一种同源2型CRISPR/Cas基因编辑系统的构建方法

技术领域

本发明涉及一种同源2型CRISPR/Cas基因编辑系统的构建方法，属于基因编辑技术领域。

背景技术

基因编辑(gene editing)技术使得针对DNA序列特定位点进行改造成为可能，比如第一代基因编辑工具锌指核酸酶(zinc finger nucleases,ZFNs)，第二代基因编辑工具类似转录激活的小型核酸酶(transcription activator-like effector nucleases,TALENs)，都可以用于改造靶向基因组，但是这些方法设计困难，不易制作，成本昂贵且普适性不强。

CRISPR(成簇的规律间隔的短回文重复序列，Clustered Regularly InterspacedShort Palindromic Repeat)/Cas(CRISPR-associated protein)系统，是来自古生菌和细菌的天然免疫系统，为第三代基因编辑工具。CRISPR/Cas系统不同于以往基因编辑工具(蛋白质-DNA识别)，其利用向导RNA(single guide RNA,sgRNA)基于核酸互补配对原理进行目标DNA碱基序列的识别，引导Cas效应蛋白进行定点切割，适用性强、设计简单、成本低、效率高。Cas蛋白包含多种不同的效应结构域(domain)，在核酸识别、稳定复合物结构、水解DNA磷酸二酯键等不同的活动中发挥作用。其中，源自化脓链球菌(Streptococcus pyogeneCas，SpCas9)的II型CRISPR/Cas9系统因其切割效率高，成为目前应用最为广泛的CRISPR/Cas系统。

大多数现有的CRISPR基因编辑系统是来自Streptococcus pyogenes中的SpCas9系统。这一系统通过识别并切割靶向的多核苷酸上的原间隔序列临近模块(ProtospacerAdjacent Motif,PAM)序列，即“NGG”，限制了其进一步的广泛应用，得到更多种具有不同的理化性质并能识别不同PAM的同源CRSIPR/Cas系统成为研究重点。与此同时，在庞大以及各色各样的宏基因组中，隐藏了尚未培养甚至尚未发现的微生物，可能存在大量的未被发现的CRISPR/Cas蛋白编辑系统，这些系统在原核生物、真核生物，以及在体外环境中的活性需要得到证实。

发明内容

本发明的目的在于克服现有技术的不足，提供一种同源2型CRISPR/Cas基因编辑系统的构建方法，通过该构建方法预测出5种新的同源CRISPR/Cas基因编辑系统。

为实现上述目的，本发明采取的技术方案为：一种同源2型CRISPR/Cas基因编辑系统的构建方法，包括如下步骤：

(1)对宏基因组测序数据进行处理，筛选得到contig序列和蛋白序列；

(2)将步骤(1)所述的蛋白序列使用软件进行聚类，再使用HMMer软件包扩大聚类；

(3)在步骤(2)扩大的聚类中，用已知的2型CRISPR/Cas系统相关的效应蛋白的隐马尔可夫模型文件搜索与2型CRISPR/Cas系统有关的聚类；

(4)将步骤(3)所述的2型CRISPR/Cas系统有关的聚类，与相关数据库进行比对，筛选得到基因；

(5)将步骤(4)筛选得到的基因所在的contig，进行CRISPR/Cas系统预测，将预测出的2型CRISPR/Cas系统的效应蛋白提取出，然后用软件分析结构域；

(6)将步骤(5)预测出的2型CRISPR/Cas系统的效应蛋白的结构域和2型CRISPR/Cas系统的效应蛋白相关的辅助蛋白做比较基因组学和进化相关分析，即可得到同源2型CRISPR/Cas基因编辑系统。

本申请的发明人通过深度分析来自各种环境的宏基因组数据，经过组装短读段序列(100-300bp)得到长DNA序列(≥4000bp)草图，在长DNA序列中挖掘出新的2型同源CRISPR/Cas基因编辑系统的方法，通过该方法预测的内容包括：Cas效应蛋白(Cas9、Cas12和Cas13)、与Cas效应蛋白发挥作用相关的辅助蛋白、与Cas效应蛋白发挥作用相关的辅助序列、Cas效应蛋白发挥作用的关键序列。基于组装长DNA序列做CRISPR-Cas相关的蛋白质及元件预测，不依赖物种的精准判断；精确判断2型CRISPR/Cas系统的编码蛋白基因、成簇规律间隔的短回文重复序列，crRNA序列及反式激活crRNA序列；进一步挖掘其生物学过程中切割DNA功能的结构预测，包括但不限于crRNA-tracrRNA二级结构预测、Cas效应蛋白功能域预测、Cas-crRNA-tracrRNA/Cas-crRNA复合物结构预测；进一步挖掘Cas9/Cas12效应蛋白识别的多种紧邻靶向序列下游/上游的原间隔序列临近模块(PAM)。

作为本发明所述的构建方法的优选实施方式，步骤(1)中，所述对宏基因组测序数据进行处理具体为：通过对宏基因组的数据进行质控、拼接和基因预测；所述宏基因组测序数据为肠道宏基因组测序数据；所述contig序列的长度为≥4000bp，所述蛋白序列的长度为≥600bp。

作为本发明所述的构建方法的优选实施方式，步骤(2)具体为：所述的蛋白序列使用orthofinder软件进行聚类，再使用HMMer软件包扩大聚类。

作为本发明所述的构建方法的优选实施方式，步骤(4)中，相关数据库为Swissprot数据库和NCBI nr数据库。

作为本发明所述的构建方法的优选实施方式，步骤(5)中，所述进行CRISPR/Cas系统预测的软件为CRISPRCasFinder，所述分析结构域的软件为HHpred。

本发明的另一目的是采用本发明所述的构建方法构建的同源2型CRISPR/Cas基因编辑系统。

作为本发明所述的同源2型CRISPR/Cas基因编辑系统的优选实施方式，所述的同源2型CRISPR/Cas基因编辑系统包括Cas核糖核蛋白复合物和CRISPR RNA。

作为本发明所述的同源2型CRISPR/Cas基因编辑系统的优选实施方式，所述的同源2型CRISPR/Cas基因编辑系统还包括辅助蛋白Cas1、辅助蛋白Cas2、辅助蛋白Cas4、辅助蛋白Csn2、辅助蛋白csx27、辅助蛋白csx28、辅助蛋白WYL或反式激活CRISPR RNA。

Cas9核酸内切酶是一种多结构域和多功能DNA核酸内切酶，其识别靶向序列需要两个重要因素：一个是结合的guide RNA与靶向序列互补链碱基互补配对；另一个是靶向序列相邻的原间隔序列临近模块(Protospacer Adjacent Motif,PAM)序列具备特异性。Cas9蛋白通过不同的核酸酶结构域在靶向序列所在链的PAM序列上游有效切割与guide RNA互补的双链DNA。其中，HNH样核酸酶结构域切割与RNA序列互补DNA链，RuvC样核酸酶结构域负责切非互补链DNA，从而形成双链DNA分子断裂(double-strand breaks，DSB)；Cas12核酸内切酶通过不同的核酸酶结构域在PAM序列的下游切割与CRISPR RNA(crRNA)互补的双链DNA，产生5个核苷酸突出的粘性末端。其中，核酸酶结构域为HNH样核酸酶结构域或RuvC样核酸酶结构域；Cas13是一种多结构域和多功能RNA核酸内切酶。它通过两个HEPN样核酸酶结构域切割与sgRNA互补的单链RNA。

Cas9蛋白系统中crRNA(CRISPR RNA)与反式激活crRNA(trans-activating，tracrRNA)形成sgRNA；Cas12和Cas13蛋白系统中crRNA无需反式激活crRNA即可发挥guideRNA的作用。CRISPR阵列(array)转录产物前体crRNA(pre-crRNA)进一步被RNA酶处理产生crRNA，其为引导Cas蛋白识别入侵的外源DNA/RNA的碱基互补配对部分，这段序列一般长约19-23bp。

通过人为设计crRNA中的目标序列，CRISPR/Cas系统原则上可以靶向基因组中任意感兴趣的DNA/RNA序列。对于DNA识别，其切割产生位点特异的DSB，进一步通过非同源末端修复DSB，从而在切割位点产生小的随机插入/缺失片段(indels)，致使目的基因失活；并通过高保真同源修复，可以使用同源修复模板在DSB位点进行精确的基因组修饰，实现庞大的基因编辑修饰功能。

作为本发明所述的同源2型CRISPR/Cas基因编辑系统的优选实施方式，所述Cas9核糖核蛋白复合物的链长度为900-1500aa，所述CRISPR RNA的长度为15-36bp。所述反式激活CRISPR RNA的复合物的链长度为70-160bp，所述Cas9/Cas12核糖核蛋白复合物识别的PAM序列长度为1-10bp。

作为本发明所述的同源2型CRISPR/Cas基因编辑系统的优选实施方式，

所述的同源2型CRISPR/Cas基因编辑系统有3大效应蛋白，分别为效应Cas9蛋白、效应Cas12蛋白和效应Cas13蛋白；

所述效应Cas9蛋白有12种，编号为C1556、C1793、C1807、C4640、C6165、Lt1、Lt2、Lt3、Lt4、Lt5、Lt6和Lt7，其蛋白序列分别如SEQ1-SEQ12所示；所述效应Cpf1蛋白有1种，编号为LtCpf1，其蛋白序列如SEQ13所示，所述效应Cas13蛋白有2种，编号为LtCas13b和LtCas13d，其蛋白序列分别如SEQ14-SEQ15所示；其中，在所述编号为C1556、C1793、C1807、C4640和C6165的同源2型CRISPR/Cas9基因编辑系统中：所述Cas9核糖核蛋白复合物的链长度分别为1144aa、1358aa、1426aa、1315aa和1152aa，所述CRISPR RNA与所述反式激活CRISPR RNA的复合物的链长度分别为126bp、124bp、141bp、152bp、120bp，所述Cas9核糖核蛋白复合物识别的PAM序列分别为W(A>T)TNTAH(A>T>C)NNAT、NATS(G>C)NY(C>T)GAT、NNNNTA、NNCGC和CGNGAGG。

与现有技术相比，本发明的有益效果为：

(1)本发明提供的一种同源2型CRISPR/Cas基因编辑系统的挖掘方法，过程简单敏感且普适性强；

(2)用本发明提供的一种同源2型CRISPR/Cas基因编辑系统的构建方法，设计简单易懂；

(3)用本发明提供的一种同源2型CRISPR/Cas基因编辑系统的构建方法，初步发现了12个Cas9，1个Cpf1和2个Cas13，其中编号为C1556、C1793、C1807、C4640和C6165的5个Cas9具有不同的理化性质并能识别不同PAM的序列，对应识别的PAM序列分别为W(A>T)TNTAH(A>T>C)NNAT、NATS(G>C)NY(C>T)GAT、NNNNTA、NNCGC和CGNGAGG。

附图说明

图1为本发明提供的一种通过宏基因组序列组装的挖掘同源2型CRISPR/Cas的方法流程图。

图2为本发明所述的编号C1556的同源2型CRISPR/Cas9基因编辑系统；其中，图2A为编号C1556的同源2型CRISPR/Cas9基因编辑系统组成图；图2B为编号C1556的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图；图2C为编号C1556的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图；图2D为编号C1556的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图；图2E为编号C1556的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图。

图3为本发明所述的为编号C1793的同源2型CRISPR/Cas9基因编辑系统，其中，图3A为编号C1793的同源2型CRISPR/Cas9基因编辑系统组成图；图3B为编号C1793的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图；图3C为编号C1793的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图；图3D为编号C1793的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图；图3E为编号C1793的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图。

图4为本发明所述的编号C1807的同源2型CRISPR/Cas9基因编辑系统，其中，图4A为编号C1807的同源2型CRISPR/Cas9基因编辑系统组成图；图4B为编号C1807的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图；图4C为编号C1807的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图；图4D为编号C1807的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图；图4E为编号C1807的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图。

图5为本发明所述的编号C4640的同源2型CRISPR/Cas9基因编辑系统，其中，图5A为编号C4640的同源2型CRISPR/Cas9基因编辑系统组成图；图5B为编号C4640的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图；图5C为编号C4640的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图；图5D为编号C4640的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图；图5E为编号C4640的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图。

图6为本发明所述的编号C6165的同源2型CRISPR/Cas9基因编辑系统，其中，图6A为编号C6165的同源2型CRISPR/Cas9基因编辑系统组成图；图6B为编号C6165的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图；图6C为编号C6165的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图；图6D为编号C6165的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图；图6E为编号C6165的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图。

具体实施方式

为更好地说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。

实施例1

本实施例为本发明提供的一种同源2型CRISPR/Cas基因编辑系统的构建方法(具体流程如图1所示)，包括如下步骤：

(1)对宏基因组测序数据进行处理，筛选得到contig序列和蛋白序列；

(2)将步骤(1)所述的蛋白序列使用orthofinder软件进行聚类，再使用HMMer软件包扩大聚类；

(3)在步骤(2)扩大的聚类中，用已知的2型CRISPR/Cas系统相关的效应蛋白的隐马尔可夫模型文件搜索与2型CRISPR/Cas系统有关的聚类；

(4)将步骤(3)所述的2型CRISPR/Cas系统有关的聚类，与Swiss prot和NCBI nr数据库进行比对，筛选得到基因；

(5)将步骤(4)筛选得到的基因所在的contig，用CRISPRCasFinder进行CRISPR/Cas系统预测，包括必要的辅助序列和辅助蛋白；

(6)将步骤(5)预测出的2型CRISPR/Cas系统的效应蛋白提取出，然后用HHpred软件分析结构域；

(7)将步骤(6)预测出的2型CRISPR/Cas系统的效应蛋白的结构域和2型CRISPR/Cas系统的效应蛋白相关的辅助蛋白做比较基因组学和进化相关分析，即可得到同源2型CRISPR/Cas基因编辑系统。

实施例2

本实施例为本发明所述的一种同源2型CRISPR/Cas9基因编辑系统的构建方法，关于如下4个步骤具体操作的介绍：1.对宏基因组测序数据进行处理，筛选得到contig序列和蛋白序列；2.对得到的长序列做CRISPR-Cas相关的CRISPR序列及Cas蛋白预测；3.预测同源2型CRISPR/Cas基因编辑系统的RNA二级结构；4.预测同源2型CRISPR/Cas基因编辑系统能识别的PAM序列。

1.初步处理宏基因组数据，得到长读长序列、潜在的编码基因及对应的蛋白序列。

(1)材料：IgAN肾病患者体内肠道样本宏基因组数据

(2)软件：fastqc(v0.11.5),fastp(v0.19.8),SOAPnuke(v1.5.2),hisat2(v2.0.4),Samtools(v1.9),GeneMark(v3.38),IDBA-UD(v1.1.3),Bowtie2(v2.3.5.1),CD-HIT(v4.6),HMMER(3.1b2),BLAST(v2.3.0+)。

(3)检测方法：本步骤的数据来源为肠道宏基因组，通过对肠道宏基因组的数据进行质控、拼接和基因预测，得到宏基因组中可能的所有的基因序列、编码蛋白序列，并且对应每一个基因序列所在的基因组长序列；

