Ser His Ile Pro Tyr Glu Glu Gly Gln Asn

1140 1145 1150

Ile Leu Asp Asp Leu Arg Glu Met Lys Asp Ile Thr Thr Ile Val Asn

1155 1160 1165

Gly Ile Leu Glu Ile Phe Trp Leu Thr Val Gln Leu Arg Asn Ser Arg

1170 1175 1180

Ile Asp Asn Pro Asp Tyr Asp Arg Ile Ile Ser Pro Val Leu Asn Lys

1185 1190 1195 1200

Asn Gly Glu Phe Phe Asp Ser Asp Glu Tyr Asn Ser Tyr Ile Asp Ala

1205 1210 1215

Gln Lys Ala Pro Leu Pro Ile Asp Ala Asp Ala Asn Gly Ala Phe Cys

1220 1225 1230

Ile Ala Leu Lys Gly Met Tyr Thr Ala Asn Gln Ile Lys Glu Asn Trp

1235 1240 1245

Val Glu Gly Glu Lys Leu Pro Ala Asp Cys Leu Lys Ile Glu His Ala

1250 1255 1260

Ser Trp Leu Ala Phe Met Gln Gly Glu Arg Gly

1265 1270 1275

<210> 15

<211> 53

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> CCR5适配体引物序列(5'-3')

<400> 15

tcgtcggcag cgtcagatgt gtataagaga cagggtattt ctgttcagat cac 53

<210> 16

<211> 55

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> CCR5适配体引物序列(5'-3')

<400> 16

gtctcgtggg ctcggagatg tgtataagag acaggcccat caattataga aagcc 55

<210> 17

<211> 53

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> DNMT1适配体引物序列(5'-3')

<400> 17

tcgtcggcag cgtcagatgt gtataagaga cagctgcaca cagcaggcct ttg 53

<210> 18

<211> 54

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> DNMT1适配体引物序列(5'-3')

<400> 18

gtctcgtggg ctcggagatg tgtataagag acagcccaat aagtggcaga gtgc 54

<210> 19

<211> 28

<212> RNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NbFTa14_1/2-2 crRNA序列(PAM位点)

<400> 19

tttggataat ttgtactctt gtcgatgt 28

<210> 20

<211> 28

<212> RNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NbFTa14_1/2-4 crRNA序列(PAM位点)

