CN114634923A - 腺苷脱氨酶、碱基编辑器融合蛋白、碱基编辑器系统及用途 - Google Patents

Abstract

本发明公开了一种腺苷脱氨酶、碱基编辑器融合蛋白、碱基编辑器系统及用途。本发明提供了一种腺苷脱氨酶，其包含以下序列中的一种或多种：(i)如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；(ii)与SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少80％同一性的氨基酸序列；(iii)在SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列等。本发明提供的腺苷脱氨酶，在构成碱基编辑器及用于碱基编辑器系统时，具有优异的编辑效率以及极窄的编辑窗口，促进了疾病的靶向治疗、以及在精准治疗中的应用。

Description

腺苷脱氨酶、碱基编辑器融合蛋白、碱基编辑器系统及用途

技术领域

本发明属于生物技术领域，更具体地，本发明涉及腺苷脱氨酶、碱基编辑器融合蛋白、碱基编辑器系统及用途。

背景技术

如何精准、高效地对基因组进行修饰是生命科学领域研究的重要目标，而CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9介导的基因编辑技术成为实现该目标的最强工具。传统的CRISPR/Cas9技术通过在靶点处产生DNA双链断裂(Double Strand Breaks,DSB)，从而诱发细胞内的同源重组(HomologousRecombination,HR)和非同源末端连接(Non-Homologous End Joining,NHEJ)修复途径，进而实现对基因组DNA的定点敲除、替换、插入等修饰。然而，DSB引发的DNA修复很难实现高效稳定的单碱基突变。

单核苷酸变异会导致大约2/3人类遗传病的发生，也是许多动植物重要性状变异的遗传基础，因此开发一种精准且能够高效实现单碱基替换的技术尤为重要，David R.Liu实验室开发的碱基编辑器就是为此而生的。David R.Liu实验室开发了三种不同的碱基编辑器，分别是胞嘧啶碱基编辑器(Cytosine Base Editor,CBE)、腺嘌呤碱基编辑器(Adenine Base Editor,ABE)和先导编辑器(Prime Editor)，这些碱基编辑器在工作时不依赖DSB的产生，也不需要供体DNA的参与。

以腺苷脱氨酶为基础的腺嘌呤碱基编辑技术主要是利用由切口酶Cas9n(D10A)或dCas9结合腺苷脱氨酶组成融合蛋白，在sgRNA的引导下，将位于碱基编辑活性窗口内的靶碱基腺嘌呤A脱氨形成次黄嘌呤I，再经DNA修复和复制后逐渐被替换为G，最终形成A向G的定向替换(A至G)。

但是目前的碱基编辑器存在位点依赖性的编辑效率的差异，并且编辑窗口较宽导致非必要的编辑，因此对现有碱基编辑器的改造十分必要。

发明内容

发明要解决的问题

本发明的目的在于克服上述缺点，提供了一种新的腺苷脱氨酶，以及包含该腺苷脱氨酶的碱基编辑器融合蛋白、碱基编辑器系统，以提高A·T到G·C的编辑效率并提高碱基编辑的精确性(缩小编辑窗口)。

用于解决问题的方案

在本发明的一些方面，提供了一种腺苷脱氨酶，其包含以下序列中的一种或多种：

(i)如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；

(ii)与SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ IDNO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性；

(iii)在SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

在一些实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：

S15、D16、H17、F19、N20、D21、E22、Y23、W24、R26、H27、L29、K33、R34、A35、V41、V43、L47、L49、N59、A61、I62、A69、E72、G80、L81、V82、L83、Q84、N85、Y86、I89、D90、A91、T92、V95、F97、I112、S113、R114、L115、F117、V119、R120、N121、K123、R124、N132、V133、L134、N135、P137、G138和M139。

在另一些实施方案中，所述的取代为在如SEQ ID NO:2所示的氨基酸序列的以下位点中的一个或多个发生的取代：

K19、A20、R21、E22、V33、L34、D35、D36、A46、I47、T48、L49、V80、T81、F82、E83、P84、I97、K98、R99、G103、V104、S105、N106、S107、K108、R109、G110、L116、N117、V118、L119、N120、Y121、P122、G123、C144、Q145、F146、Y147、Q148、Q149、P150、R151、E152、V153、F154、N155。