具体操作如下：

(a)首先用fastqc软件进行指控，检查数据为原始数据还是清理过的数据，若为原始数据，则接下来使用fastpc软件对原始数据进行清理以及去除接头，若为清理过的数据，则直接进入下一步骤；

(b)使用hisat2软件将宏基因组的reads比对至人参考基因组(hg19)上，形成(Binary Alignment/Map)BAM格式的文件，再使用Samtools提取出不能比对至人参考基因组上的reads，得到无宿主(人)基因污染的数据部分的序列文件；

(c)然后进行长序列的组装和基因的预测，使用IDBA-UD对清理过的read数据进行组装，得到长序列；

(d)去除掉过短的长序列以后，再使用GeneMark软件中的MetaGeneMark，进行基因预测，得到的基因进行翻译并过滤掉小于600氨基酸的序列；

(e)对于Cas13的查找，在候选序列上的蛋白质序列中寻找存在HEPN domain模型(E……RxxxxH)的候选蛋白；对于Cas9和Cpf1的寻找，使用CD-HIT和Orthofinder对所有得到的蛋白序列进行聚类，用已知的2型CRISPR-Cas系统相关的序列制作而成的HMM profile对每一个聚类进行搜索，得到可能与CRISPR-Cas系统相关的蛋白序列；

(f)再比对主流数据库(nr，swiss prot)得到未被注释为CRISPR-Cas系统相关蛋白的基因，在此步骤中我们得到了12个Cas9蛋白序列(SEQ ID NO.1-SEQ ID NO.12)、1个Cpf1蛋白序列(SEQ ID NO.13)和2个Cas13蛋白序列(SEQ ID NO.14-SEQ ID NO.15)；

(g)通过基因名称中的标签，找回对应的长序列，在下一步骤中备用。

2.对得到的长序列做CRISPR/Cas系统相关的CRISPR序列及Cas蛋白预测

(1)材料：上述1(初步处理宏基因组数据)中得到的可能与CRISPR-Cas系统相关的长序列

(2)软件：改造后的CRISPRCasFinder(v4.2.19),python(v3.7.4),bedtools(v2.25.0)

(3)检测方法：本步骤是对得到的可能与2型CRISPR-Cas系统相关的长序列进行分析，得到预测的与CRISPR-Cas系统相关的蛋白和相关的元件。

具体操作：

(a)使用CRISPRCasFinder对得到的可能与2型CRISPRCasFinder系统相关的长序列进行分析和预测，得到Cas蛋白，Spacer，direct repeat等CRISPR-Cas系统相关的蛋白和元件；

(b)注释候选CRISPR-Cas系统相关蛋白，对于Cas13蛋白系统：收集Cas13a，Cas13b，Cas13c及Cas13d辅助蛋白的HMM文件，对长序列进行HMMER比对，确定是否存在Cas1，Cas2，csx27，csx28，WYL辅助蛋白；对于Cas9/Cpf1蛋白系统，收集Cas9/Cpf1辅助蛋白的HMM文件，对长序列进行HMMER比对，确定是否存在Cas1,Cas2,Cas4及Csn辅助蛋白。

(c)对于Cas9蛋白，在已预测出来的元件互补的部分中寻找anti-repeat并确认tracrRNA对应的DNA序列；最后确认包含所有必须元件的序列范围，以便后期实验合成。

3.预测RNA二级结构

(1)材料：预测的repeat或tracrRNA序列

(2)软件：NUPACK(http://www.nupack.org/partition/new)

(3)预测方法：对于Cas9蛋白，通过模拟tracrRNA和repeat转录出的RNA在体外37℃下各1μl的相互作用过程，进行二级结构预测；对于Cas12/Cas13蛋白，通过模拟体外37℃CRISPR RNA 1μl的环境，得到的RNA进行二级结构预测

4.预测Cas9/Cas12的PAM序列

(1)材料：上述3(预测RNA二级结构)中得到的可能与CRISPR-Cas系统相关的Spacer序列

(2)软件：CRISPRCasFinder(v4.2.19),python(v3.7.4),bedtools(v2.25.0)

(3)数据库：NCBI(Phage/Virus/Plasmid)数据库

具体操作：将Spacer序列与同该长序列相关的物种的核酸数据库进行比对，得到其上下游序列，再通过保守性的原则，用weblogo预测出对应该CRISPR/Cas系统的PAM序列。

实施例3

本实施例为编号C1556的同源2型CRISPR/Cas9基因编辑系统，其是从病人97A肠道宏基因组数据中挖掘预测的同源CRISPR/Cas9系统，该系统所包含的内容如图2所示。

图2A为编号C1556的同源2型CRISPR/Cas9基因编辑系统组成图，该系统包含Cas蛋白基因组座(核酸内切酶Cas9、Cas1蛋白、Cas2蛋白)、CRISPR array及反式激活crRNA(tracrRNA)所在的前导序列。

图2B为编号C1556的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图，具体包含Pox A22、BH、HNH4共3个结构域；

图2C为编号C1556的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图，CRISPRarray由36bp的重复序列以及29/30个间隔序列为基本单位组成；

图2D为编号C1556的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图，sgRNA(crRNA-tracrRNA)结构包含上游的两个bulge结构以及下游的两个stem loop。

图2E为编号C1556的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图，该系统能够识别的PAM序列为W(A>T)TNTAH(A>T>C)NNAT。

实施例4

本实施例为编号C1793的同源2型CRISPR/Cas9基因编辑系统，其是从病人147A肠道宏基因组数据中挖掘预测的同源CRISPR/Cas9系统，该系统所包含的内容如图3所示。

图3A为编号C1793的同源2型CRISPR/Cas9基因编辑系统组成图，该系统组成包含核酸内切酶Cas9、CRISPR array及反式激活crRNA(tracrRNA)所在的前导序列；

图3B为编号C1793的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图，核酸内切酶Cas9蛋白包含RuvX、BH、REC、HNH4、RuvCⅢ、P1共6个结构域；

图3C为编号C1793的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图，CRISPRarray由36bp的重复序列以及30个间隔序列为基本单位组成；

图3D为编号C1793的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图，sgRNA(crRNA-tracrRNA)结构包含上游的三个bulge结构以及下游的三个stem loop；

图3E为编号C1793的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图，该系统能够识别的PAM序列为NATS(G>C)NY(C>T)GAT。

实施例5

本实施例为编号C1807的同源2型CRISPR/Cas9基因编辑系统，其是从病人1A肠道宏基因组数据中挖掘预测的同源CRISPR/Cas9系统，该系统所包含的内容如图4所示。

图4A为编号C1807的同源2型CRISPR/Cas9基因编辑系统组成图，该系统组成包含核酸内切酶Cas9、CRISPR array及反式激活crRNA(tracrRNA)所在的前导序列；

图4B为编号C1807的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图，核酸内切酶Cas9蛋白包含RuvX、REC、HNH4、RuvCⅢ共4个结构域；

图4C为编号C1807的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图，CRISPRarray由47bp的重复序列以及29-31个间隔序列为基本单位组成；

图4D为编号C1807的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图，sgRNA(crRNA-tracrRNA)结构包含上游的五个bulge结构以及下游的两个stem loop；

图4E为编号C1807的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图，该系统能够识别的PAM序列为NNNNTA。

实施例6

本实施例为编号C4640的同源2型CRISPR/Cas9基因编辑系统，其是从病人152A肠道宏基因组数据中挖掘预测的同源CRISPR/Cas9系统，该系统所包含的内容如图5所示。

图5A为编号C4640的同源2型CRISPR/Cas9基因编辑系统组成图，该系统组成包含Cas蛋白基因组座(核酸内切酶Cas9和Csn2蛋白)、CRISPR array及反式激活crRNA(tracrRNA)所在的前导序列；

图5B为编号C4640的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图，核酸内切酶Cas9蛋白包含RuvX、BH、REC、HNH4、RuvcⅢ、P1共6个结构域；

图5C为编号C4640的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图，CRISPRarray由37bp的重复序列以及28/29个间隔序列为基本单位组成；

图5D为编号C4640的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图，sgRNA(crRNA-tracrRNA)结构包含上游的两个bulge结构以及下游的三个stem loop；

图5E为编号C4640的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图，该系统能够识别的PAM序列为NNCGC。

实施例7

本实施例为编号C6165的同源2型CRISPR/Cas9基因编辑系统，其是从病人135A肠道宏基因组数据中挖掘预测的同源CRISPR/Cas9系统，该系统所包含的内容如图6所示。

图6A为编号C6165的同源2型CRISPR/Cas9基因编辑系统组成图，该系统组成包含Cas蛋白基因组座(核酸内切酶Cas9、Cas1蛋白、Cas2蛋白)、CRISPR array及反式激活crRNA(tracrRNA)所在的前导序列；

图6B为编号C6165的同源2型CRISPR/Cas9基因编辑系统中核酸内切酶Cas9蛋白结构图，核酸内切酶Cas9蛋白包含REC、HNH4、RuvCⅢ共4个结构域；

图6C为编号C6165的同源2型CRISPR/Cas9基因编辑系统中CRISPR序列图，CRISPRarray由47bp的重复序列以及29/30个间隔序列为基本单位组成；

图6D为编号C6165的同源2型CRISPR/Cas9基因编辑系统识别的guide RNA分子的RNA二级结构预测图，sgRNA(crRNA-tracrRNA)结构包含上游的四个bulge结构以及下游的三个stem loop；

图6E为编号C6165的同源2型CRISPR/Cas9基因编辑系统的保守型PAM序列一致性示意图，该系统能够识别的PAM序列为CGNGAGG。

最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

SEQUENCE LISTING

<110> 中山大学

<120> 一种同源2型CRISPR/Cas基因编辑系统的构建方法

<130> 2020.11.18

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Ser Phe Arg His Met Leu Ser Ala Gly Thr Leu Ser Glu Glu Gln Val

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Gly Arg Trp Leu Glu Ser His Tyr Pro Val Leu Ser Glu Gln Asn Arg