<400> 20

tttagtccac aaacagctaa gcccacat 28

<210> 21

<211> 20

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NGS NbFTa14_1正向引物

<400> 21

tgagctgaag atggattatg 20

<210> 22

<211> 20

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NGS NbFTa14_1反向引物

<400> 22

tcatgcttaa gataaaagag 20

<210> 23

<211> 20

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NGS NbFTa14_2正向引物

<400> 23

tcatgagctt aagatggatc 20

<210> 24

<211> 20

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> NGS NbFTa14_2反向引物

<400> 24

gtttaagcta aaagaactac 20

<210> 25

<211> 43

<212> RNA

<213> 人工序列（Artificial Sequence）

<220>

<223> LsXTb12 crRNA #1

<400> 25

aatttctact aagtgtagat tcttcatcct caattccatc acc 43

<210> 26

<211> 43

<212> RNA

<213> 人工序列（Artificial Sequence）

<220>

<223> LsXTb12 crRNA #2

<400> 26

aatttctact aagtgtagat gcaagcctgt aactctggaa gac 43

<210> 27

<211> 1504

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> HsCCR5线性DNA

<400> 27

ggtggtggct gtgtttgcgt ctctcccagg aatcatcttt accagatctc aaaaagaagg 60

tcttcattac acctgcagct ctcattttcc atacagtcag tatcaattct ggaagaattt 120

ccagacatta aagatagtca tcttggggct ggtcctgccg ctgcttgtca tggtcatctg 180

ctactcggga atcctaaaaa ctctgcttcg gtgtcgaaat gagaagaaga ggcacagggc 240

tgtgaggctt atcttcacca tcatgattgt ttattttctc ttctgggctc cctacaacat 300

tgtccttctc ctgaacacct tccaggaatt ctttggcctg aataattgca gtagctctaa 360

caggttggac caagctatgc aggtgacaga gactcttggg atgacgcact gctgcatcaa 420

ccccatcatc tatgcctttg tcggggagaa gttcagaaac tacctcttag tcttcttcca 480

aaagcacatt gccaaacgct tctgcaaatg ctgttctatt ttccagcaag aggctcccga 540

gcgagcaagc tcagtttaca cccgatccac tggggagcag gaaatatctg tgggcttgtg 600

acacggactc aagtgggctg gtgacccagt cagagttgtg cacatggctt agttttcata 660

cacagcctgg gctgggggtg gggtgggaga ggtctttttt aaaaggaagt tactgttata 720

gagggtctaa gattcatcca tttatttggc atctgtttaa agtagattag atcttttaag 780

cccatcaatt atagaaagcc aaatcaaaat atgttgatga aaaatagcaa cctttttatc 840

tccccttcac atgcatcaag ttattgacaa actctccctt cactccgaaa gttccttatg 900

tatatttaaa agaaagcctc agagaattgc tgattcttga gtttagtgat ctgaacagaa 960

ataccaaaat tatttcagaa atgtacaact ttttacctag tacaaggcaa catataggtt 1020

gtaaatgtgt ttaaaacagg tctttgtctt gctatgggga gaaaagacat gaatatgatt 1080

agtaaagaaa tgacactttt catgtgtgat ttcccctcca aggtatggtt aataagtttc 1140

actgacttag aaccaggcga gagacttgtg gcctgggaga gctggggaag cttcttaaat 1200

gagaaggaat ttgagttgga tcatctattg ctggcaaaga cagaagcctc actgcaagca 1260

ctgcatgggc aagcttggct gtagaaggag acagagctgg ttgggaagac atggggagga 1320

aggacaaggc tagatcatga agaaccttga cggcattgct ccgtctaagt catgagctga 1380

gcagggagat cctggttggt gttgcagaag gtttactctg tggccaaagg agggtcagga 1440

aggatgagca tttagggcaa ggagaccacc aacagccctc aggtcagggt gaggatggcc 1500

tctg 1504

<210> 28

<211> 1119

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> HsDNMT1线性DNA

<400> 28

gctgctctcg aactcctggc ctcaactaat ccacctgcct tggcctccca aagtgctggg 60

attacaggcg tgagccactg ctcccagccc cacgtgtctt tgtctcaagt ctttctgaag 120

ctcttcaaag gcccagtgac ttgtggctgt ggggcgggat gatgggccag ttggagggtc 180