在一些具体的实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：S15T、D16E、H17K、F19Y、N20Q、E22D、Y23F、W24F、R26K、H27R、L29I、K33R、R34K、A35S、G80A、L81N、V82A、L83I、Q84N、N85S、Y86W、I89L、D90G、A91T、T92D、I112L、S113K、R114K、N132K、V133I、L134F、N135H、P137F、G138A和M139L。

在一些更具体的实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点的组合发生的取代：

(1)S15T、D16E、H17K、F19Y、N20Q；

(2)E22D、Y23F、W24F、R26K、H27R、L29I；

(3)K33R、R34K、A35S；

(4)G80A、L81N、V82A、L83I、Q84N、N85S、Y86W；

(5)I89L、D90G、A91T、T92D；

(6)I112L、S113K、R114K；

(7)N132K、V133I、L134F、N135H；和/或

(8)P137F、G138A、M139L。

在本发明的一些方面，提供了一种碱基编辑器融合蛋白，其包含上述的腺苷脱氨酶，以及核酸可编程核苷酸结合结构域。

在一些实施方案中，所述核酸可编程核苷酸结合结构域为Cas蛋白或AGO蛋白。

在一些实施方案中，所述碱基编辑器融合蛋白中还包括至少一条核定位信号序列。

在一些可选的实施方案中，所述碱基编辑器融合蛋白中还包括接头。在一些任选的实施方案中，所述接头包含如SEQ ID NO:3～12所示的序列中的一种或多种。

在一些具体的实施方案中，所述碱基编辑器融合蛋白包含以下序列中的一种或多种：

(i)如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列；

(ii)与SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；

(iii)在SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

在一些更具体的实施方案中，所述的碱基编辑器融合蛋白包含SEQ ID NO:46～53所示序列中的任一种。

在本发明的一些方面，提供了一种多核苷酸，其编码上述的腺苷脱氨酶或者编码上述的碱基编辑器融合蛋白。

在本发明的一些方面，提供了一种载体，其包含上述的多核苷酸。

在本发明的一些方面，提供了一种细胞，其包含上述的腺苷脱氨酶、上述的碱基编辑器融合蛋白、上述的多核苷酸和上述的载体中的一种或多种。

在本发明的一些方面，提供了一种碱基编辑器系统，其包含上述的腺苷脱氨酶、核酸可编程核苷酸结合结构域、以及引导多核苷酸；或者，其包含上述的碱基编辑器融合蛋白，以及引导多核苷酸。

在本发明的一些方面，提供了一种药物组合物，其包含上述的腺苷脱氨酶、上述的碱基编辑器融合蛋白、上述的多核苷酸、上述的载体、上述的细胞和上述的碱基编辑器系统中的一种或多种，以及药学上可接受的载体。

在本发明的一些方面，提供了一种试剂盒，其包含上述的腺苷脱氨酶、上述的碱基编辑器融合蛋白、上述的多核苷酸、上述的载体、上述的细胞和上述的碱基编辑器系统中的一种或多种。

在本发明的一些方面，提供了一种递送系统，其包含上述的腺苷脱氨酶、上述的碱基编辑器融合蛋白、上述的多核苷酸、上述的载体、上述的细胞和上述的碱基编辑器系统中的一种或多种，以及递送介质。

在本发明的一些方面，提供了一种核酸的碱基编辑方法，其包括将待被编辑碱基的核酸与上述的碱基编辑器系统相接触的步骤。

在本发明的一些方面，提供了上述的腺苷脱氨酶、上述的碱基编辑器融合蛋白、上述的多核苷酸、上述的载体、上述的细胞、上述的碱基编辑器系统、上述的药物组合物或上述的递送系统在制备用于治疗与点突变相关或由点突变引起的疾病的药物中的用途。

发明的效果

本发明提供的新的腺苷脱氨酶，在构成碱基编辑器及用于碱基编辑器系统时，具有优异的编辑效率以及极窄的编辑窗口，促进了疾病的靶向治疗，并且促进了碱基编辑器在精准治疗中的应用。