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

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

1220 1225 1230

Leu Asn Glu Lys Tyr Asp His Ile Ser Arg Glu Gln Asn Val Glu

1235 1240 1245

Leu Tyr Thr Leu Leu Ser Gln Lys Leu Lys His Ser Ile Phe Ala

1250 1255 1260

Lys Cys Pro Gly Ser Ile Ala Arg Thr Val Glu Asp Gly Tyr Glu

1265 1270 1275

Lys Phe Ser Gln Leu Asp Pro Glu Asp Gln Ile Gly Cys Leu Met

1280 1285 1290

Gly Ile Val Ser Trp Phe Gly Ser Gln Ser Asn Gly Ile Asp Leu

1295 1300 1305

Thr Leu Ile Gly Gly Lys Ala Ala Thr Gly Thr Lys Leu Ile Asn

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Leu Leu Gly Leu Leu

1355

<210> 3

<211> 1426

<212> PRT

<213> C1807蛋白序列

<400> 3

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

1 5 10 15

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

20 25 30

Phe Ser Val Val Thr Phe Pro Cys Gly Val Gly Thr Gly Lys Thr Gly

35 40 45

Glu Phe Ser Tyr Ala Ala Glu Arg Thr Gln Lys Arg Ser Gln Arg Lys

50 55 60

Leu Tyr Lys Val Arg Arg Tyr Arg Lys Trp Ser Thr Leu Ala Leu Leu

65 70 75 80

Ile Gln Tyr Asp Phe Cys Pro Leu Ser Leu Glu Glu Leu Asp Arg Trp

85 90 95

Arg Val Tyr Asp Lys Glu Arg Gly Leu Lys Arg Gln Tyr Pro Val Glu

100 105 110

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

115 120 125

Met Pro Asp Cys Thr Pro Tyr Gln Leu Arg Lys Ser Leu Val Glu Thr

130 135 140

Lys Leu Asp Leu Ser Leu Pro Ser Asp Arg Tyr Lys Val Gly Arg Ala

145 150 155 160

Leu Tyr His Ile Ala Gln Arg Arg Gly Phe Lys Ser Ser Arg Gly Glu

165 170 175

Thr Ala Lys Glu Gln Glu Ser Ala Glu Gln Ala Asp Asp Ser Asn Glu

180 185 190

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

195 200 205

Asp Phe Met Lys Ala His Asp Cys Pro Thr Val Gly Tyr Ala Leu Ala

210 215 220

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

225 230 235 240

Val Arg Ser Gln Tyr Met Glu Glu Val Glu Thr Ile Phe Arg Phe Gln

245 250 255

Gly Met Tyr Glu Arg Phe Pro Glu Phe Cys Gln Gly Ile Leu Ser Arg

260 265 270

Lys Lys Gly Glu Gly Thr Ile Phe Tyr Lys Arg Pro Leu Arg Ser Gln

275 280 285

Arg Gly Leu Val Gly Lys Cys Thr Leu Glu Pro Asn Lys Pro Arg Cys

290 295 300

Pro Ile Ala His Pro Asp Phe Glu Glu Phe Arg Ala Leu Val Phe Leu

305 310 315 320

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

325 330 335

Asp Ser Asp Met Lys Glu Glu Leu Met Ala Ser Lys Phe Tyr Arg Cys

340 345 350

Gln Thr Thr Phe Pro Phe Lys Glu Ile Arg Glu Trp Leu Glu Lys Lys

355 360 365

Thr Gly Trp Ser Leu Ser Lys Lys Gly Lys Thr Val Asn Tyr Ser Asp

370 375 380

Arg Asp Ser Val Ser Ala Ser Pro Val Thr Ala Arg Leu Arg His Leu

385 390 395 400

Leu Gly Asp Asp Trp Lys Asn Trp Thr Phe Val Ser Glu Phe Glu Lys

405 410 415

Cys Gly His Asp Lys Val Ala Arg Lys Ala Val Tyr Thr Ala Tyr Asp

420 425 430

Ile Trp His Val Cys Tyr Asp Cys Asp Asp Glu Asp Phe Leu Cys Asp

435 440 445

Phe Ala Ala Asn Lys Leu Ser Phe Asp Ser Gln Gln Thr Lys Lys Leu

450 455 460

Val Arg Leu Tyr Glu Glu Met Arg Gln Gly Tyr Ala Met Leu Ser Leu

465 470 475 480

Lys Ala Ile Arg Asn Ile Leu Pro Phe Leu Arg Lys Gly Tyr Ile Tyr

485 490 495

Ser Thr Ala Val Met Leu Ala Lys Ile Pro Glu Ile Val Gly Arg Glu

500 505 510

Lys Trp Asn Glu Tyr Glu Lys Asp Phe Val Ala Gln Val His Leu Leu

515 520 525

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

530 535 540

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

545 550 555 560

His Asn Tyr Asp Tyr Val Leu Asp Asp Ser Asp Arg Ala Asp Val Leu

565 570 575

Lys Ala Ala Val Gly Cys Ile Gly Gln Lys Thr Trp Glu Ser Lys Pro

580 585 590

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

595 600 605

Ala Phe Phe Ala Asp His Lys Arg Lys Tyr Tyr Glu Met Pro Lys Met

610 615 620

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

625 630 635 640

Ala Lys Ala Phe Asp Arg Leu Tyr His Pro Ser Asp Phe Glu Val Tyr

645 650 655

Pro His Ser Ala Thr Gly Leu Leu Gly Ser Pro Val Thr Gly Ala Leu

660 665 670

Lys Asn Pro Met Ala Met Arg Val Leu Tyr Ser Leu Arg Arg Glu Val

675 680 685

Asn Ala Leu Leu Lys Lys Gly Ile Ile Asp Ala Asp Thr Arg Ile Val

690 695 700

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

705 710 715 720

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

725 730 735

Ile Leu Val Glu Leu Leu Gly Lys Arg Asp Tyr Ser Asp Thr Glu Val

740 745 750

Asp Lys Thr Arg Phe Leu Leu Glu Gln His Asp Ile Leu Asp Val Ala

755 760 765

Ala Met Pro Gln Ala Glu Lys Lys Thr Lys Lys Asn Thr Glu Lys Gln

770 775 780

Lys Ala Glu Lys Phe Asp Arg Asp Thr Thr Lys Tyr Arg Leu Trp Leu

785 790 795 800

Glu Gln Gly Cys Arg Cys Ile Tyr Thr Gly Lys Val Ile Asn Ile Thr

805 810 815

Ser Leu Phe Asp Asp Asn Leu Tyr Glu Ile Glu His Thr Ile Pro Arg

820 825 830

Ser Leu Ser Phe Asp Asn Ser Gln Ala Asn Leu Thr Val Cys Asp Ala

835 840 845

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

850 855 860

Asn Tyr Asp Glu Ile Tyr Met Arg Ile Thr Pro Trp Val Glu Lys Val

865 870 875 880

Glu Gln Leu Ala Asp Arg Val Lys Ala Trp Thr Asp Ala Ala Lys Arg

885 890 895

Ala Gln Asp Lys Asp Arg Lys Asp Tyr Cys Ile Arg Gln Arg His Gln

900 905 910

Trp Gln Met Glu Leu Asp Tyr Trp Arg Asp Lys Val Asn Arg Phe Thr

915 920 925

Met Thr Glu Val Thr Asp Gly Phe Arg Asn Ser Gln Leu Val Asp Thr

930 935 940

Arg Ile Ile Thr Lys Tyr Ala Tyr His Phe Leu Lys Thr Val Phe Glu

945 950 955 960

Arg Val Asp Val Gln Lys Gly Ala Val Thr Ala Asp Phe Arg Lys Met

965 970 975

Leu Gly Val Gln Ser Leu Glu Glu Lys Lys Asn Arg Asp Arg His Ser

980 985 990

His His Ala Ile Asp Ala Thr Val Leu Thr Leu Val Pro Ala Ala Asn

995 1000 1005

Val Arg Asp Arg Leu Leu Gln Leu Phe Tyr Glu Lys Glu Glu Ala

1010 1015 1020

Glu Arg Phe Ser Gly Asp Ala Gly Leu Lys Glu Arg Ala Phe Leu

1025 1030 1035

Ser Glu Leu Lys Arg Leu Asn Phe Lys Gly Val Ser Arg Leu Pro

1040 1045 1050

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

1055 1060 1065

Arg Ala Leu Val Pro Ala Ser Arg Lys Leu Arg Arg Arg Gly Lys

1070 1075 1080

Glu Val Leu Phe Asp Gly Lys Asn Arg Arg Met Thr Gly Asp Cys

1085 1090 1095

Ile Arg Gly Gln Leu His Gly Asp Thr Phe Tyr Gly Ala Ile Arg

1100 1105 1110

Gln Tyr Cys Phe Asp Asp Asp His Arg Val Ile Arg Asp Ala Asp

1115 1120 1125

Gly Lys Pro Cys Glu Thr Asp Val Met Tyr Val Val Arg Lys Glu

1130 1135 1140

Phe Arg Ala Lys Gly Asn Ser Gly Asp Ala Gly Gly Phe Ala Ser

1145 1150 1155

Trp Ala Asp Val Glu Lys Ser Ile Val Asp Lys Ala Leu Tyr Arg

1160 1165 1170

Met Met Arg Ser Gln Phe Ala Asp Asp Val Ser Phe Lys Asp Ala

1175 1180 1185

Tyr Ala Gln Gly Ile Tyr Met Leu Asp Arg Ala Gly Arg Arg Val

1190 1195 1200

Asn Lys Ile Arg His Ile Arg Cys Trp Ala Ser Arg Val Asn Asn

1205 1210 1215

Pro Leu Val Val Arg Lys His Thr His Leu Ser Asp Arg Glu Tyr

1220 1225 1230

Lys Gln Asn Tyr Tyr Ala Val Asn Gly Glu Asn Thr Tyr Leu Ala

1235 1240 1245

Ile Tyr Trp Asp Gly Val Ser Asn Asn Asn Asp Phe Asp Cys Lys

1250 1255 1260

Ser Leu Met Asp Leu Ala Arg Met Lys Pro Thr Asp Arg Asn Pro

1265 1270 1275

Leu His Phe Phe Pro Pro Gln Lys Lys Val Gly Lys Gly Lys Lys

1280 1285 1290

Ala Val Phe Met Pro Leu Tyr Ala Val Leu Lys Pro Gly Thr Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

Gln Leu Met Arg Arg Leu Tyr His Met Val Gly Phe Asp Ser Gly

1340 1345 1350

Asp Gly Arg Ile Gln Phe Lys Tyr His Leu Glu Ala Arg Asn Asp

1355 1360 1365

Asn Arg Leu Met Glu Ala Phe Pro Glu Asn Lys Tyr Gly Lys Val

1370 1375 1380

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

1385 1390 1395

Lys Leu Arg Leu Ser Arg Ser Asn Tyr Tyr Phe Leu Ile Glu Gly

1400 1405 1410

Lys Asp Phe Glu Ile Asp Gly Asp Thr Ile Val Phe Lys

1415 1420 1425

<210> 4

<211> 1314

<212> PRT

<213> C4640蛋白序列

<400> 4

Met Glu Asn Tyr Tyr Leu Gly Leu Asp Leu Gly Thr Asp Ser Ile Gly

1 5 10 15

Trp Ala Val Thr Asp Lys Asn Tyr Asn Ile Pro Lys Phe Lys Gly Asn

20 25 30

Ser Met Trp Gly Ile Arg Leu Leu Glu Gly Gly Asn Thr Ala Val Glu

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Asp Lys Asn Val Ser Gly Lys Tyr Ser Leu Phe Asn Asp Pro Asp Tyr

100 105 110

Thr Asp Lys Asp Tyr Tyr Lys Lys Tyr Pro Thr Ile Tyr His Leu Arg

115 120 125

Lys Glu Leu Ile Glu Ser Val Glu Pro His Asp Val Arg Leu Val Phe

130 135 140

Leu Ala Leu His His Ile Ile Lys Asn Arg Gly His Phe Leu Phe Asp

145 150 155 160

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

165 170 175

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

180 185 190

Gly Phe Ser Cys Asn Asp Leu Lys Pro Val Glu Asp Ile Ile Lys Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Tyr Asp Asp Thr Glu Gly Lys Ser Ile Cys Phe Lys Ser Gly Tyr Asp

260 265 270

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

275 280 285

Ile Glu Lys Leu Lys Ala Ile Tyr Asp Trp Ala Ile Leu Ala Asp Ile

290 295 300

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

305 310 315 320

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

325 330 335

Val Pro Glu Lys Tyr Ser His Ile Phe Asn Glu Asn Ser Asp Lys Val

340 345 350

Ser Asn Tyr Leu Ser Tyr Ser Gly Tyr Ser Ser Lys Asn Pro Val Met

355 360 365

Lys Lys Cys Asp Gln Glu Thr Phe Cys Asp Phe Leu Arg Lys Gln Leu

370 375 380

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

385 390 395 400

Glu Thr Ser Ser Phe Met Pro Lys Ala Val Thr Lys Asp Asn Ser Val

405 410 415

Ile Pro Met Gln Leu Asn Arg Ala Glu Leu Glu Ala Ile Leu Ser Asn

420 425 430

Ala Lys Asn Tyr Leu Pro Phe Leu Ala Asp Arg Asp Ser Ser Gly Lys

435 440 445

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

450 455 460

Tyr Val Gly Pro Leu Asn Lys His Ser Glu Lys Ser Trp Leu Val Arg

465 470 475 480

Thr Gly Glu Lys Ile Tyr Pro Trp Asn Phe Glu Asn Val Val Asp Ala

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Asn Gly Tyr Met Asn Ile Glu Ile Thr Gly Ile Asp Thr Thr Ile Lys

580 585 590

Gly Ser Leu Lys Pro Phe Ile Asp Leu Gln Asn Ile Asp Leu Ser Tyr

595 600 605

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

610 615 620

Lys Lys Leu Leu Lys Asn Arg Leu Lys Arg Leu Tyr Gly Asp Arg Leu

625 630 635 640

Thr Ala Asp Asp Ile Lys Lys Ile Ser Lys Leu Lys Tyr Thr Gly Trp

645 650 655

Ser Arg Leu Ser Glu Lys Leu Leu Thr Gly Ile Glu Ala Val Leu Pro

660 665 670

Ser Thr Gly Glu Tyr Thr Asn Ile Ile His Ala Leu Trp Glu Thr Asn

675 680 685

Asp Asn Leu Met Gln Leu Leu Ser Ser Asn Tyr Asp Phe Arg Lys Lys

690 695 700

Ile Asp Glu Glu Asn Gly Asp Ser Thr Phe Thr Ser Leu Arg Glu Glu

705 710 715 720

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

725 730 735

Ala Met Gln Ile Val Glu Glu Ile Val Lys Ile Gln Gly His Asp Pro

740 745 750

Lys Lys Ile Phe Ile Glu Val Ala Arg Asp Glu Gly Glu Lys Lys Arg

755 760 765

Thr Val Ser Arg Lys Gln Lys Leu Ile Glu Leu Tyr Lys Tyr Cys Lys

770 775 780

Lys Asp Glu Gln Lys Leu Tyr Glu Gln Leu Cys Asn Thr Asp Glu Asn

785 790 795 800

Asp Phe Arg Arg Asp Ala Leu Tyr Leu Tyr Tyr Thr Gln Leu Gly Lys

805 810 815

Cys Met Tyr Thr Gly Lys Pro Ile Glu Leu Ser Glu Ile Tyr Asn Asn

820 825 830

Asn Ile Tyr Asp Ile Asp His Ile Phe Pro Arg Ser Lys Ile Lys Asp

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Phe Glu Arg Leu Val Arg Asn Gln Pro Leu Thr Asp Glu Glu Leu Ser

900 905 910

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

915 920 925

Val Ala Gln Leu Leu Lys Lys Arg Tyr His Asp Thr Ala Val Glu Tyr

930 935 940

Ile Lys Ala Ser Leu Val Ser Asp Phe Arg Gln Glu Asn Asp Phe Val

945 950 955 960

Lys Ser Arg Asp Val Asn Asp Phe His His Ala Lys Asp Ala Tyr Leu

965 970 975

Asn Ile Val Val Gly Asn Val Tyr Thr Val Arg Ser His Leu Ala His

980 985 990

Phe Ile Asp Asn Val Gln Ser Gly Lys Trp Ser Val Asn Lys Met Phe

995 1000 1005

Asp Tyr Thr Thr Asn Gly Ala Trp Ile Val Glu Asn Asn Lys Ser

1010 1015 1020

Ile Asn Ile Val Lys Ser Asn Met Ala Lys Asn Asn Ile Arg Phe

1025 1030 1035

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

1040 1045 1050

Pro Leu Lys Lys Gly Leu Gly Gln Val Pro Arg Lys Lys Asp Met

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Ser Phe Glu Pro Val Asp Leu Tyr Ala Glu Lys Glu Tyr Ile Arg