caaggatctt gtgctggaag ggttttgggc ccatgtgagc aggaccagaa cccttcccca 240

aggggtgcaa tgcccaggtt gtcctccatc tgagcagggg ctggcagtac acctgccccc 300

gggccttggg cctgggtgtc cacatcaggc attgcccttc tcccctcctg caggtgggca 360

atgccgtgcc accgcccctg gccaaagcca ttggcttgga gatcaagctt tgtatgttgg 420

ccaaagcccg agagagtgcc tcaggtatgg tggggtgggc caggcttcct ctggggcctg 480

actgccctct gggggtacat gtgggggcag ttgctggcca ccgttttggg ctctgggact 540

caggcgggtc acctacccac gttcgtggcc ccatctttct caaggggctg ctgtgaggat 600

tgagtgagtt gcacgtgtca agtgcttaga gcaggcgtgc tgcacacagc aggcctttgg 660

tcaggttggc tgctgggctg gccctggggc cgtttccctc actcctgctc ggtgaatttg 720

gctcagcagg cacctgcctc agctgctcac ttgagcctct gggtctagaa ccctctgggg 780

accgtttgag gagtgttcag tctccgtgaa cgttccctta gcactctgcc acttattggg 840

tcagctgtta acatcagtac gttaatgttt cctgatggtc catgtctgtt actcgcctgt 900

caagtggcgt gacaccgggc gtgttcccca gagtgacttt tccttttatt tcccttcagc 960

taaaataaag gaggaggaag ctgctaagga ctagttctgc cctcccgtca cccctgtttc 1020

tggcaccagg aatccccaac atgcactgat gttgtgtttt taacatgtca atctgtccgt 1080

tcacatgtgt ggtacatggt gtttgtggcc ttggctgac 1119

<210> 29

<211> 1460

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> HsEMX1线性DNA

<400> 29

gtggggacag aaggtctgga gctgcccgtg aagggcagaa tgctgccctc agacccgctt 60

cctccctgtc cttgtctgtc caaggagaat gaggtctcac tggtggattt cggactaccc 120

tgaggagctg gcacctgagg gacaaggccc cccacctgcc cagctccagc ctctgatgag 180

gggtgggaga gagctacatg aggttgctaa gaaagcctcc cctgaaggag accacacagt 240

gtgtgaggtt ggagtctcta gcagcgggtt ctgtgccccc agggatagtc tggctgtcca 300

ggcactgctc ttgatataaa caccacctcc tagttatgaa accatgccca ttctgcctct 360

ctgtatggaa aagagcatgg ggctggcccg tggggtggtg tccactttag gccctgtggg 420

agatcatggg aacccacgca gtgggtcata ggctctctca tttactactc acatccactc 480

tgtgaagaag cgattatgat ctctcctcta gaaactcgta gagtcccatg tctgccggct 540

tccagagcct gcactcctcc accttggctt ggctttgctg gggctagagg agctaggatg 600

cacagcagct ctgtgaccct ttgtttgaga ggaacaggaa aaccaccctt ctctctggcc 660

cactgtgtcc tcttcctgcc ctgccatccc cttctgtgaa tgttagaccc atgggagcag 720

ctggtcagag gggaccccgg cctggggccc ctaaccctat gtagcctcag tcttcccatc 780

aggctctcag ctcagcctga gtgttgaggc cccagtggct gctctggggg cctcctgagt 840

ttctcatctg tgcccctccc tccctggccc aggtgaaggt gtggttccag aaccggagga 900

caaagtacaa acggcagaag ctggaggagg aagggcctga gtccgagcag aagaagaagg 960

gctcccatca catcaaccgg tggcgcattg ccacgaagca ggccaatggg gaggacatcg 1020

atgtcacctc caatgactag ggtgggcaac cacaaaccca cgagggcaga gtgctgcttg 1080

ctgctggcca ggcccctgcg tgggcccaag ctggactctg gccactccct ggccaggctt 1140

tggggaggcc tggagtcatg gccccacagg gcttgaagcc cggggccgcc attgacagag 1200

ggacaagcaa tgggctggct gaggcctggg accacttggc cttctcctcg gagagcctgc 1260

ctgcctgggc gggcccgccc gccaccgcag cctcccagct gctctccgtg tctccaatct 1320

cccttttgtt ttgatgcatt tctgttttaa tttattttcc aggcaccact gtagtttagt 1380

gatccccagt gtcccccttc cctatgggaa taataaaagt ctctctctta atgacacggg 1440

catccagctc cagccccaga 1460

<210> 30

<211> 5311

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 一体化载体(HsCCR5)