附图说明

图1A和图1B分别为PHK09质粒图谱(图1A)以及p004V1-nCas9结构图(图1B)。

图2为p004V1-nCas9在位点1处A·T到G·C的编辑效率，其包括A～C，图2中的A为p004V1-nCas9和ABE8e在位点1的编辑效率的比较，编辑位置为sgRNA 5’端起+3，+5，+7，+8的腺嘌呤脱氧核苷酸，误差线表示平均值±SEM，每组样品3个生物学重复；图2中的B为碱基编辑器p004V1-nCas9转染后在位点1的测序结果；图2中的C为碱基编辑器ABE8e转染后在位点1的测序结果。

图3为p004V1-nCas9在位点17处A·T到G·C的编辑效率，其包括A～C，图3中的A为p004V1-nCas9和ABE8e在位点17的编辑效率的比较，编辑位置为sgRNA 5’端起+3，+4，+5，+7的腺嘌呤脱氧核苷酸，误差线表示平均值±SEM，每组样品3个生物学重复；图3中的B为碱基编辑器p004V1-nCas9转染后在位点17的测序结果；图3中的C为碱基编辑器ABE8e转染后在位点17的测序结果。

图4为p004V1-nCas9在位点18处A·T到G·C的编辑效率，其包括A～C，图4中的A为p004V1-nCas9和ABE8e在位点18的编辑效率的比较，编辑位置为sgRNA 5’端起+3，+5，+7，+9的腺嘌呤脱氧核苷酸，误差线表示平均值±SEM，每组样品3个生物学重复；图4中的B为碱基编辑器p004V1-nCas9转染后在位点18的测序结果；图4中的C为碱基编辑器ABE8e转染后在位点18的测序结果。

图5为p004V1-nCas9在PCSK9位点处A·T到G·C的编辑效率，其包括A～C，图5中的A为p004V1-nCas9和ABE8e在PCSK9位点的编辑效率的比较，编辑位置为sgRNA 5’端起+6的腺嘌呤脱氧核苷酸，误差线表示平均值±SEM，每组样品3个生物学重复；图5中的B为碱基编辑器p004V1-nCas9转染后在PCSK9位点的测序结果；图5中的C为碱基编辑器ABE8e转染后在PCSK9位点的测序结果。

图6为004V1各个突变体-nCas9在不同位点的编辑效率。

图7为p005V1-nCas9质粒图谱。

图8为p005V1-nCas9在位点1处A·T到G·C的编辑效率，其包括A～C，图8中的A为p005V1-nCas9和ABE8e在位点1的编辑效率的比较，编辑位置为sgRNA 5’端起+3，+5，+7，+8的腺嘌呤脱氧核苷酸，误差线表示平均值±SEM，每组样品3个生物学重复；图8中的B为碱基编辑器p005V1-nCas9转染后在位点1的测序结果；图8中的C为碱基编辑器ABE8e转染后在位点1的测序结果。

具体实施方式

为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。

本说明书中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。

本说明书中，使用“基本上”或“实质上”表示与理论模型或理论数据的标准偏差在5％、优选为3％、更优选为1％范围以内。

本说明书中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。

本说明书中，“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生，并且该描述包括该事件发生的情况和该事件不发生的情况。

本说明书中，所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如，特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中，并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外，应理解，所述要素可以任何合适的方式组合在各种实施方案中。

如本公开所使用的，术语“核酸”和“核酸分子”是指包含核碱基和酸性部分的化合物，例如核苷、核苷酸或核苷酸的聚合物。通常，聚合核酸，例如包含三个或更多个核苷酸的核酸分子是线性分子，其中相邻核苷酸通过磷酸二酯键相互连接。在一些实施方案中，“核酸”是指单个核酸残基(例如核苷酸和/或核苷)。在一些实施方案中，“核酸”是指包含三个或更多个单独核苷酸残基的寡核苷酸链。如本文所用，术语“寡核苷酸”和“多核苷酸”可互换使用以指核苷酸的聚合物(例如，至少三个核苷酸的串)。在一些实施方案中，“核酸”包括RNA以及单链和/或双链DNA。核酸可以是天然存在的，例如在基因组、转录物、mRNA、tRNA、rRNA、siRNA、snRNA、质粒、粘粒、染色体、染色单体或其他天然存在的核酸分子的上下文中。另一方面，核酸分子可以是非天然存在的分子，例如重组DNA或RNA、人工染色体、工程基因组或其片段，或合成的DNA、RNA、DNA/RNA杂交体、或包括非天然存在的核苷酸或核苷。此外，术语“核酸”、“DNA”、“RNA”和/或类似术语包括核酸类似物，例如具有除磷酸二酯骨架之外的其他骨架的类似物。核酸可以从天然来源纯化、使用重组表达系统产生和任选地纯化、化学合成等。在合适的情况下，例如在化学合成分子的情况下，核酸可以包含核苷类似物，例如具有化学修饰碱基的类似物或糖和骨架修饰。