1100 1105 1110

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

1115 1120 1125

Lys Ile Ile Ile Pro Cys Val Lys Tyr Asn Ala Leu Ile Ser Ile

1130 1135 1140

Asn Gly Phe Arg Met His Ile Ser Ser Lys Ser Ser Gly Gly Ala

1145 1150 1155

Asn Leu Ile Cys Lys Pro Ala Val Gln Leu Val Val Ser Gln Glu

1160 1165 1170

Gln Glu Lys Tyr Ile Lys Lys Ile Ser Ser Tyr Leu Ala Lys Cys

1175 1180 1185

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

1190 1195 1200

Thr Glu Lys Asp Asn Ala Asp Leu Tyr Glu Ala Leu Lys His Lys

1205 1210 1215

Thr Asn His Thr Ile Tyr Asn Val Lys Phe Ser Lys Leu Ala Asn

1220 1225 1230

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

1235 1240 1245

Glu Gln Cys Tyr Ile Leu Met Gln Ile Ile Asn Ile Leu His Ala

1250 1255 1260

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

1265 1270 1275

Ser Gly Val Thr Thr Ile Ser Asn Lys Met Gln Pro Ser Tyr Lys

1280 1285 1290

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

1295 1300 1305

Glu Ile Asp Leu Leu Lys

1310

<210> 5

<211> 1151

<212> PRT

<213> C6165蛋白序列

<400> 5

Met Lys Arg Asn Gly Thr Thr Ala Glu Gln Lys Ala Glu Asn Tyr Arg

1 5 10 15

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

20 25 30

Ile Val Leu Gln Lys Ile Asn Gly Gln Ile His Gly Thr Ser Gly Tyr

35 40 45

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

50 55 60

Thr Val Gly Gln Tyr Gln Met Gln Leu Leu Glu Lys Asp Pro Asn Thr

65 70 75 80

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

85 90 95

Glu Arg Ile Trp Glu Thr Gln Ala Thr Tyr His Pro Glu Leu Thr Ala

100 105 110

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

115 120 125

Leu Lys Ser Gln Lys Ser Leu Val Gly Tyr Cys Glu Leu Glu Ser Lys

130 135 140

Pro Lys Glu Val Val Val Asp Gly Lys Lys Lys Thr Ile Thr Thr Gly

145 150 155 160

Leu Arg Val Cys Pro Lys Ser Ser Pro Leu Phe Gln Ser Phe Lys Ile

165 170 175

Trp Gln Thr Leu Asn Asn Val Gln Val Ser Gly Asn Ile Ile Pro Glu

180 185 190

Lys Gln Leu Asp Leu Phe Gly Thr Ala Ala Thr Tyr Lys Tyr Gly Ser

195 200 205

Arg Cys Leu Thr Glu Glu Glu Lys Gln Thr Leu Tyr Lys Glu Leu Ser

210 215 220

Leu Lys Glu Arg Met Ser Ala Ala Glu Ile Leu Lys Leu Leu Phe Lys

225 230 235 240

Ser Gly Lys Gly Leu Ser Leu Asn Phe Arg Glu Val Asp Gly Asn His

245 250 255

Thr Met Ala Ala Phe Val Lys Ala Cys Gln Thr Ile Ile Val Met Ser

260 265 270

Gly His Asn Glu Tyr Asp Phe Ala Lys Leu Ser Tyr Glu Thr Val Val

275 280 285

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

290 295 300

Leu Asn Phe Asp Pro Cys Leu Glu Gly Lys Ala Phe Glu His Gln Pro

305 310 315 320

Ala Tyr Gln Leu Trp His Leu Leu Tyr Ser Tyr Ala Gly Asp Asn Ser

325 330 335

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

340 345 350

Met Glu His Asn Tyr Ala Ala Val Phe Ala Thr Ile Thr Phe Met Pro

355 360 365

Asp Tyr Gly Asn Leu Ser Ala Lys Ala Met Arg Arg Ile Leu Pro Tyr

370 375 380

Met Met Asp Gly Asn Glu Tyr Ser Val Ala Cys Glu Tyr Ala Gly Tyr

385 390 395 400

Arg His Ser Arg Arg Ser Leu Thr Lys Glu Glu Leu Asp Lys Lys Pro

405 410 415

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

420 425 430

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

435 440 445

Ser Ala Gln Tyr Gly Lys Pro Asp Glu Ile Arg Ile Glu Met Ala Arg

450 455 460

Glu Leu Lys Lys Ser Ala Lys Glu Arg Glu Gln Met Thr Ala Asp Ile

465 470 475 480

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

485 490 495

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

500 505 510

Leu Trp Leu Glu Leu Lys Asp Asn Gly Tyr Lys Thr Leu Tyr Ser Gln

515 520 525

Thr Tyr Ile Pro Arg Glu Lys Leu Phe Ser Lys Glu Phe Asp Val Glu

530 535 540

His Ile Ile Pro Gln Ala Arg Leu Phe Asp Asp Ser Phe Ser Asn Lys

545 550 555 560

Thr Leu Glu Ala Arg Gln Ala Asn Leu Glu Lys Ser Asn Ala Thr Ala

565 570 575

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

580 585 590

Lys Glu Arg Ile Asp Tyr Leu Tyr Ala Asp Gly Val Ile Ser Lys Thr

595 600 605

Lys His Asp Lys Leu Leu Met Lys Glu Ala Asp Ile Pro Glu Gly Phe

610 615 620

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

625 630 635 640

Glu Ile Leu Glu Ser Met Val Arg Val Val Val Pro Thr Thr Gly Ala

645 650 655

Val Thr Asp Arg Leu Arg Asp Asp Trp Gln Leu Val Asp Ile Met Lys

660 665 670

Glu Leu Asn Trp Asp Lys Tyr Asn Lys Leu Gly Leu Thr Glu Thr Phe

675 680 685

Lys Asp His Asp Gly Arg Gln Ile Lys Arg Ile Lys Asp Trp Thr Lys

690 695 700

Arg Asn Asp His Arg His His Ala Met Asp Ala Leu Thr Ile Ala Phe

705 710 715 720

Thr Gln His Ser Phe Ile Gln Tyr Leu Asn Asn Leu Asn Ala Arg Ser

725 730 735

Asn Lys Ser Gly Ser Ile Tyr Ala Ile Glu Gln Thr Cys Leu Tyr Arg

740 745 750

Asp Ala His Gly Lys Leu Arg Phe Met Pro Pro Met Pro Leu Asp Val

755 760 765

Phe Arg Ala Glu Ala Arg Arg Gln Leu Gln Asp Ile Leu Val Ser Thr

770 775 780

Lys Ala Lys Asn Lys Val Val Thr Arg Asn Ile Asn Ser Ile Lys Arg

785 790 795 800

Arg Gly Glu Lys Lys Gln Thr Val Gln Leu Thr Pro Arg Cys Gln Leu

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Lys Thr Val Thr Tyr Glu Thr Ile Phe Thr Gln Arg Lys Pro Ile Asp

900 905 910

Lys Asp Leu Lys Val Asp Lys Val Ile Asp Glu His Val Lys Glu Ile

915 920 925

Leu Glu Ala Arg Leu Lys Glu Phe Gly Gly Asp Ala Lys Lys Ala Phe

930 935 940

Ser Asn Leu Glu Glu Asn Pro Ile Trp Leu Asn Arg Glu Arg Gly Ile

945 950 955 960

Gln Ile Lys Arg Val Thr Ile Arg Gly Val Ser Asn Ala Val Ala Leu

965 970 975

His Asp Lys His Asp Val Gln Gly Lys Pro Met Cys Asp Ser Glu Gly

980 985 990

Arg Arg Met Pro Ser Asp Tyr Val Ser Thr Ser Asn Asn His His Val

995 1000 1005

Ala Ile Phe Arg Asp Ala Asn Gly Asn Leu Gln Glu His Val Val

1010 1015 1020

Ser Tyr Phe Glu Ala Thr Ala Arg Ala Ile Gln His Leu Pro Ile

1025 1030 1035

Val Asp Arg Asp Tyr Asn Lys Asp Glu Gly Trp Gln Phe Leu Phe

1040 1045 1050

Thr Met Lys Arg Asn Glu Tyr Phe Val Phe Pro Asn Glu Lys Thr

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Lys Asn Tyr Phe Phe Arg His His Leu Glu Thr Asn Val Glu Thr

1100 1105 1110

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

1115 1120 1125

Lys Gly Ile Ala Gly Ile Val Lys Val Arg Val Asn Asn Ile Gly

1130 1135 1140

Gln Ile Val Ala Val Gly Glu Tyr

1145 1150

<210> 6

<211> 1372

<212> PRT

<213> Lt1Cas9蛋白序列

<400> 6

Met Cys Thr Lys Glu Ser Glu Lys Leu Asn Lys Asn Ala Asp Tyr Tyr

1 5 10 15

Ile Gly Leu Asp Met Gly Thr Ser Ser Ala Gly Trp Ala Val Ser Asp

20 25 30

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

35 40 45

Arg Gln Phe Glu Glu Ala Lys Thr Ala Ala Glu Arg Arg Gly Phe Arg

50 55 60

Val Ala Arg Arg Arg Lys Gln Arg Gln Gln Val Arg Asn Arg Leu Leu

65 70 75 80

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

85 90 95

Lys Arg Met Glu Asp Ser Arg Phe Val Ile Ser Asp Lys Arg Val Pro

100 105 110

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

115 120 125

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

130 135 140

Ser Asn Glu Arg Phe Asp Ile Arg Leu Val Phe Leu Gly Ile His Ser

145 150 155 160

Leu Phe Gln His Pro Gly His Phe Leu Asp Lys Gly Asp Val Asp Thr

165 170 175

Asp Asn Thr Gly Pro Glu Glu Leu Ile Gln Phe Leu Glu Asp Cys Met

180 185 190

Asn Glu Ile Gln Ile Ser Ile Pro Leu Val Ser Asn Gln Lys Val Leu

195 200 205

Thr Asp Ile Leu Thr Asp Ser Arg Ile Thr Arg Arg Asp Lys Glu Gln

210 215 220

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

225 230 235 240

Ser Gln Phe Val Lys Val Leu Thr Gly Gln Lys Ala Lys Leu Gly Asp

245 250 255

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

260 265 270

Ser Phe Arg Glu Lys Thr Leu Glu Glu Ile Leu Pro Asp Ile Glu Gly

275 280 285

Val Ile Asp Gly Leu Ala Leu Glu Tyr Ile Glu Ser Ile Tyr Ser Leu

290 295 300

Tyr Ser Trp Ser Leu Leu Asn Ser Tyr Met Lys Asp Thr Leu Thr Gly

305 310 315 320

His Tyr Tyr Ser Tyr Leu Ala Glu Ala Arg Val Ala Ala Tyr Asp Lys

325 330 335

His His Ser Asp Leu Val Lys Leu Lys Thr Leu Phe Arg Glu Tyr Ile

340 345 350

Pro Glu Glu Tyr Asp Asn Phe Phe Arg Lys Met Glu Lys Ala Asn Tyr

355 360 365

Ser His Tyr Ile Gly Ser Thr Glu Tyr Asp Gly Glu Lys Arg Cys Arg

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Phe Val Pro Asn Gln Leu Gln Leu Lys Glu Leu Arg Lys Ile Leu Gln

435 440 445

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

450 455 460

Met Thr Ala Ile Asp Arg Ile Glu Ala Leu Phe Ser Phe Arg Ile Pro

465 470 475 480

Tyr Tyr Ile Gly Pro Leu Lys Asn Thr Asp Asn Gln Gly His Gly Trp

485 490 495

Ala Val Arg Arg Asp Gly His Glu Gln Ile Pro Val Arg Pro Trp Asn

500 505 510

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

515 520 525

Asn Leu Val Asn Ser Cys Thr Tyr Leu Arg Thr Glu Lys Val Leu Pro

530 535 540

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

545 550 555 560

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

565 570 575

Lys Arg Met Ile Phe Glu Gln Cys Phe Tyr Ser Gly Lys Lys Val Thr

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Asn Asp Thr Gln Lys Val Met Ala Glu Gln Ile Ile Glu Trp Ser Thr

645 650 655

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

660 665 670

Tyr Pro Glu Leu Thr Asp Gln Gln Ile Arg Arg Ile Ala Gly Phe Lys

675 680 685

Phe Ser Glu Trp Gly Asn Leu Ser Arg Ala Phe Leu Glu Met Glu Gly

690 695 700

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

705 710 715 720

Asp Thr Gln Lys Asn Leu Met Gln Leu Leu Ser Asn Asp Ser Ala Phe

725 730 735

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

740 745 750

Ser Ile Glu Pro Asp Asp Leu Asp Gly Met Tyr Leu Ser Ala Pro Val

755 760 765

Arg Arg Met Ile Trp Gln Thr Phe Leu Ile Leu Arg Glu Val Val Asp

770 775 780

Thr Ile Gly Tyr Ser Pro Lys Lys Ile Phe Met Glu Met Ala Arg Gly

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Ser Lys Asp Phe Asn Asn His Ile Lys Gln Asp Val Tyr Pro Ile Pro

900 905 910

Glu Gln Ile Gln Ser Arg Gln Lys Gly Phe Trp Ala Met Leu Leu Lys

915 920 925

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

930 935 940

Pro Phe Thr Asp Glu Glu Leu Ala Glu Phe Val Asn Arg Gln Leu Val

945 950 955 960

Glu Thr Arg Gln Gly Thr Lys Ala Ile Ile Ser Leu Ile Asn Gln Cys

965 970 975

Phe Pro Asp Ser Glu Val Val Tyr Val Lys Ala Gly Asn Thr Ser Asp

980 985 990

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

995 1000 1005

His His Ala Val Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Val

1010 1015 1020

Tyr Asp Thr Lys Phe Thr Lys Asn Pro Ile Asn Phe Ile Lys Lys

1025 1030 1035

Met Arg Lys Ser Gly Asn Leu His Ser Tyr Ser Leu Arg Arg Met

1040 1045 1050

Tyr Asp Phe Asn Val Gln Arg Gly Asp Gln Thr Ala Trp Val Ala

1055 1060 1065

Glu Asn Asp Thr Thr Leu Lys Thr Val Lys Lys Thr Ala Phe Lys

1070 1075 1080

Thr Ser Pro Met Val Thr Lys Arg Thr Tyr Glu Arg Lys Gly Gly

1085 1090 1095

Leu Ala Asp Ser Val Leu Ile Ala Ala Lys Lys Ala Lys Pro Gly

1100 1105 1110

Val His Leu Pro Val Lys Thr Ser Asp Ser Arg Phe Ala Asn Gln

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

Glu Asn Val Pro Ile His Leu Lys Glu Lys Leu Lys Thr Lys Glu

1160 1165 1170

Glu Leu Glu His Tyr Cys Ala Gln Val Leu Gly Met Val Gln Pro

1175 1180 1185

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

1190 1195 1200

Asp Gly Tyr Tyr Tyr Tyr Leu Thr Gly Arg Thr Gly Gly Asn Leu

1205 1210 1215

Ser Leu Ser Asn Ala Val Glu Leu Cys Leu Pro Ala Lys Glu Gln

1220 1225 1230

Ala His Ile Arg Met Ile Ser Lys Ile Ala Gly Gly Arg Ser Thr

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Phe Ser Lys Arg Lys Asn Pro Ile Gly Pro Lys Leu Leu Lys Tyr

1280 1285 1290

Arg Glu Ala Phe Val Lys Gln Thr Ile Glu Asn Gln Cys Lys Val

1295 1300 1305

Ile Leu Gln Ile Leu Lys Leu Thr Ser Thr Asn Cys Lys Thr Ser

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Leu Ile Cys Gln Ser Pro Ser Gly Ile Tyr Glu Thr Arg Lys Asn