<400> 30

cttccgcttc ctcgctcact gattcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480

actatcgtct tgaatccaac ccggtaagac acgacttatc gccactggca gcagccactg 540

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctgg ctccccgtcg 1020

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080

gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800

tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 1860

cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 1920

ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 1980

ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 2040

ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc 2100

gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2160

accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2220

gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2280

ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2340

ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2400

ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc 2460

ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2520

gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2580

agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2640

atcagagctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2700

tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2760

tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2820

tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2880

tcagcaccgt ttctgcggac tggctttcta cgtgaaaagg atctaggtga agatcctttt 2940

tgataatctc atgcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat 3000

gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact 3060

ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag 3120

ttcacgggag actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg 3180

ccccggcgtg tcaataatat cactctgtac atccacaaac agacgataac ggctctctct 3240

tttataggtg taaaccttaa actgccgtac gtataggctg cgcaactgtt gggaagggcg 3300

atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 3360

attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga 3420

attgtaatac gattcactat agggcgaatt gggccctcta gatgcatgct cgagcggccg 3480

ccagtgtgat ggatatctgc agaattcgcc cttggtggtg gctgtgtttg cgtctctccc 3540

aggaatcatc tttaccagat ctcaaaaaga aggtcttcat tacacctgca gctctcattt 3600

tccatacagt cagtatcaat tctggaagaa tttccagaca ttaaagatag tcatcttggg 3660

gctggtcctg ccgctgcttg tcatggtcat ctgctactcg ggaatcctaa aaactctgct 3720

tcggtgtcga aatgagaaga agaggcacag ggctgtgagg cttatcttca ccatcatgat 3780

tgtttatttt ctcttctggg ctccctacaa cattgtcctt ctcctgaaca ccttccagga 3840

attctttggc ctgaataatt gcagtagctc taacaggttg gaccaagcta tgcaggtgac 3900

agagactctt gggatgacgc actgctgcat caaccccatc atctatgcct ttgtcgggga 3960

gaagttcaga aactacctct tagtcttctt ccaaaagcac attgccaaac gcttctgcaa 4020

atgctgttct attttccagc aagaggctcc cgagcgagca agctcagttt acacccgatc 4080

cactggggag caggaaatat ctgtgggctt gtgacacgga ctcaagtggg ctggtgaccc 4140

agtcagagtt gtgcacatgg cttagttttc atacacagcc tgggctgggg gtggggtggg 4200

agaggtcttt tttaaaagga agttactgtt atagagggtc taagattcat ccatttattt 4260

ggcatctgtt taaagtagat tagatctttt aagcccatca attatagaaa gccaaatcaa 4320

aatatgttga tgaaaaatag caaccttttt atctcccctt cacatgcatc aagttattga 4380

caaactctcc cttcactccg aaagttcctt atgtatattt aaaagaaagc ctcagagaat 4440

tgctgattct tgagtttagt gatctgaaca gaaataccaa aattatttca gaaatgtaca 4500

actttttacc tagtacaagg caacatatag gttgtaaatg tgtttaaaac aggtctttgt 4560

cttgctatgg ggagaaaaga catgaatatg attagtaaag aaatgacact tttcatgtgt 4620

gatttcccct ccaaggtatg gttaataagt ttcactgact tagaaccagg cgagagactt 4680

gtggcctggg agagctgggg aagcttctta aatgagaagg aatttgagtt ggatcatcta 4740

ttgctggcaa agacagaagc ctcactgcaa gcactgcatg ggcaagcttg gctgtagaag 4800

gagacagagc tggttgggaa gacatgggga ggaaggacaa ggctagatca tgaagaacct 4860

tgacggcatt gctccgtcta agtcatgagc tgagcaggga gatcctggtt ggtgttgcag 4920

aaggtttact ctgtggccaa aggagggtca ggaaggatga gcatttaggg caaggagacc 4980

accaacagcc ctcaggtcag ggtgaggatg gcctctgaag ggcgaattcc agcacactgg 5040

cggccgttac tagtggatcc gagctcggta ccaagcttgg cgtaatcatg gtcatagctg 5100

tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 5160

aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 5220

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 5280

gcggggagag gcggtttgcg tattgggcgc t 5311

<210> 31

<211> 4926

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 一体化载体(HsDNMT1)