如本公开所使用的，术语“多肽”、“肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。

如本公开所使用的，“融合蛋白”是指包含来自至少两种不同蛋白质的蛋白质结构域的杂化多肽。一种蛋白质可位于融合蛋白的氨基-末端(N-末端，N端)部分或羧基-末端(C-末端，C端)蛋白处，因此分别形成“氨基-末端融合蛋白”或“羧基-末端融合蛋白”。本文提供的任何蛋白质可通过本领域已知的任何方法产生。例如，本文提供的蛋白质可经由重组蛋白质表达和纯化来产生，这尤其适合于包含肽接头的融合蛋白。用于重组蛋白质表达和纯化的方法是公知的，并且包括以下中所述的那些：Green and Sambrook,MolecularCloning:A Laboratory Manual(4th ed.,Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.(2012))，其全部内容通过引用并入本文。

如本公开所使用的，术语“CRISPR”是指成簇、规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats)，其来自微生物的免疫系统。

如本公开所使用的，术语“Cas蛋白”是指CRISPR-associated蛋白，Cas蛋白与CRISPR序列共同构成CRISPR/Cas系统，Cas蛋白具有与核酸酶相关的功能结构域，通过识别PAM(protospacer adjacent motif)在特定位置切割靶序列。

如本公开所使用的，术语“crRNA”包含重复序列(repeat)和间隔序列(spacer)，CRISPR转录形成长链的pre-CRISPR RNA(pre-crRNA)，pre-crRNA加工后得到包含一段重复区序列和一段间隔区序列的短的crRNA。在一些CRISPR/Cas系统中，crRNA由Cas蛋白作用于pre-crRNA得到。在另外一些CRISPR/Cas系统中，crRNA由Cas蛋白与tracrRNA(trans-activating crRNA)共同作用于pre-crRNA得到。

在不同的CRISPR/Cas系统中，crRNA可以单独作为引导RNA(guide RNA，gRNA)引导Cas蛋白定位到位于PAM序列附近的靶序列，或者crRNA与tracrRNA合并成为单一引导RNA(single guide RNA，sgRNA)引导Cas蛋白定位到位于PAM序列附近的靶序列。

如本公开所使用的，“crRNA的引导序列”是指crRNA中与目标核酸的靶序列杂交的序列，其对应由crRNA的间隔序列(spacer)形成。

如本公开所使用的，术语“靶序列”是指目标核酸中与crRNA互补或至少部分互补的核苷酸序列，Cas蛋白、crRNA与靶序列形成三元复合物后，Cas蛋白发挥对目标核酸中靶核酸链和/或非核苷酸链的特异性切割活性。在本公开中，“靶序列”与“靶核酸”、“靶多核苷酸”、“目标序列”、“目标核酸序列”可以互换地使用。

如本公开所使用的，术语“靶标链”(target strand)是指目标核酸中与crRNA杂交的核苷酸链；术语“非靶标链”(non-target strand)是指目标核酸中与crRNA不发生杂交配对的核苷酸链。