1355 1360 1365

Leu Leu Thr Ile

1370

<210> 7

<211> 1351

<212> PRT

<213> Lt2Cas9蛋白序列

<400> 7

Met Lys Lys Gln Phe Gly Glu Tyr Tyr Leu Ala Phe Asp Ile Gly Thr

1 5 10 15

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

20 25 30

Leu Asn Gly Lys Tyr Met Trp Gly Thr Arg Leu Phe Glu Ala Gly Lys

35 40 45

Thr Ala Ala Glu Arg Arg Ser Phe Arg Ala Ala Arg Arg Arg Leu Gln

50 55 60

Arg Arg Thr Gln Arg Ile Arg Leu Leu Gln Glu Ile Phe Ala Glu Glu

65 70 75 80

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

85 90 95

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

100 105 110

Ala Asn Phe Lys Asp Lys Asp Tyr His Ala Lys Phe Pro Thr Ile Phe

115 120 125

His Leu Arg Lys Thr Leu Ile Glu Ser Lys Glu Lys Phe Asp Val Arg

130 135 140

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

145 150 155 160

Leu Phe Glu Gly Gln Arg Met Glu Asn Val Thr Ser Phe Asp Ala Ala

165 170 175

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

180 185 190

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

195 200 205

Leu Gly Val Lys Asp Lys Asn Lys Tyr Leu Asn Asp Leu Leu Gly Ala

210 215 220

Asp Thr Lys Gln Lys Lys Glu Met Val Asn Leu Ile Cys Gly Gly Thr

225 230 235 240

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

245 250 255

Ile Asp Lys Leu Cys Phe Lys Thr Met Asp Tyr Ala Asp Ile His Asp

260 265 270

Lys Leu Leu Asp Thr Ile Gly Asp Leu Ser Lys Ile Glu Cys Leu Asp

275 280 285

Arg Leu Lys Ser Ile Phe Asp Trp Ser Leu Leu Ala Glu Ile Lys Lys

290 295 300

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

305 310 315 320

Lys Gln Asp Ile Lys Leu Leu Lys Ser Leu Ile Lys Lys Tyr Tyr Asp

325 330 335

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

340 345 350

Tyr Val Ala Tyr Val Gly Met Thr Lys Lys Asn Asn Lys Lys Gln Val

355 360 365

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

370 375 380

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

385 390 395 400

Lys Ser Lys Cys Ala Asn Val Ser Phe Ala Pro Lys Gln Ile Asn Arg

405 410 415

Asp Asn Gly Val Ile Pro Tyr Gln Met His Leu Leu Glu Leu Glu Lys

420 425 430

Ile Leu Glu Asn Ala Lys Arg Tyr Leu Pro Phe Leu Asn Glu Lys Asp

435 440 445

Ala Ser Gly Tyr Thr Ala Ala Glu Lys Ile Val Lys Ile Met Thr Phe

450 455 460

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

465 470 475 480

Asp Gly Gln Lys Gly His Cys Trp Ile Val Lys Asn Ser Asp Glu Lys

485 490 495

Ile Arg Pro Trp Asn Phe Glu Arg Val Val Asp Gln Glu Ala Cys Ala

500 505 510

Glu Ala Phe Ile Arg Arg Met Thr Asn Lys Cys Thr Tyr Met His Asp

515 520 525

Ala Asp Val Leu Pro Lys Asp Ser Leu Leu Tyr Ser Lys Phe Val Val

530 535 540

Leu Asn Glu Leu Asn Asn Val Cys Val Asn Gly Asp Arg Leu Pro Thr

545 550 555 560

Asp Leu Lys Gln His Ile Tyr Arg Glu Leu Phe Met Gln Gln Lys Lys

565 570 575

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

580 585 590

Arg Glu Thr Asp Ser Leu Ser Gly Phe Asp Gly Asp Phe Lys Gly Ser

595 600 605

Leu Gly Ser Leu Ile Asp Phe Arg Lys Ile Ile Gly Val Lys Ala Gly

610 615 620

Asp Asn Lys Met Val Glu Glu Ile Ile Lys Trp Ile Val Leu Phe Gly

625 630 635 640

Asp Thr Lys Lys Leu Leu Lys Asp Arg Ile Asn Lys Phe Tyr Gly Asp

645 650 655

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

660 665 670

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

675 680 685

Ile Pro Gly Phe Ala Asn Glu Leu Gly Ile Ile Thr Ala Met Tyr Glu

690 695 700

Thr Pro Asn Asn Leu Met Glu Leu Leu Ser Gly Gln Tyr Gln Tyr Leu

705 710 715 720

Asp Lys Leu Gln Ala Tyr Asn Asn Thr Met His Glu Ala Thr Gly Ser

725 730 735

Leu Thr Tyr Glu Thr Val Ala Asp Leu Tyr Val Ser Pro Ala Val Lys

740 745 750

Arg Ser Ile Trp Gln Thr Leu Val Leu Ala Glu Glu Ile Lys Asn Val

755 760 765

Met Gly His Glu Pro Lys Lys Val Phe Ile Glu Met Thr Arg Ser Asp

770 775 780

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

785 790 795 800

Leu Tyr Lys Ala Cys Lys Gly Glu Ala Arg Asp Trp Leu Val Glu Leu

805 810 815

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

820 825 830

Tyr Thr Gln Met Gly Arg Cys Met Tyr Thr Gly Glu Arg Ile Glu Ile

835 840 845

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

850 855 860

Glu His Ile Phe Pro Gln Ser Lys Ile Lys Asp Asp Ser Ile Asp Asn

865 870 875 880

Arg Val Leu Val Lys Ala Gly Ala Asn Arg Ala Lys Gly Asp Gln Tyr

885 890 895

Pro Val Pro Gln Glu Tyr Arg Asn Lys Cys Gly Gly Ile Trp Lys Met

900 905 910

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

915 920 925

Arg Asn Thr Pro Phe Ser Glu Asp Glu Leu Ala Gly Phe Ile Ala Arg

930 935 940

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

945 950 955 960

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

965 970 975

Val Ser Ala Phe Arg Gln Gln Asn Asp Phe Val Lys Cys Arg Glu Ile

980 985 990

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

995 1000 1005

Asn Val Tyr Asn Thr Lys Phe Thr His Ser Pro Ile Asn Phe Ile

1010 1015 1020

Lys Ser Lys Asn Asn Lys Tyr Ser Leu Asn Lys Met Tyr Asp Phe

1025 1030 1035

Lys Val Glu Arg Asn Gly Ser Val Ala Trp Glu Pro Gly Asp Asn

1040 1045 1050

Gly Thr Ile Val Thr Val Lys Arg Thr Met His Asn Asn Arg Val

1055 1060 1065

Leu Phe Thr Arg Tyr Ala Ser Gln Ala Lys Gly Lys Leu Phe Glu

1070 1075 1080

Val Thr Leu Leu Lys Lys Gly Lys Gly Lys His Pro Ile Lys Ser

1085 1090 1095

Asp Met Asn Asp Pro Ile Ser Asn Ile Glu Asn Tyr Gly Gly Tyr

1100 1105 1110

Asp Ser Ile Phe Gly Ala Tyr Phe Phe Leu Val Glu His Thr Leu

1115 1120 1125

Lys Asn Lys Arg Ile Arg Thr Ile Glu Tyr Val Pro Ile Leu Lys

1130 1135 1140

Ala Ala Met Leu Gln Gly Asn Ala Ala Ala Leu Leu Gln Tyr Cys

1145 1150 1155

Leu Asp Asp Leu Asn Leu His Glu Pro Arg Ile Leu Leu Ala Glu

1160 1165 1170

Ile Lys Phe Asn Thr Leu Phe Lys Val Asp Gly Phe Lys Met His

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Leu Thr Asp Phe Asp Lys Ile Asp Lys Asp Asn Asn Val Glu Leu

1235 1240 1245

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

1250 1255 1260

Arg Leu Gly Glu Gln Ile Lys Lys Leu Glu Lys Gly Arg Glu Ile

1265 1270 1275

Phe Ile Asn Leu Gly Ile Glu Glu Gln Val Ile Thr Leu Phe Glu

1280 1285 1290

Ile Leu His Leu Phe Gln Cys Asn Arg Ile Lys Ser Asn Leu Thr

1295 1300 1305

Lys Val Gly Gly Ser Ala Asn Ala Gly Thr Leu Leu Thr Ala Lys

1310 1315 1320

Lys Ile Ser Val Leu Lys Lys Ile Ser Ile Ile Asn Gln Ser Pro

1325 1330 1335

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

1340 1345 1350

<210> 8

<211> 1353

<212> PRT

<213> Lt3Cas9蛋白序列

<400> 8

Met Gln Thr Lys Lys Val Asp Glu Tyr Tyr Val Gly Phe Asp Ile Gly

1 5 10 15

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

20 25 30

Lys His Gly Gly Glu Pro Met Trp Gly Ser His Val Phe Glu Ala Ala

35 40 45

Ser Thr Ala Gln Glu Arg Arg Thr Phe Arg Thr Ala Arg Arg Arg Asn

50 55 60

Asp Arg Lys Lys Gln Arg Ile Ala Leu Val Ser Glu Ile Phe Ala Pro

65 70 75 80

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

85 90 95

Ala Leu Phe Arg Asp Asp Val Asp Ile Lys Asp Arg Tyr Val Val Phe

100 105 110

Asn Asp Asp Asp Phe Thr Asp Lys Asp Tyr Tyr Asp Ile Tyr Pro Thr

115 120 125

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

130 135 140

Ile Arg Leu Val Tyr Met Ala Cys Ala Tyr Leu Val Ala His Arg Gly

145 150 155 160

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

165 170 175

Phe Asp Val Val Tyr Cys Asn Phe Leu Asn Val Met Asp Asn Tyr Ala

180 185 190

Glu Ile Pro Trp Lys Cys Asp Ile Ser Lys Phe Lys Glu Ile Leu Lys

195 200 205

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

210 215 220

Asn Glu Gly Lys Lys Phe Lys Thr Ser Glu Glu Asp Asp Val Ser Arg

225 230 235 240

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

245 250 255

Leu Phe Pro Lys Leu Thr Phe Glu Glu Lys Val Ser Val Ser Phe Asn

260 265 270

Met Ala Glu Glu Asp Phe Ala Met Val Leu Gln Gln Leu Gly Asp Glu

275 280 285

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

290 295 300

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

305 310 315 320

Val Tyr Glu Gln His Lys Lys Asp Leu Ser Phe Leu Lys Tyr Phe Val

325 330 335

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

340 345 350

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

355 360 365

Asn Val Ser Lys Phe Lys Gly Ala Lys Lys Asp Val Phe Cys Asp Tyr

370 375 380

Ile Lys Lys Val Val Lys Asp Ile Lys Val Asp Asp Glu Asp Lys Val

385 390 395 400

Glu Tyr Glu Asp Met Met Phe Arg Leu Asp Thr Tyr Ser Phe Ile Pro

405 410 415

Lys Gln Val Glu Asn Asp Asn Arg Val Ile Pro Tyr Gln Leu Tyr Tyr

420 425 430

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

435 440 445

Leu Asp Glu Lys Asp Met Asp Gly Tyr Thr Ser Arg Glu Lys Leu Leu

450 455 460

Ser Ile Met Glu Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Arg Thr

465 470 475 480

Asp Asn Gly Gln His Gly Trp Met Lys Arg Lys Ala Glu Gly Arg Ile

485 490 495

Tyr Pro Trp Asn Phe Glu Asp Lys Val Asp Leu Asp Ala Ser Glu Gln

500 505 510

Glu Phe Ile Asn Arg Met Thr Asn Ser Cys Thr Tyr Leu Pro Gly Glu

515 520 525

Thr Val Val Pro Lys Tyr Ser Leu Leu Tyr Cys Lys Phe Asn Val Leu

530 535 540

Asn Glu Ile Asn Asn Ile Lys Ile Asn Asp Cys Ser Ile Pro Ile Glu

545 550 555 560

His Lys Gln Gly Ile Tyr Lys Leu Phe Glu Arg Tyr Arg Lys Val Thr

565 570 575

Pro Lys Lys Ile Lys Asp Phe Leu Ile Ser Asn Asn Leu Leu His Pro

580 585 590

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

595 600 605

Ser Tyr His Asp Phe Lys Lys Leu Leu Glu Ser Cys Val Leu Lys Glu

610 615 620

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

625 630 635 640

Gly Arg Ile Leu Arg Trp Leu His Met Glu Phe Pro Asp Leu Ser Asp

645 650 655

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

660 665 670

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

675 680 685

Gly Glu Val Asp Ser Ile Met Gly Met Leu Trp Ser Thr Asn Asp Asn

690 695 700

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

705 710 715 720

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

725 730 735

Met Leu Asp Glu Met Tyr Val Ser Asn Ala Val Arg Arg Pro Ile His

740 745 750

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

755 760 765

Pro Ser Lys Ile Phe Val Glu Met Ala Arg Gly Gly Gly Glu Lys Gly

770 775 780

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

785 790 795 800

Met Asp Lys Ala Glu Val Arg Glu Leu Ser Glu Gln Leu Glu Gly Lys

805 810 815

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

820 825 830

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

835 840 845

Lys Thr Asn Ile Tyr Asn Val Asp His Ile Tyr Pro Gln Ser Tyr Val

850 855 860

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

865 870 875 880

Gly Gln Lys Gly Asp Lys Tyr Pro Ile Ser Lys Asp Ile Arg Glu Lys

885 890 895

Met Gln Pro Phe Trp Tyr Arg Leu Leu Ser Asn Lys Leu Ile Ser Glu

900 905 910

Glu Lys Tyr Arg Arg Leu Thr Arg Cys Thr Ser Phe Thr Glu Glu Glu

915 920 925

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

930 935 940

Lys Ala Val Thr Thr Val Phe Arg Thr Leu Phe Pro Asp Val Glu Ile

945 950 955 960

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

965 970 975

Met Leu Lys Thr Arg Ser Val Asn Asp Leu His His Ala Lys Asp Ala

980 985 990

Tyr Leu Asn Ile Val Val Gly Asn Val Tyr His Cys Arg Phe Thr Lys

995 1000 1005

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

1010 1015 1020

Phe Thr His Ser Val Lys Leu Gly Asp Asp Val Ile Trp Asn Gly

1025 1030 1035

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

1040 1045 1050

Ile His Tyr Thr Lys Tyr Pro Phe Met Arg Lys Gly Gly Leu Phe

1055 1060 1065

Asp Gln Met Pro Val Lys Ala Ala Ala Gly Leu Ile Pro Arg Lys

1070 1075 1080

Thr Gly Leu Asp Thr Glu Lys Tyr Gly Gly Tyr Asn Lys Ser Thr

1085 1090 1095

Ala Thr Ala Phe Leu Leu Val Lys Tyr Lys Glu Lys Gly Lys Gln

1100 1105 1110

Glu Ala Met Ile Met Pro Val Asp Tyr Met Tyr Ser Glu Lys Val

1115 1120 1125

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

1130 1135 1140

Lys Lys Ile Trp Gly Arg Thr Glu Asp Gln Val Ile Asp Val Ser

1145 1150 1155

Leu Pro Leu Gly Leu Arg Pro Ile Lys Ile Asn Thr Met Leu Ser

1160 1165 1170

Phe Asp Gly Phe Arg Ala Cys Ile Thr Gly Lys Ala Asn Ala Gly

1175 1180 1185

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

1190 1195 1200

Glu Trp Glu Asn Tyr Ile Lys Lys Ile Asp Asn Tyr Ile Glu Lys

1205 1210 1215

Lys Gly Lys Asn Lys Asn Ile Thr Leu Asn Glu Lys Asn Asp Gly

1220 1225 1230

Ile Cys Gly Glu Lys Asn Glu Lys Leu Tyr Cys Ile Leu Thr Asp

1235 1240 1245

Lys Ile Ile Asn Asn Ile Tyr Ser Ile Pro Phe Asn Ser Gln Gln

1250 1255 1260

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

1265 1270 1275

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

1280 1285 1290

Ser Gly Arg Ala Gly Ser Cys Asp Met Ser Ala Ile Gly Gly Ser

1295 1300 1305

Lys Asn Ala Ala Thr Phe Ala Phe Gly Ser Lys Leu Ser Leu Trp

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

<210> 9

<211> 1383

<212> PRT

<213> Lt4Cas9蛋白序列

<400> 9

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

1 5 10 15

Thr Ser Val Gly Trp Ala Ala Thr Asp Glu Asn Tyr Glu Ile Ile Lys

20 25 30

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

35 40 45

Thr Ala Ala Asp Arg Arg Met His Arg Ile Ala Arg Arg Arg Ile Glu

50 55 60

Arg Arg Ser Arg Arg Ile Asp Leu Leu Gln Glu Leu Phe Ala Gln Glu

65 70 75 80

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

85 90 95

Leu Tyr Glu Glu Asp Lys Thr Val His Gln Lys Asn Ser Leu Phe Asn

100 105 110

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

115 120 125

Tyr His Leu Arg Tyr Asp Leu Met Thr Lys Asp Arg Pro Phe Asp Val

130 135 140

Arg Leu Val Tyr Leu Ala Val His His Ile Leu Lys His Arg Gly His

145 150 155 160

Phe Leu Phe Asp His Phe Gln Val Asp Glu Asn Gly Val Ser Gly Phe

165 170 175

Glu Glu Ser Phe Ala Ala Phe Gly Asp Ala Leu Glu His Ile Lys Gly

180 185 190

Glu Ser Phe Asp Met Gly Lys Glu Glu Glu Met Lys Ala Leu Cys Arg

195 200 205

Asp Lys Lys Leu Gly Val Arg His Lys Ala Leu Ala Leu Ala Gln Cys

210 215 220

Leu Gly Arg Ser Lys Asp Lys Asp Phe Lys Ala Met Met Thr Leu Ala

225 230 235 240

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

245 250 255

Lys Asp Phe Ser Lys Asn Lys Val Ser Phe Ser Asp Ser Gln Phe Glu

260 265 270

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

275 280 285

Ile Ala Ala Leu His Gly Leu Tyr Asn Trp Ser Phe Leu Ala Glu Leu

290 295 300

Met Arg Gly His Lys Tyr Ile Ser Glu Ala Lys Ile Glu Ile Tyr Asp

305 310 315 320

Lys His Lys Glu Asp Leu Ala Leu Leu Lys Lys Val Leu Lys Gln Asp

325 330 335

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

340 345 350

Ile Asn Tyr Ser Ala Tyr Val Lys Ala Cys Lys Thr Asn Gly Lys Lys

355 360 365

Leu Pro Leu Pro Tyr Gly Lys Phe Lys Tyr Glu Glu Phe Ile Lys Thr

370 375 380

Val Lys Phe Cys Leu Lys Asn Leu Pro Asp Ser Pro Asp Lys Lys Asn

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Arg Ile Pro Tyr Tyr Val Gly Pro Ile Asn Glu His Ala Gly Ser Cys