<400> 31

cttccgcttc ctcgctcact gattcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480

actatcgtct tgaatccaac ccggtaagac acgacttatc gccactggca gcagccactg 540

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctgg ctccccgtcg 1020

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080

gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800

tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 1860

cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 1920

ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 1980

ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 2040

ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc 2100

gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2160

accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2220

gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2280

ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2340

ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2400

ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc 2460

ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2520

gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2580

agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2640

atcagagctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2700

tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2760

tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2820

tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2880

tcagcaccgt ttctgcggac tggctttcta cgtgaaaagg atctaggtga agatcctttt 2940

tgataatctc atgcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat 3000

gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact 3060

ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag 3120

ttcacgggag actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg 3180

ccccggcgtg tcaataatat cactctgtac atccacaaac agacgataac ggctctctct 3240

tttataggtg taaaccttaa actgccgtac gtataggctg cgcaactgtt gggaagggcg 3300

atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 3360

attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga 3420

attgtaatac gattcactat agggcgaatt gggccctcta gatgcatgct cgagcggccg 3480

ccagtgtgat ggatatctgc agaattcgcc cttgctgctc tcgaactcct ggcctcaact 3540

aatccacctg ccttggcctc ccaaagtgct gggattacag gcgtgagcca ctgctcccag 3600

ccccacgtgt ctttgtctca agtctttctg aagctcttca aaggcccagt gacttgtggc 3660

tgtggggcgg gatgatgggc cagttggagg gtccaaggat cttgtgctgg aagggttttg 3720

ggcccatgtg agcaggacca gaacccttcc ccaaggggtg caatgcccag gttgtcctcc 3780

atctgagcag gggctggcag tacacctgcc cccgggcctt gggcctgggt gtccacatca 3840

ggcattgccc ttctcccctc ctgcaggtgg gcaatgccgt gccaccgccc ctggccaaag 3900

ccattggctt ggagatcaag ctttgtatgt tggccaaagc ccgagagagt gcctcaggta 3960

tggtggggtg ggccaggctt cctctggggc ctgactgccc tctgggggta catgtggggg 4020

cagttgctgg ccaccgtttt gggctctggg actcaggcgg gtcacctacc cacgttcgtg 4080

gccccatctt tctcaagggg ctgctgtgag gattgagtga gttgcacgtg tcaagtgctt 4140

agagcaggcg tgctgcacac agcaggcctt tggtcaggtt ggctgctggg ctggccctgg 4200

ggccgtttcc ctcactcctg ctcggtgaat ttggctcagc aggcacctgc ctcagctgct 4260

cacttgagcc tctgggtcta gaaccctctg gggaccgttt gaggagtgtt cagtctccgt 4320

gaacgttccc ttagcactct gccacttatt gggtcagctg ttaacatcag tacgttaatg 4380

tttcctgatg gtccatgtct gttactcgcc tgtcaagtgg cgtgacaccg ggcgtgttcc 4440

ccagagtgac ttttcctttt atttcccttc agctaaaata aaggaggagg aagctgctaa 4500

ggactagttc tgccctcccg tcacccctgt ttctggcacc aggaatcccc aacatgcact 4560

gatgttgtgt ttttaacatg tcaatctgtc cgttcacatg tgtggtacat ggtgtttgtg 4620

gccttggctg acaagggcga attccagcac actggcggcc gttactagtg gatccgagct 4680

cggtaccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 4740

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 4800

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 4860

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 4920

ggcgct 4926

<210> 32

<211> 5267

<212> DNA

<213> 人工序列（Artificial Sequence）

<220>

<223> 一体化载体(HsEMX1)