术语“Cas9”或“Cas9结构域”是指RNA引导的核酸酶，其包含Cas9蛋白或其片段(例如，包含Cas9的活性、无活性或部分活性的DNA切割结构域的蛋白，和/或gRNA Cas9的结合结构域)。Cas9核酸酶有时也称为CRISPR相关核酸酶9。如前所述，CRISPR是一种适应性免疫系统，可针对移动遗传元件(病毒、转座元件和接合质粒)提供保护。CRISPR簇包含众多短而保守的重复序列区(repeat)和间隔区(spacer)。CRISPR簇被转录并加工成pre-crRNA。在第II型CRISPR/cas9系统中，正确处理pre-crRNA需要转编码的小RNA(tracrRNA)、内源性核糖核酸酶3(RNase III)和Cas9蛋白。tracrRNA作为核糖核酸酶3辅助处理pre-crRNA的引导。随后，Cas9/crRNA/tracrRNA核酸内切切割与间隔序列互补的线性或环状dsDNA靶标。与crRNA不互补的靶标链通过核酸内切方式切割。在自然界中，DNA结合和切割通常需要蛋白质和两种RNA。然而，可以对单一引导RNA(single guide RNA,sgRNA)进行工程改造，以便将crRNA和tracrRNA的各个方面整合到单个RNA种类中。参见，如，Jinek M.等人，Science337:816-821(2012)，其全部内容以引用方式并入本文。Cas9识别CRISPR重复序列(PAM或原间隔序列临近基序)中的一个短基序，以帮助区分自我与非自我。Cas9核酸酶序列和结构是本领域技术人员众所周知的(参见，如“Complete genome sequence of an M1 strain ofStreptococcus pyogenes.”Ferretti等，Proc.Natl.Acad.Sci.U.S.A.98:4658-4663(2001)；“CRISPR RNA maturation by trans-encoded small RNA and host factorRNase III.”Deltcheva E.等，Nature471:602-607(2011)；和“Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.”Jinek M.等，Science337:816-821(2012))。Cas9直向同源物已在各种物种中得到描述，包括但不限于化脓链球菌和嗜热链球菌。基于本公开内容，其他合适的Cas9核酸酶和序列对本领域技术人员来说是显而易见的，并且此类Cas9核酸酶和序列包括来自Chylinski,Rhun和Charpentier,“ThetracrRNA and Cas9 families of type II CRISPR-Cas immunity systems”(2013)RNABiology10:5,726-737中公开的生物体和基因座的Cas9序列；其全部内容在此引入作为参考。

核酸酶灭活的Cas9蛋白可互换地称为“dCas9”蛋白(核酸酶-“死的”Cas9或无核酸酶活性的Cas9)或催化灭活的Cas9。用于产生具有无活性DNA切割结构域的Cas9蛋白(或其片段)是已知的(参见，如Jinek等,Science.337:816-821(2012)；Qi等人“RepurposingCRISPR as an RNA-Guided Platform for Sequence-Specific Control of GeneExpression”(2013)Cell.28；152(5):1173-83)。例如，已知Cas9的DNA切割结构域包括两个子结构域，HNH核酸酶子结构域和RuvC子结构域。HNH子结构域切割与gRNA互补的链，而RuvC子结构域切割非互补链。这些子结构域内的突变可以使Cas9的核酸酶活性沉默。例如，突变D10A和H840A使化脓性链球菌Cas9的核酸酶活性完全灭活(Jinek等人,Science.337:816-821(2012)；Qi等人,Cell.28:152(5):1173-83(2013))。基于本领域的知识，另外的合适的无核酸酶活性的Cas9结构域对于本领域技术人员而言将是显而易见的。此类另外的示例性合适的无核酸酶活性的Cas9结构域包括但不限于D10A/H840A、D10A/D839A/H840A和D10A/D839A/H840A/N863A突变体结构域(参见例如Prashant等，Nature Biotechnology.2013；31(9)：833-838)。

Cas9切口酶能够切割双链DNA的一条链。可以通过将失活的突变引入到HNH子结构域或RuvC子结构域中来生成Cas9切口酶。例如，可以在酿脓链球菌Cas9的RuvC结构域中引入失活的突变(D10A)，而HNH结构域保持有活性，即位置840处的残基保持为组氨酸。此类Cas9变体能够基于gRNA确定的靶序列在特定位置处生成单链DNA断裂(切口)。本领域技术人员能够鉴定任何已知Cas9蛋白的RuvC和HNH结构域中的催化残基并引入失活的突变以生成相应的dCas9或nCas9。

同样地，对于其他Cas蛋白，本领域技术人员可以通过同样的方式获得相应的无核酸酶活性的Cas蛋白和切割双链DNA的一条链的切口酶。

如本公开所使用的，术语“脱氨酶”或“脱氨酶结构域”是指催化脱氨反应的蛋白质或酶。在一些实施方案中，脱氨酶是腺苷脱氨酶，其催化腺嘌呤水解脱氨为次黄嘌呤。在一些实施方案中，脱氨酶是腺苷脱氨酶，其催化腺苷或腺嘌呤(A)水解脱氨为肌苷(I)。在一些实施方案中，脱氨酶或脱氨酶结构域是分别催化腺苷或脱氧腺苷水解脱氨为肌苷或脱氧肌苷的腺苷脱氨酶。在一些实施方案中，腺苷脱氨酶催化脱氧核糖核酸(DNA)中腺苷的水解脱氨。