465 470 475 480

Trp Val Val Lys Lys Asp Lys Gln Gly Lys Val Tyr Pro Trp Asn Phe

485 490 495

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

500 505 510

Leu Thr Asn Lys Cys Thr Tyr Leu Ile Gly Glu Asp Val Leu Pro Lys

515 520 525

Asn Ser Leu Leu Tyr Ser Glu Phe Thr Val Leu Asn Glu Leu Asn Asn

530 535 540

Val Arg Ile Gly Glu Asn Ala Gln Lys Leu Ser Pro Glu Leu Lys Glu

545 550 555 560

Lys Val Leu Glu Asn Leu Phe Lys Lys His Lys His Val Ser Arg Arg

565 570 575

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

580 585 590

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

595 600 605

Leu Ile Asp Met Lys His Ile Leu Gly Asn Asp Phe Ser Arg Glu Asp

610 615 620

Ala Glu Lys Met Ile Lys Asp Ile Thr Ile Phe Gly Gly Asp Lys Lys

625 630 635 640

Met Leu Lys Lys Arg Leu His Arg Glu Phe Ser Tyr Leu Thr Ser Glu

645 650 655

Gln Leu Thr Ser Leu Thr Arg Leu Ser Tyr Asp Gly Trp Gly Arg Leu

660 665 670

Ser Lys Glu Leu Leu Val Asn Leu Leu Pro Val Glu Lys Ser Thr Gly

675 680 685

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

690 695 700

Ala Met Glu Gln Thr Ser Tyr Asn Leu Met Glu Leu Leu Ser Ser Arg

705 710 715 720

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

725 730 735

Asn Gly Thr Ile Ser Tyr Gln Asp Val Glu Asp Met Tyr Ile Ser Pro

740 745 750

Ala Val Lys Arg Pro Leu Trp Gln Ala Leu Lys Ile Val Arg Glu Ile

755 760 765

Val Lys Ile Leu Gly Lys Glu Pro Ser Lys Ile Phe Ile Glu Met Ala

770 775 780

Arg Glu Asn Gly Glu Lys Gly Lys Arg Thr Ile Ser Arg Lys Ala Arg

785 790 795 800

Leu Gln Glu Leu Tyr Lys Lys Cys Arg Asp Asp Ser Arg Asp Trp Ala

805 810 815

Lys Glu Leu Ala Glu Lys Pro Glu Glu Asp Phe Arg Ser Asp Arg Leu

820 825 830

Tyr Leu Tyr Tyr Thr Gln Met Gly Arg Ser Met Tyr Thr Gly Lys Pro

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Gly Ser Ala Leu Asp Gly Cys His Ile Gln Gly Glu Ile His Ile Gln

900 905 910

Asp Ile Gln Arg Glu Met Arg Pro Phe Trp His Met Leu Leu Glu Lys

915 920 925

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

930 935 940

Leu Ser Asp Thr Glu Lys Ala Ala Phe Ile Gly Arg Gln Leu Val Glu

945 950 955 960

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

965 970 975

Pro Gln Ala Arg Ile Val Tyr Thr Lys Ala Gly Asn Ala Ser Arg Phe

980 985 990

Arg Gln Tyr Gly Gly Phe Ile Lys Val Arg Asp Met Asn Asp Tyr His

995 1000 1005

His Ala Lys Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Val Phe

1010 1015 1020

Asp Thr Arg Phe Thr Ala Asn Pro Leu His Phe Leu Lys Gly Asn

1025 1030 1035

His Pro Val Tyr Ser Leu Asn Thr Glu Ala Leu Tyr Gly His Lys

1040 1045 1050

Val Ser Arg Gly Gly Val Asp Ala Trp Ile Pro Pro Glu Lys Asp

1055 1060 1065

Asp Glu Gly His Ile Met Ala Gly His Glu Gly Thr Met Gly Thr

1070 1075 1080

Val Arg Lys Trp Met Arg Lys Asn Asn Ile Leu Phe Thr Arg Met

1085 1090 1095

Pro Leu Glu Gly Lys Gly Gly Leu Phe Asp Gln Thr Ile Met Lys

1100 1105 1110

Lys Gly Lys Gly Gln Val Pro Leu Lys Gly Asp Ser Pro Val Ser

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

Lys Asp Lys Glu Asp Tyr Ile Lys Arg His Trp Lys Asp His Gly

1175 1180 1185

Lys Lys Met Val Asn Pro His Ile Cys Tyr Gly His Ile Pro Val

1190 1195 1200

Gln Ser Leu Leu Glu Ile Asn Gly Phe Lys Val His Leu Thr Gly

1205 1210 1215

Lys Ser Gly Lys Asp Phe Lys Leu Arg Asn Ala Glu Gln Leu Cys

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Thr Pro Phe Asp Asn Ile Gln Glu Val Asp Leu Asn Arg Leu Tyr

1265 1270 1275

Gln Val Phe Glu Asp Lys Leu Thr Asn Gln Val Tyr Lys Val Lys

1280 1285 1290

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

1295 1300 1305

Asn Glu Leu Pro Leu Glu Val Lys Cys Arg Val Ile Gly Glu Ile

1310 1315 1320

Leu His Leu Phe Gln Cys Asn Ala Ala Ile Ala Asp Leu Arg Leu

1325 1330 1335

Ile Gly Gly Ala Lys Asn Ala Gly Ala Leu Thr Met Asn Pro Arg

1340 1345 1350

Val Ser Pro Glu Asp His Val Tyr Leu Ile Glu Gln Ser Val Thr

1355 1360 1365

Gly Phe Phe Glu Lys Arg Ile Leu Leu Ala Pro Tyr Gly Gly Lys

1370 1375 1380

<210> 10

<211> 1381

<212> PRT

<213> Lt5Cas9蛋白序列

<400> 10

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

1 5 10 15

Ser Val Gly Trp Thr Ala Thr Asp Glu Asn Tyr Glu Val Ile Lys Lys

20 25 30

Asn Gly Lys Ala Leu Trp Gly Ile Arg Leu Phe Asp Glu Ala Gln Thr

35 40 45

Ala Glu Asp Arg Arg Met His Arg Ile Ala Arg Arg Arg Ile Glu Arg

50 55 60

Arg Ser Arg Arg Ile Asp Leu Leu Gln Glu Leu Phe Ala Gln Glu Ile

65 70 75 80

Cys Lys Lys Asp Pro Gly Phe Tyr Glu Arg Leu Asn Glu Ser Gly Leu

85 90 95

Tyr Glu Glu Asp Lys Thr Val His Gln Thr Asn Ser Leu Phe Asn Asp

100 105 110

Val Asp Phe Asn Asp Lys Ala Tyr Tyr Lys Lys Tyr Pro Thr Ile Tyr

115 120 125

His Leu Arg His Ala Leu Met Thr Glu Asn His Pro Phe Asp Val Arg

130 135 140

Leu Val Tyr Leu Ala Ile His His Ile Leu Lys His Arg Gly His Phe

145 150 155 160

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

165 170 175

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

180 185 190

Ser Pro Asp Val Arg Lys Ala Asp Ser Met Lys Asp Ile Leu Lys Asp

195 200 205

Lys Lys Leu Gly Val Lys Glu Lys Ala Ala Ser Leu Leu Gln Cys Leu

210 215 220

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

225 230 235 240

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

245 250 255

Asp Phe Glu Lys Asn Lys Val Asn Phe Ser Ser Ala Gln Phe Glu Glu

260 265 270

Asn Glu Pro Asp Ile Met Ala Glu Leu Gly Asp Arg Tyr Asp Leu Ile

275 280 285

Ala Ala Leu His Gly Phe Tyr Asn Trp Ser Leu Leu Ala Glu Leu Met

290 295 300

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

305 310 315 320

His Lys Ala Asp Leu Lys Val Leu Lys Arg Val Leu Lys Gln Arg Pro

325 330 335

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

340 345 350

Asn Tyr Ser Ala Tyr Val Gly Val Cys Lys Val Lys Gly Lys Lys Ala

355 360 365

Ala Ile Glu Lys Cys Ser Tyr Glu Asp Phe Thr Lys Thr Leu Lys Pro

370 375 380

Cys Leu Lys Asp Met Pro Asp Ser Asn Asp Lys Asp Tyr Ile Ser Arg

385 390 395 400

Glu Leu Asn Met Gly Thr Phe Leu Pro Lys Ser Val Ser Lys Glu Asn

405 410 415

Gly Val Ile Pro Tyr Gln Leu His Leu Gln Glu Leu Lys Ile Ile Leu

420 425 430

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

435 440 445

Gly Thr Val Ser Asp Lys Ile Ile Ser Leu Phe Thr Phe Arg Ile Pro

450 455 460

Tyr Tyr Val Gly Pro Ile Asn Glu His Ala Gly Ser Cys Trp Val Val

465 470 475 480

Lys Lys Asp Lys Arg Gly Lys Val Tyr Pro Trp Asn Phe Thr Glu Lys

485 490 495

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

500 505 510

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

515 520 525

Leu Tyr Ser Glu Phe Thr Val Leu Asn Glu Leu Asn Asn Val Arg Ile

530 535 540

Gly Glu Thr Met Gln Lys Leu Pro Leu Arg Leu Lys Glu Lys Val Met

545 550 555 560

Asp Asn Leu Phe Ser Arg Tyr Lys His Val Ser Arg Thr Lys Phe Ile

565 570 575

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

580 585 590

Ser Gly Leu Asp Gly Asp Phe Lys Ser Ser Leu Ser Ser Leu Ile Asp

595 600 605

Met Lys His Ile Leu Gly Asn Asp Phe Ser Arg Glu Asn Ala Glu Lys

610 615 620

Met Ile Gln Asp Ile Thr Ile Phe Gly Gly Asp Lys Lys Met Leu Lys

625 630 635 640

Asn Arg Leu His Arg Glu Phe Ser Tyr Leu Thr Pro Glu Gln Leu Thr

645 650 655

Ser Leu Thr Gln Leu Ser Tyr Asp Gly Trp Gly Arg Leu Ser Lys Glu

660 665 670

Phe Leu Val Asn Leu Leu Pro Ala Glu Gly Asp Ser Cys Glu Val Leu

675 680 685

Val Asp His Thr Ser Gly Glu Val Leu Asn Ile Ile Ser Ala Met Arg

690 695 700

Gln Thr Ser Tyr Asn Leu Met Glu Leu Leu Gly Ser Arg Phe Gly Tyr

705 710 715 720

Gly Gln Ala Ile Glu Glu Arg Asn Lys Lys Glu Glu Gly Gln Gly Arg

725 730 735

Ile Thr Tyr Lys Asp Val Glu Asp Leu Tyr Ile Ser Pro Ala Val Arg

740 745 750

Arg Pro Leu Trp Gln Ala Leu Lys Ile Val Arg Glu Ile Val Lys Ile

755 760 765

Thr Gly Lys Glu Pro Ser Lys Ile Phe Ile Glu Met Ala Arg Glu Asn

770 775 780

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

785 790 795 800

Leu Tyr Lys Lys Cys Arg Asp Asp Thr Arg Asp Trp Ala Lys Glu Leu

805 810 815

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

820 825 830

Tyr Thr Gln Met Gly Arg Ser Met Tyr Thr Gly Lys Pro Ile Asp Ile

835 840 845

Asn Arg Leu Phe Asp Arg Asn Val Tyr Asp Ile Asp His Ile Tyr Pro

850 855 860

Gln Ser Leu Thr Gly Asp Asp Ser Leu Asp Asn Arg Val Leu Val Glu

865 870 875 880

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

885 890 895

Leu Asp Gly Cys Tyr Ile Ser Gly Gln Gln Ile Arg Ile Gln Asp Ile

900 905 910

Gln Lys Glu Met Arg Pro Phe Trp His Met Leu Leu Glu Lys Glu Leu

915 920 925

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

930 935 940

Asp Ala Glu Lys Ala Ala Phe Ile Gly Arg Gln Leu Val Glu Thr Arg

945 950 955 960

Gln Ser Thr Lys Ala Cys Ala Glu Leu Leu Ser Lys Ala Tyr Pro Gln

965 970 975

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

980 985 990

Tyr Gly Gly Phe Ile Lys Val Arg Asp Met Asn Asp Tyr His His Ala

995 1000 1005

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

1010 1015 1020

Arg Phe Thr Ala Asn Pro Leu His Phe Leu Lys Gly Asn His Gln

1025 1030 1035

Ala Tyr Ser Leu Asn Thr Glu Ala Leu Tyr Gly His Lys Val Ser

1040 1045 1050

Arg Asn Gly Val Asp Ala Trp Ile Pro Ala Glu Lys Asp Glu Lys

1055 1060 1065

Gly Gln Val Met Ala Gly His Glu Gly Thr Met Gly Thr Val Arg

1070 1075 1080

Lys Trp Met Arg Lys Asn Asn Ile Leu Phe Thr Arg Met Pro Tyr

1085 1090 1095

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

1100 1105 1110

Lys Gly Gln Val Pro Ile Lys Gly Asp Ser Pro Ile Ser Asn Ile

1115 1120 1125

Lys Lys Tyr Gly Gly Tyr Asn Lys Ala Lys Val Ala Tyr Phe Val

1130 1135 1140

Leu Thr Gln Ser Lys Leu Asn Lys Lys Thr Val Tyr Thr Leu Glu

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

Met Glu His Pro Ile Val Cys Leu Gly His Ile Pro Val Gln Ser

1190 1195 1200

Leu Leu Glu Ile Asn Gly Phe Lys Val His Leu Ser Gly Lys Asn

1205 1210 1215

Gly Lys Asp Ile Leu Leu Arg Asn Ala Glu Gln Leu Cys Ile Asn

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

Leu Ser Pro Glu Gln Lys Cys Lys Leu Ile Gly Glu Ile Leu His

1310 1315 1320

Leu Cys Gln Cys Lys Ala Thr His Ala Asp Leu Arg Leu Ile Gly

1325 1330 1335

Gly Ala Lys Lys Ala Gly Ile Leu Thr Met Gly Thr Gln Ile Tyr

1340 1345 1350

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

1355 1360 1365

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

1370 1375 1380

<210> 11

<211> 1368

<212> PRT

<213> Lt6Cas9蛋白序列

<400> 11

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

1 5 10 15

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

20 25 30

Gln Lys Ala Leu Trp Gly Val Arg Leu Phe Asp Gly Ala Lys Thr Ala

35 40 45

Ala Glu Arg Arg Gly Phe Arg Thr Ser Arg Arg Arg Val Glu Arg Arg

50 55 60

Asn Gln Arg Leu Asn Trp Leu Gln Asn Val Phe Ser Glu Glu Ile Ala

65 70 75 80

Lys Lys Asp Pro Gly Phe Phe Gln Arg Leu Lys Glu Ser Met Phe Ser

85 90 95

Pro Glu Asp Lys Arg Ser Asp Phe Glu Glu Gln Pro Gly Arg Phe Ser

100 105 110

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

115 120 125

Pro Thr Ile Ser His Leu Lys Val Asp Leu Leu Glu Lys Asp Lys Lys

130 135 140

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

145 150 155 160

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

165 170 175

Phe Asp Ile Gly Phe Asp Ser Leu Asn Asp Tyr Leu Glu Asp Cys Gly

180 185 190

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

195 200 205

Leu Asp Lys Lys Ile Lys Val Lys Asp Lys Thr Lys Leu Leu Leu Glu

210 215 220

His Ala Gly Tyr Ser Lys Lys Asp Lys Gln Lys Glu Ala Leu Phe Thr

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Arg Ile Lys Ala Val Tyr Asp Trp Ala Leu Leu Glu Asp Ile Leu Gln