<400> 32

cttccgcttc ctcgctcact gattcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60

cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120

acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180

ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240

ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300

gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360

gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420

ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480

actatcgtct tgaatccaac ccggtaagac acgacttatc gccactggca gcagccactg 540

gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600

ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660

ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720

gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780

tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840

tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900

aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960

aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctgg ctccccgtcg 1020

tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080

gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140

agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200

aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260

gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320

caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380

cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440

ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500

ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560

gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620

cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680

gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740

caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800

tactcttcct ttttcaattc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg 1860

cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag 1920

ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag 1980

ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca 2040

ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc 2100

gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag 2160

accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg 2220

gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt 2280

ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag 2340

ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt 2400

ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc 2460

ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga 2520

gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg 2580

agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg 2640

atcagagctt gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac 2700

tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc 2760

tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt 2820

tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatccgggg 2880

tcagcaccgt ttctgcggac tggctttcta cgtgaaaagg atctaggtga agatcctttt 2940

tgataatctc atgcctgaca tttatattcc ccagaacatc aggttaatgg cgtttttgat 3000

gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga ccggcacact 3060

ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca ccgggtaaag 3120

ttcacgggag actttatctg acagcagacg tgcactggcc agggggatca ccatccgtcg 3180

ccccggcgtg tcaataatat cactctgtac atccacaaac agacgataac ggctctctct 3240

tttataggtg taaaccttaa actgccgtac gtataggctg cgcaactgtt gggaagggcg 3300

atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 3360

attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga 3420

attgtaatac gattcactat agggcgaatt gggccctcta gatgcatgct cgagcggccg 3480

ccagtgtgat ggatatctgc agaattcgcc cttgtgggga cagaaggtct ggagctgccc 3540

gtgaagggca gaatgctgcc ctcagacccg cttcctccct gtccttgtct gtccaaggag 3600

aatgaggtct cactggtgga tttcggacta ccctgaggag ctggcacctg agggacaagg 3660

ccccccacct gcccagctcc agcctctgat gaggggtggg agagagctac atgaggttgc 3720

taagaaagcc tcccctgaag gagaccacac agtgtgtgag gttggagtct ctagcagcgg 3780

gttctgtgcc cccagggata gtctggctgt ccaggcactg ctcttgatat aaacaccacc 3840

tcctagttat gaaaccatgc ccattctgcc tctctgtatg gaaaagagca tggggctggc 3900

ccgtggggtg gtgtccactt taggccctgt gggagatcat gggaacccac gcagtgggtc 3960

ataggctctc tcatttacta ctcacatcca ctctgtgaag aagcgattat gatctctcct 4020

ctagaaactc gtagagtccc atgtctgccg gcttccagag cctgcactcc tccaccttgg 4080

cttggctttg ctggggctag aggagctagg atgcacagca gctctgtgac cctttgtttg 4140

agaggaacag gaaaaccacc cttctctctg gcccactgtg tcctcttcct gccctgccat 4200

ccccttctgt gaatgttaga cccatgggag cagctggtca gaggggaccc cggcctgggg 4260

cccctaaccc tatgtagcct cagtcttccc atcaggctct cagctcagcc tgagtgttga 4320

ggccccagtg gctgctctgg gggcctcctg agtttctcat ctgtgcccct ccctccctgg 4380

cccaggtgaa ggtgtggttc cagaaccgga ggacaaagta caaacggcag aagctggagg 4440

aggaagggcc tgagtccgag cagaagaaga agggctccca tcacatcaac cggtggcgca 4500

ttgccacgaa gcaggccaat ggggaggaca tcgatgtcac ctccaatgac tagggtgggc 4560

aaccacaaac ccacgagggc agagtgctgc ttgctgctgg ccaggcccct gcgtgggccc 4620

aagctggact ctggccactc cctggccagg ctttggggag gcctggagtc atggccccac 4680

agggcttgaa gcccggggcc gccattgaca gagggacaag caatgggctg gctgaggcct 4740

gggaccactt ggccttctcc tcggagagcc tgcctgcctg ggcgggcccg cccgccaccg 4800

cagcctccca gctgctctcc gtgtctccaa tctccctttt gttttgatgc atttctgttt 4860

taatttattt tccaggcacc actgtagttt agtgatcccc agtgtccccc ttccctatgg 4920

gaataataaa agtctctctc ttaatgacac gggcatccag ctccagcccc agaaagggcg 4980

aattccagca cactggcggc cgttactagt ggatccgagc tcggtaccaa gcttggcgta 5040

atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 5100

acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 5160

aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 5220

atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgct 5267

Claims (20)