如本公开所使用的，术语“核酸可编程核苷酸结合结构域”、“核酸可编程DNA结合蛋白(Nucleic acid programmable DNA-binding protein,napDNAbp)”是指与核酸(例如，DNA或RNA)，例如引导多核苷酸(例如，gRNA)结合的蛋白质，所述核酸例如通过与靶核酸序列杂交将napDNAbp引导至特定核酸序列。例如，Cas9蛋白可以与引导RNA结合，该引导RNA将Cas9蛋白引导至与引导RNA互补的特定DNA序列。在一些实施方案中，napDNAbp是Cas9结构域，例如，核酸酶活性Cas9，Cas9切口酶(nCas9)或无核酸酶活性Cas9(dCas9)。核酸可编程DNA结合蛋白的实例包括但不限于Cas9(例如，dCas9和nCas9)、CasX、CasY、Cpf1、C2c1、C2c2、C2c3和Argonaute蛋白(AGO)。然而，应当理解，核酸可编程DNA结合蛋白也包括结合RNA的核酸可编程蛋白。例如，napDNAbp可以与将napDNAbp引导至RNA的核酸结合。其他核酸可编程DNA结合蛋白也在本公开内容的范围内，尽管它们可能未在本公开内容中具体描述。

如本公开所使用的，“碱基编辑器(Base Editor,BE)”或“核碱基编辑器”是指结合多核苷酸并具有核碱基修饰活性的试剂。在各种实施方案中，碱基编辑器包含核碱基修饰多肽(例如，脱氨酶)和与引导多核苷酸(例如，引导RNA)结合的核酸可编程核苷酸结合结构域(例如，核酸可编程DNA结合蛋白)。在各种实施方案中，所述试剂是包含具有碱基编辑活性的蛋白质结构域的生物分子复合物，即能够修饰核酸分子(例如，DNA、RNA)内的碱基(例如，A、T、C、G或U)。在一些实施方案中，所述多核苷酸可编程DNA结合结构域与脱氨酶结构域融合或连接。在一个实施方案中，所述试剂是包含具有碱基编辑活性的结构域的融合蛋白。在一些实施方案中，具有碱基编辑活性的结构域能够使核酸分子内的碱基脱氨基。在一些实施方案中，所述碱基编辑器能够使DNA分子内的一个或多个碱基脱氨基。在一些实施方案中，所述碱基编辑器是腺苷碱基编辑器(ABE)。

如本公开所使用的，“碱基编辑活性”是指用于化学改变多核苷酸内的碱基。在一个实施方案中，第一个碱基被转化为第二个碱基。在一个实施方案中，所述碱基编辑活性是腺苷或腺嘌呤脱氨酶活性，例如将靶标A·T转化为C·G。

在一些实施例中，碱基编辑活性通过编辑效率来评估。碱基编辑效率可以通过任何合适的方式来测量，例如，通过sanger测序或二代测序。在一些实施方案中，碱基编辑效率通过具有受碱基编辑器影响的核碱基转化的总测序读数的百分比来测量，例如，具有转化为A·T碱基对的目标C·G碱基对的总测序读数的百分比。在一些实施方案中，当在细胞群中进行碱基编辑时，碱基编辑效率通过具有受碱基编辑器影响的核碱基转化的总细胞的百分比来测量。

如本公开所使用的，术语“碱基编辑器系统”是指用于编辑目标核苷酸序列的核碱基的系统。在各种实施方案中，所述碱基编辑器系统包含(1)核酸可编程核苷酸结合结构域(例如Cas9)；(2)用于使所述核碱基脱氨基的脱氨酶结构域(例如腺苷脱氨酶)；(3)一种或多种引导多核苷酸(例如引导RNA)。