290 295 300

Gly Glu Thr Tyr Ile Ser Thr Ala Arg Lys Lys Ile Tyr Glu Lys His

305 310 315 320

Ala Lys Asp Leu Lys Leu Leu Lys Gln Leu Val Arg Lys Tyr Leu Thr

325 330 335

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

340 345 350

Asn Tyr Thr Ala Tyr Ser Gly Asn Tyr Ser Asp Lys Ser Gln Lys Ser

355 360 365

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

370 375 380

Tyr Lys Tyr Ile Lys Lys Lys Phe Lys Pro Phe Glu Asp Lys Asp Asp

385 390 395 400

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

405 410 415

Gln Val Gly Ala Glu Asn Gly Leu Ile Pro His Gln Ile Asn Glu Arg

420 425 430

Glu Leu Lys Lys Ile Leu Glu Asn Ser Ser Lys Tyr Tyr Pro Phe Leu

435 440 445

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

450 455 460

Gln Val Phe Asn Phe Lys Ile Pro Tyr Tyr Ile Gly Pro Leu Asn Lys

465 470 475 480

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

485 490 495

Trp Asn Phe Ser Asp Val Val Asp Thr Lys Lys Ser Ala Glu Asn Phe

500 505 510

Ile Val Arg Met Thr Ala Lys Cys Thr Tyr Thr Gly Ala Asp Val Leu

515 520 525

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

530 535 540

Asn Lys Leu Lys Ile Asn Gly Glu Pro Ile Thr Val Glu Gln Lys Gln

545 550 555 560

Ser Met Tyr Asn Asp Leu Phe Met Arg Tyr Lys Thr Val Lys Thr Lys

565 570 575

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

580 585 590

Val Val Ser Gly Ile Asp Met Glu Thr Gly Ile Lys Ala Ser Leu Ser

595 600 605

Ser Tyr His Ala Phe Arg Asn Ile Leu Glu Asn Tyr Lys Asp Glu Glu

610 615 620

Met Val Glu Asp Ile Ile Arg His Ile Val Leu Phe Gly Asp Asp Lys

625 630 635 640

Lys Leu Ile Lys Ser Tyr Ile Ser Glu Lys Tyr Gly His Ile Leu Ser

645 650 655

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

660 665 670

Arg Leu Ser Lys Glu Leu Leu Thr Lys Ile Tyr His Val Asn Arg Glu

675 680 685

Thr Gly Glu Ala Lys Ser Ile Ile Thr Ser Met Trp Glu Asp Asn Lys

690 695 700

Asn Leu Met Glu Leu Leu Ser Cys Glu Tyr Asp Tyr Met Asp Lys Ala

705 710 715 720

Ile Glu Tyr Lys Lys Gln His Met Pro His Gly Ala Asn Ser Val Lys

725 730 735

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

740 745 750

Ile Gln Ala Thr Gly Ile Ile Asn Glu Ile Glu Lys Ile Met Lys Ala

755 760 765

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

770 775 780

Lys Lys Lys Lys Lys Asn Glu Ala Lys Arg Lys Pro Arg Lys Asp Gln

785 790 795 800

Leu Ile Glu Leu Tyr Lys Lys Cys Lys Glu Glu Glu Pro Glu Leu Phe

805 810 815

Ala Ser Leu Gln Asp Thr Ser Glu Glu Lys Leu Arg Lys Asp Ser Leu

820 825 830

Tyr Phe Tyr Tyr Ala Gln Leu Gly Arg Cys Met Tyr Ser Gly Glu Arg

835 840 845

Ile Ser Ile Glu Arg Leu Ser Ser Asp Tyr Asp Ile Asp His Ile Tyr

850 855 860

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

865 870 875 880

Lys Lys Thr Ile Asn Ser Asp Ile Lys Lys Asp Asn Tyr Pro Ile Asp

885 890 895

His Asn Ile Gln Lys Asn Met Lys Asn Phe Trp Asn Glu Leu Glu Lys

900 905 910

Lys Gly Met Ile Ser Lys Glu Lys Tyr Lys Arg Leu Thr Arg Thr Ser

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

Asp Phe Arg Lys Gly Ser Val Asn Lys Lys Thr Gly Glu Val Glu Arg

980 985 990

Lys Ala Phe Val Lys Cys Arg Asp Ile Asn Asp Tyr His His Ala Lys

995 1000 1005

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

1010 1015 1020

Phe Thr Lys Ser Pro Val Asn Phe Ile Lys Glu Leu Lys Lys Ser

1025 1030 1035

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

1040 1045 1050

Arg Gly Gly Glu Thr Ala Trp Val Pro Gly Lys Asp Gly Thr Phe

1055 1060 1065

Ala Thr Val Glu Arg Gln Met Ser Lys Asn Asn Ile Leu Phe Thr

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

Arg Ile Arg Ser Ile Glu Pro Val Phe Ile Leu Asn Lys Asn Val

1145 1150 1155

Phe Glu Ala Asp Pro Val Lys Tyr Cys Thr Glu Val Leu Asn Leu

1160 1165 1170

Lys Asn Pro Val Val Leu Ile Lys Gln Ile Lys Ile Asp Ser Leu

1175 1180 1185

Ile Ser Ile Asp Gly Phe Arg Gly His Ile Ser Gly Arg Thr Gly

1190 1195 1200

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

1205 1210 1215

Leu Trp His Asp Tyr Val Lys Lys Leu Ser Lys Tyr Leu Asp Arg

1220 1225 1230

Cys Lys Asp Thr Gln Lys Glu Leu Pro Ile Thr Lys Phe Asp Gly

1235 1240 1245

Ile Thr Lys Glu Glu Asn Leu Met Leu Tyr Asp Val Ile Thr Gln

1250 1255 1260

Lys Leu Asn Thr Lys Leu Tyr Glu Ile Lys Tyr Gly Thr Gln Tyr

1265 1270 1275

Asp Thr Leu Lys Asn Asn Arg Asn Lys Phe Asp Gly Leu Ser Ile

1280 1285 1290

His Asp Gln Ala Val Ile Leu Lys Asn Asn Leu Asn Met Leu Lys

1295 1300 1305

Cys Asn Ala Val Asn Ala Asp Phe Thr Leu Leu Thr Gly Ala Gly

1310 1315 1320

Cys Val Gly Arg Ile Lys Ile Thr Lys Asn Ile Ser Pro Gln Ile

1325 1330 1335

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

1340 1345 1350

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

1355 1360 1365

<210> 12

<211> 1327

<212> PRT

<213> Lt7Cas9蛋白序列

<400> 12

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

1 5 10 15

Gly Trp Ala Val Leu Thr Glu Asp Tyr Glu Leu Ile Lys Arg Lys Met

20 25 30

Lys Ile Ser Gly Asn Thr Gln Lys Lys Ala Val Lys Lys Asn Phe Trp

35 40 45

Gly Val Arg Leu Phe Glu Gln Gly Glu Thr Ala Glu Gly Arg Arg Leu

50 55 60

Lys Arg Thr Thr Arg Arg Arg Ile Ala Arg Arg Arg Gln Arg Ile Gln

65 70 75 80

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

85 90 95

Phe Phe Ala Arg Leu Asp Glu Ser Phe Ser Ile Ile Asp Glu Lys Glu

100 105 110

Asn Glu Arg His Pro Ile Phe Gly Asn Val Ala Glu Glu Ala Ala Tyr

115 120 125

His Glu Gln Phe Pro Thr Ile Tyr His Leu Arg Glu His Leu Ala Asn

130 135 140

Ser Ser Glu Gln Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His

145 150 155 160

Ile Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Glu Leu Asn Thr

165 170 175

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

180 185 190

Asn Glu Thr Phe Asn Val Glu Lys Ala Leu Asp Leu Thr Val Asp Leu

195 200 205

Asp Gly Ile Ala Glu Gln Lys Ile Ser Arg Met Lys Arg Ala Glu Leu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ile Leu Lys Val Thr Asp Ala Thr Thr Arg Ala Lys Leu Ser Ser Ser

305 310 315 320

Met Val Val Arg Phe Thr Asp His Lys Glu Asp Leu Lys Thr Leu Lys

325 330 335

Arg Phe Val Arg Glu Asn Leu Pro Asp Glu Tyr Asp Asp Leu Phe Lys

340 345 350

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

355 360 365

Gln Glu Ala Phe Tyr Lys Tyr Leu Lys Lys Thr Leu Ala Lys Ala Thr

370 375 380

Gly Ala Glu Gly Phe Leu Ala Lys Met Glu Gln Glu Asp Phe Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Val Ile Pro Tyr Gln Leu His Leu

405 410 415

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

420 425 430

Leu Asp Glu Glu Lys Ile Ser Gln Leu Met Thr Phe Arg Ile Pro Tyr

435 440 445

Tyr Val Gly Pro Leu Ala Lys Glu Lys Gly Gln Phe Ala Trp Leu Thr

450 455 460

Arg Lys Glu Met Gly Lys Ile Thr Pro Trp Asn Leu Asn Glu Lys Val

465 470 475 480

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

485 490 495

Asp Ser Tyr Leu Pro Met Glu Lys Val Leu Pro Lys His Ser Leu Leu

500 505 510

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

515 520 525

Asp Asp Asn Gly Arg Ala Gln Asn Phe Ser Ser Lys Glu Lys Arg Gln

530 535 540

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

545 550 555 560

Leu Glu Ala Phe Leu Lys Asn Glu Tyr Gly Ile Glu Asn Pro Lys Val

565 570 575

Glu Gly Ile Glu Lys Ser Phe Asn Ala Cys Leu Gly Thr Tyr His Asp

580 585 590

Leu Ile Lys Leu Gly Ile Arg Pro Glu Leu Phe Glu Gln Pro Glu His

595 600 605

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

610 615 620

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

625 630 635 640

Glu Glu Glu Gln Lys Gln Leu Glu Arg Lys His Tyr Lys Gly Trp Gly

645 650 655

Arg Leu Ser Ala Lys Leu Ile His Gly Ile Val Asp Gln Lys Thr Gln

660 665 670

Lys Thr Ile Leu Asp Tyr Leu Ile Asp Asp Asp Asp Leu Pro Lys Asn

675 680 685

Arg Asn Arg Asn Phe Met Gln Leu Ile Asn Asp Glu Asn Leu Ser Phe

690 695 700

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

705 710 715 720

Glu Ile Val Gln Glu Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

725 730 735

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

740 745 750

Leu Pro Thr Asn Ile Val Val Glu Met Ala Arg Glu Asn Gln Thr Thr

755 760 765

Ala Gln Gly Asn Arg Ala Ser Lys Ala Arg Met Lys Tyr Leu Glu Glu

770 775 780

Ser Ile Lys Lys Leu Gly Ser Ser Ile Leu Glu Asp Glu Pro Ile Ser

785 790 795 800

Lys Asp Ala Asn Leu Leu Arg Asn Asp Arg Leu Phe Leu Tyr Tyr Leu

805 810 815

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

820 825 830

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

835 840 845

Asp Asp Ser Ile Asp Asn Arg Val Leu Thr Thr Ser Ser Met Asn Arg

850 855 860

Gly Lys Ser Asn Thr Val Pro Ala Glu Ser Val Val Lys Lys Met Arg

865 870 875 880

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

885 890 895

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

900 905 910

Lys Ala His Phe Ile Gln Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

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

930 935 940

Ser Glu Lys Pro Val Arg Val Ile Thr Leu Lys Ser Ala Leu Ala Ser

945 950 955 960

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

965 970 975

Tyr His His Ala Gln Asp Ala Tyr Leu Asn Gly Val Ile Ala Gln Ala

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

Met Val Ala Asn Glu Glu Thr Gly Glu Val Leu Trp Asn Arg Gln

1040 1045 1050

Arg Asp Ile Lys Thr Ile Lys Lys Val Met Asn Tyr His Gln Met

1055 1060 1065

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

1070 1075 1080

Glu Ser Ile Leu Pro Lys Gly Pro Ser Lys Lys Leu Ile Ala Arg

1085 1090 1095

Lys Asn Asn Trp Ala Pro Val Asn Tyr Gly Gly Val Asp Ser Pro

1100 1105 1110

Thr Val Ala Tyr Ser Val Ile Ile Thr His Glu Lys Gly Lys Ala

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

Phe Ile His Pro Lys Val Gln Leu Lys Leu Pro Lys Tyr Ser Leu

1160 1165 1170

Tyr Gln Phe Ala Asp Gly Arg Arg Arg Leu Leu Ala Ser Ala Glu

1175 1180 1185

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

1190 1195 1200

Glu Leu Leu Tyr His Ala Lys His Val Ser Asp Ser Ser Gly Lys

1205 1210 1215

Ser Leu Glu Tyr Leu Asn Glu His Arg His Glu Phe Ala Glu Leu

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Asp Met Arg Glu Leu Ala Ser Ser Phe Ile Gln Leu Leu Gln

1265 1270 1275

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

1280 1285 1290

Thr Ile Pro Arg Arg Arg Tyr Lys Asn Thr Ala Glu Ile Val Asp

1295 1300 1305

Ala Thr Phe Ile Asn Gln Ser Ile Thr Gly Leu Tyr Glu Thr Gln

1310 1315 1320

Arg Arg Leu Val

1325

<210> 13

<211> 1296

<212> PRT

<213> LtCpf1蛋白序列

<400> 13

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

1 5 10 15

Thr Leu Arg Phe Ala Leu Lys Pro Glu Pro Lys Thr Glu Glu Asn Leu

20 25 30

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

35 40 45

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

50 55 60

Glu Gly Leu Lys Ser Leu Glu Asn Gln Asn Ala Leu Asp Trp Thr Glu

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Glu Thr Leu Pro Leu Gly Lys Glu Glu Lys Glu Ala Val Lys Val