1.一种Cas12a蛋白，具有如SEQ ID NO：1所示的氨基酸序列。

2.根据权利要求1所述的Cas12a蛋白，其中，所述Cas12a蛋白具有SEQ ID NO：1所示氨基酸序列，其由SEQ ID NO：2所示的核苷酸序列编码。

3.根据权利要求1所述的Cas12a蛋白，其中所述蛋白具有核酸内切酶活性。

4.根据权利要求1所述的Cas12a蛋白，其中，所述具有SEQ ID NO：1所示的氨基酸序列的Cas12a蛋白在pH 7.0至pH 7.9下具有最佳活性。

5.一种Cas12a蛋白，其具有如SEQ ID NO：1所示的氨基酸序列，其中925位的赖氨酸(Lys)被另一氨基酸取代。

6.根据权利要求5所述的Cas12a蛋白，其中，所述其他氨基酸选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任何一种。

7.一种Cas12a蛋白，其具有如SEQ ID NO：3所示的氨基酸序列。

8.根据权利要求7所述的Cas12a蛋白，其中，所述Cas12a蛋白具有SEQ ID NO：3所示氨基酸序列，由SEQ ID NO：4所示的核苷酸序列编码。

9.根据权利要求7所述的Cas12a蛋白，其中所述蛋白具有核酸内切酶活性。

10.根据权利要求7所述的Cas12a蛋白，其中，所述具有SEQ ID NO：3所示的氨基酸序列的Cas12a蛋白在pH 7.0至pH 7.9下具有最佳活性。

11.一种Cas12a蛋白，其具有如SEQ ID NO：1所示的氨基酸序列，其中930位的赖氨酸(Lys)被另一氨基酸取代。

12.根据权利要求11所述的Cas12a蛋白，其中，所述其他氨基酸选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任何一种。

13.一种Cas12a蛋白，其具有如SEQ ID NO：1所示的氨基酸序列，其中877位的天冬氨酸(Asp)被另一氨基酸取代。

14.如权利要求13所述的Cas12a蛋白，其中所述其他氨基酸选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任何一种。

15.根据权利要求13所述的Cas12a蛋白，其中所述蛋白具有降低的核酸内切酶活性。

16.一种Cas12a蛋白，其具有如SEQ ID NO：3所示的氨基酸序列，其中873位的天冬氨酸(Asp)被另一个氨基酸取代。

17.根据权利要求16所述的Cas12a蛋白，其中，所述其他氨基酸选自下组的精氨酸(Arg)、组氨酸(His)、天冬氨酸(Asp)、谷氨酸(Glu)、丝氨酸(Ser)、苏氨酸(Thr)、天冬酰胺(Asn)、谷氨酰胺(Gln)、酪氨酸(Tyr)、丙氨酸(Ala)、异亮氨酸(Ile)、亮氨酸(Leu)、缬氨酸(Val)、苯丙氨酸(Phe)、蛋氨酸(Met)、色氨酸(Trp)、甘氨酸(Gly)、脯氨酸(Pro)和半胱氨酸(Cys)的任何一种。

18.根据权利要求16所述的Cas12a蛋白，其中所述蛋白具有降低的核酸内切酶活性。

19.一种用于治疗癌症的药物组合物，其包含作为活性成分：

mgCas12a；和

靶向特异性存在于癌细胞中的核酸序列的crRNA。

20.根据权利要求19所述的药物组合物，其中所述mgCas12a具有选自下组的任一种氨基酸序列：SEQ ID NO：1、SEQ ID NO：3、SEQ ID NO：5、以及SEQ ID NO：6。

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