130 135 140

Phe Ala Gly Phe Gly Gly Phe Phe Asn Asn Tyr Asn Lys Asn Arg Glu

145 150 155 160

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

165 170 175

Val Asn Glu Asn Phe Ser Lys His Phe Ser Asn Val Glu Ile Val Thr

180 185 190

Lys Ile Gln Lys Glu Val Pro Glu Leu Ile Gln Ile Val Glu Ala Gln

195 200 205

Phe Lys Gly Tyr Asp Ala Ile Phe Thr Val Asn Gly Tyr Asn Met Ala

210 215 220

Leu Ser Gln Ala Gly Ile Asp Thr Tyr Asn Glu Met Val Ala Ile Trp

225 230 235 240

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

245 250 255

Gly His Pro Leu Lys Lys Lys Arg Asn Tyr Leu Leu Ser Ala Leu Phe

260 265 270

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

275 280 285

Asp Gly Asp Glu Glu Val Ile Glu Ala Leu Thr Gly Val Lys Lys Met

290 295 300

Leu Gln Glu Ala Asp Val Phe Glu Lys Leu Asn Met Leu Val Glu Asp

305 310 315 320

Met Glu Asn Trp Asp Tyr Ser Lys Ile Tyr Leu Ser Ala Gln Ser Leu

325 330 335

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

340 345 350

Asn Ser Trp Asn Tyr Leu Asp Asn Val Leu Arg Glu Lys Trp Gln Ile

355 360 365

Glu Leu Gln Gly Lys Lys Lys Gly Thr Asp Leu Glu Glu Ala Ile Arg

370 375 380

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

385 390 395 400

Asn Ala Leu Glu Glu Thr Asp Lys Cys Tyr Ser Val Ser Lys Trp Leu

405 410 415

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

420 425 430

Ala Glu Asp Phe Cys Thr Lys Trp Lys Thr Glu Arg Asn Pro Leu Lys

435 440 445

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

450 455 460

Leu Ala Arg Tyr Cys Lys Ser Phe Tyr Ala Asn Gly Ile Glu Lys Lys

465 470 475 480

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

485 490 495

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

500 505 510

Lys Lys Pro Tyr Ser Leu Glu Lys Ile His Leu Lys Phe Gly His Val

515 520 525

Thr Leu Gly Asn Gly Trp His Ile Asn Gln Glu Lys Asp Asn Gly Thr

530 535 540

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

545 550 555 560

Leu Asn Lys Lys Ile Cys Ile Pro Ser Gln Ile Glu Gly Thr Gly Asn

565 570 575

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

580 585 590

Met Leu Ile Pro Lys Cys Thr Thr Glu Arg Lys Asn Val Glu Ser Cys

595 600 605

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

610 615 620

Phe Val Lys Pro Phe Lys Val Leu Arg Glu Glu Tyr Glu Leu Asn Lys

625 630 635 640

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

645 650 655

Thr Lys Asp Glu Lys Gly Tyr Lys Glu Ala Val Ala Lys Trp Ile Arg

660 665 670

Phe Cys Met Arg Phe Leu Gln Ser Tyr Lys Ser Thr Ala Ile Tyr Asp

675 680 685

Tyr Ser Thr Leu Gln Gln Pro Glu Glu Tyr Glu Thr Val Asp Ser Phe

690 695 700

Tyr Gln Asp Val Gly Lys Ile Thr Tyr Glu Cys His Phe Glu Tyr Val

705 710 715 720

Pro Thr Ser Glu Ile Glu Arg Leu Glu Asn Glu Gly Ser Ile Phe Leu

725 730 735

Phe Gln Ile Tyr Asn Lys Asp Phe Ser Glu Asn Arg Arg Pro Asp Ser

740 745 750

Lys Lys Asn Leu His Thr Leu Tyr Trp Glu Ala Leu Phe Ser Glu Glu

755 760 765

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

770 775 780

Arg Arg Glu Lys Ser Ile Glu Asn Pro Ile Val His Lys Ala Gly Glu

785 790 795 800

Val Leu Val Asn Lys Arg Thr Lys Lys Gly Glu Pro Ile Pro Asp Asp

805 810 815

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

820 825 830

Glu Lys Glu Asp Tyr Lys Glu Tyr Leu Asp Lys Val Tyr Thr Ser Thr

835 840 845

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

850 855 860

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

865 870 875 880

Tyr Leu Asp Gln Lys Ile Leu Arg Met Leu Arg Asp Asn Pro Asp Val

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Lys Asn Arg Asp Lys Glu Arg Lys Thr Trp Lys Asn Ile Glu Thr Ile

945 950 955 960

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

965 970 975

Asp Met Ala Ile Arg Asn Asn Ala Ile Ile Val Met Glu Asp Leu Asn

980 985 990

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

995 1000 1005

Lys Phe Glu Leu Ala Leu Leu Lys Lys Leu His Tyr Leu Val Thr

1010 1015 1020

Asp Lys Thr Glu Gly Lys Ala Met Leu Lys Pro Gly Gly Val Leu

1025 1030 1035

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

1040 1045 1050

Gly Lys Gln Cys Gly Cys Val Phe Tyr Val Pro Pro Gly Tyr Thr

1055 1060 1065

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

1070 1075 1080

Ser Gly Val Thr Asn Arg Glu Lys Lys Lys Ala Phe Phe Glu Lys

1085 1090 1095

Phe Asp Asn Met Phe Tyr Asp Glu Lys Arg Asp Met Phe Gly Phe

1100 1105 1110

Ser Phe Asn Tyr Glu Lys Phe Ala Thr Tyr Gln Ser Ser His Arg

1115 1120 1125

Asn Asp Trp Ile Val Tyr Ser Asn Gly Ser Lys Tyr Val Trp Asn

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

Asp Phe Trp Asn Lys Leu Phe Trp Tyr Phe Arg Val Leu Leu Arg

1190 1195 1200

Ile Arg Asn Ser Ser Gly Glu Leu Asp Gln Ile Ile Ser Pro Val

1205 1210 1215

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

1220 1225 1230

Glu Lys Ser Tyr Leu Ser Asp Tyr Pro Met Asp Ala Asp Thr Asn

1235 1240 1245

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

1250 1255 1260

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

1265 1270 1275

Asp Phe Tyr Lys Ile Ser Asn Ala Glu Trp Phe Arg Phe Arg Gln

1280 1285 1290

Lys Glu Lys

1295

<210> 14

<211> 986

<212> PRT

<213> LtCas13b蛋白序列

<400> 14

Met Lys Thr Asn Gln Pro Lys Thr Ile Tyr Tyr Asn Trp Lys Asp Lys

1 5 10 15

Ala Asp Phe Ala Tyr Phe Ala Phe Tyr Thr Ser Gln Ala Leu Asn Asn

20 25 30

Thr Ser Ile Ile Leu Lys Asn Ile Ser Glu Ile Ile Glu Asn Lys Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Leu Asp Ser Asn Leu Pro Phe Ser Val Lys Ser Ile Ser Asp Pro

85 90 95

Phe Leu Val Lys Asn Arg Leu Ser Tyr Phe Leu Trp Leu Leu Lys Asn

100 105 110

Phe Arg Asn Glu Tyr Ala His Tyr His Glu Arg Glu Leu Asp Ser Arg

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Lys Ile Ser Ser Arg Gln Asn Phe Thr Glu Glu Asp Phe Ile Phe Phe

195 200 205

Leu Cys Leu Phe Leu Ser Thr Lys Glu Thr Met Gln Leu Leu Asn Gly

210 215 220

Ile Lys Gly Lys Lys Asn Thr Thr Thr Glu Glu Phe Gln Trp Ile Arg

225 230 235 240

Arg Val Phe Thr Ile Phe Asn Ala Lys Arg Phe Gln Asn Lys Ile Lys

245 250 255

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

260 265 270

Ala Lys Ile Pro Ile His Leu Lys Lys Tyr Leu Thr Glu Glu Ala Lys

275 280 285

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

290 295 300

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

305 310 315 320

Ser Tyr Arg Cys Leu Gln Tyr Leu Glu Leu Phe Gly Phe Ala Lys Asn

325 330 335

Ile Asn Phe Asn Ile Asn Leu Gly Lys Val Phe Ile Asn Lys Pro Tyr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys Gln Pro Leu Lys Phe Tyr Ser Pro Lys Tyr His Phe Ser Asn Asn

405 410 415

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

420 425 430

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

435 440 445

Tyr Phe Leu Ser Glu Asn Ala Ile Pro Tyr Phe Thr Tyr Cys Met Ile

450 455 460

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

465 470 475 480

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

485 490 495

Val Gln Glu Ile Glu Gln Asn Tyr Ser Leu Lys Ile Gly Trp Ile Pro

500 505 510

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

515 520 525

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

530 535 540

Leu Ile Glu Asn Asn Asn Lys Lys Pro Glu Asp Arg Asp Arg Asn Ile

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Leu Ala Phe Tyr Gly Lys His Glu Glu Asp Leu Lys Leu Leu Phe Lys

610 615 620

Lys Trp Asn Leu Asn Glu Arg His Pro Phe Leu Lys Asp Val Ser Met

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Leu Glu Asn Ser Asn Phe Ala Val Glu Lys Ala Asn Thr Asn Phe Leu

725 730 735

Ile Ser Lys Asn Leu Glu Tyr Phe Gly Asn Gln Trp Phe Tyr Asn Lys

740 745 750

Glu Asn Tyr Gln Gln Gly Leu Asp Thr Tyr Gln Ser His Leu Arg Asp

755 760 765

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

770 775 780

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

785 790 795 800

Asn Asn Leu Ala Ala Tyr Asn Ser Glu Gln Thr Leu Leu Glu Lys Gln

805 810 815

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

820 825 830

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

835 840 845

Ile Lys Asp Phe Gly Ile Phe Arg His Leu Ile Lys Asp Arg Arg Val

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Asn Leu Ile Gly Lys Asn Ile Ser Ile His Glu Lys Phe Ser Glu Asn

915 920 925

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

930 935 940

Glu Ile Arg Asn Lys Leu Leu His Asn Lys Val Pro Glu Ile Asn Leu

945 950 955 960

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

965 970 975

Lys Ser Cys Asn Glu Leu Glu Arg Leu Ile

980 985

<210> 15

<211> 957

<212> PRT

<213> LtCas13d蛋白序列

<400> 15

Met Ile Leu Ile Leu Gly Glu Gly Thr Ile Arg Met Ala Lys Lys Lys

1 5 10 15

Asn Ala Arg Gln Arg Arg Glu Glu Glu Lys Asn Arg Ile Lys Ala Ile

20 25 30

Ile Glu Lys Ile Lys Asn Lys Val Val Glu Lys Glu Glu Thr Glu Glu

35 40 45

Ile Val Glu Asn Asn Glu Thr Lys Asn Val Glu Ser Ile Val Val Glu

50 55 60

Pro Lys Lys Lys Ser Leu Ala Lys Ala Ser Gly Val Lys Ser Val Phe

65 70 75 80

Ile Asn Asn Asp Glu Ile Ile Met Thr Ser Phe Gly Arg Gly Asn Asp

85 90 95

Ala Val Ile Glu Lys Ile Ile Lys Asp Asn Asn Ile Asp Asn Glu Asn

100 105 110

Lys Asp Lys Pro Val Tyr Asp Val Val Ala Ile Glu Asn Glu Gly Asn

115 120 125

Ile Ile Lys Val Gln Ser Glu Arg Phe Lys Ala Ile Glu Ser Ala Asn

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Leu Leu Glu Glu Phe Ile Glu Asn Gly Asp Arg Leu Gly Tyr Phe Gly

245 250 255

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

260 265 270

Tyr Asn Ile Leu Gly Leu Leu Gly Ser Leu Arg Gln Phe Cys Phe His

275 280 285

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

290 295 300

Tyr Lys Ser Asn Ser Trp Leu Tyr Asn Leu Gly Gln Leu Phe Asp Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Tyr Asp Phe Ile Ile Ser Lys Lys His Lys Asn Met Gly Phe Ser Ile

370 375 380

Lys Lys Ile Arg Glu Tyr Met Phe Asp Ile Tyr Glu Ala Phe Asp Ile

385 390 395 400

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

405 410 415

Asp Phe Ile Ile Tyr Tyr Ser Phe Ile His Tyr Lys Asn Asp Ile Ala

420 425 430

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

435 440 445

Asp Lys Val Tyr Glu Glu Ile Ala Arg Asp Thr Trp Asn Glu Tyr Lys

450 455 460

Asp Gln Ile Asn Lys Leu Lys Glu Leu Leu Thr Lys Arg Ile Gly Glu

465 470 475 480

Phe Ser Asp Ala Lys Asn Lys Asn Val Tyr Tyr Lys Glu Phe Glu Ser

485 490 495

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

500 505 510

Tyr Phe Cys Lys Leu Met Tyr Leu Leu Thr Leu Phe Leu Asp Gly Lys

515 520 525

Glu Ile Asn Asp Leu Leu Thr Thr Leu Ile Asn Lys Phe Asp Asn Ile

530 535 540

Arg Ser Phe Ile Glu Ile Met Glu Glu Lys Gln Ile Glu Cys Asn Phe

545 550 555 560

Asp Glu Lys Phe Ser Phe Phe Asp Glu Ser Lys Asn Val Cys Asp Thr

565 570 575

Leu Arg Glu Val Asn Ser Phe Ala Arg Met Gln Arg Pro Leu Asp Asn

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Phe Ile Ile Lys Asn Ile Ile Lys Ser Asn Arg Phe Ile Tyr Leu Ile

645 650 655

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

660 665 670

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

675 680 685

Tyr Tyr Asn Ser Cys Gly Leu Pro Leu Glu Glu Asp Asn Asn Val Gln

690 695 700

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

705 710 715 720

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

725 730 735

Ala Val Val Thr Leu Tyr Leu Thr Ile Leu Tyr Ile Leu Thr Lys Asn

740 745 750

Leu Val Asn Val Asn Ser Arg Tyr Val Ile Ala Leu His Cys Leu Glu

755 760 765

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

770 775 780

Pro Ser Lys Tyr His Lys Leu Thr Gln Tyr Phe Ile Asp Asn Arg Tyr

785 790 795 800

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

805 810 815

Lys Ile Ser Gly Tyr Ile Glu Lys Asn Met Lys Asn Tyr Ile Glu Cys

820 825 830

Glu Gln Ile Glu Thr Thr Glu Gln Tyr Lys Glu Thr Gly Val Asp Met

835 840 845

Phe Ile Asn Tyr Arg Asn Ser Ile Ala His Leu Asn Thr Val Arg Lys

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Asp Lys Asn Asn Thr Arg Lys Thr Lys Lys Ser Ser Leu

945 950 955