“引导多核苷酸”、“引导RNA”或“gRNA”是指可以特异性靶向目标序列并且可以与核酸可编程核苷酸结合结构域蛋白(例如Cas9)形成复合物的多核苷酸。在一个实施方案中，引导多核苷酸是引导RNA(gRNA)。gRNA可以作为两个或多个RNA的复合物存在，也可以作为单个RNA分子存在。以单个RNA分子形式存在的gRNA可称为单一引导RNA(sgRNA)，但“gRNA”可互换使用以指以单个分子或两个或更多个分子的复合物形式存在的引导RNA。通常，作为单个RNA种类存在的gRNA包括两个结构域：(1)与目标核酸具有同源性的结构域(例如，引导Cas9复合物与目标核酸的结合)；(2)结合Cas9蛋白的结构域。在一些实施方案中，结构域(2)对应于称为tracrRNA的序列，并且包括茎环结构。例如，在一些实施方案中，结构域(2)与Jinek et al.,Science 337:816-821(2012)中提供的tracrRNA相同或同源。gRNA的其他实例可以为在2013年9月6日提交的美国临时专利申请U.S.S.N.61/874,682(发明名称为“可切换的Cas9核酸酶及其用途(Switchable Cas9 Nucleases and Uses Thereof)”)和在2013年9月6日提交的美国临时专利申请U.S.S.N.61/874,746发明名称为“功能性核酸酶递送系统(Delivery System For Functional Nucleases)”中公开的。在一些实施方案中，gRNA包括结构域(1)和(2)中的两个或更多个，并且可以被称为“延伸的gRNA”。延伸的gRNA将结合两个或更多个Cas9蛋白并在两个或更多个不同区域结合目标核酸。gRNA包括与目标位点互补的核苷酸序列，其介导核酸酶/RNA复合物与所述目标位点的结合，提供核酸酶：RNA复合物的序列特异性。

根据本发明，所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。

根据本发明，氨基酸“添加”指在氨基酸序列的C端或N端添加氨基酸。根据本发明，氨基酸“缺失”指可以从氨基酸序列中删除1、2或3个以上氨基酸。根据本发明，氨基酸“插入”指在氨基酸序列中的适当位置插入氨基酸残基，插入的氨基酸残基也可以全部或部分彼此相邻，或插入的氨基酸之间都不彼此相邻。

根据本发明，氨基酸“取代”指在氨基酸序列中的某个位置的某个氨基酸残基被其他氨基酸残基替代；其中，“取代”可以是保守氨基酸取代。

根据本发明，“保守修饰”、“保守取代”或“保守置换”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见Watson等(1987),MolecularBiology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。

示例性氨基酸保守取代

原始残基	保守取代
		Ala(A)	Gly(G)；Ser(S)
Arg(R)	Lys(K)；His(H)
		Asn(N)	Gln(Q)；His(H)；Asp(D)
Asp(D)	Glu(E)；Asn(N)
		Cys(C)	Ser(S)；Ala(A)；Val(V)
Gln(Q)	Asn(N)；Glu(E)
		Glu(E)	Asp(D)；Gln(Q)
Gly(G)	Ala(A)
		His(H)	Asn(N)；Gln(Q)
Ile(I)	Leu(L)；Val(V)
		Leu(L)	Ile(I)；Val(V)
Lys(K)	Arg(R)；His(H)
		Met(M)	Leu(L)；Ile(I)；Tyr(Y)
Phe(F)	Tyr(Y)；Met(M)；Leu(L)
		Pro(P)	Ala(A)
Ser(S)	Thr(T)
		Thr(T)	Ser(S)
Trp(W)	Tyr(Y)；Phe(F)
		Tyr(Y)	Trp(W)；Phe(F)
Val(V)	Ile(I)；Leu(L)

又如，具有类似特征的氨基酸如下所示：

酸性残基	Asp(D)和Glu(E)
		碱性残基	Lys(K)、Arg(R)和His(H)
亲水性不带电荷的残基	Ser(S)、Thr(T)、Asn(N)和Gln(Q)
		脂肪族不带电荷的残基	Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)
非极性不带电荷的残基	Cys(C)、Met(M)和Pro(P)
		芳香族残基	Phe(F)、Tyr(Y)和Trp(W)

如本文可互换使用的术语“编码序列”或“蛋白质编码序列”是指编码蛋白质的多核苷酸片段。该区域或序列在靠近5'端的地方有一个起始密码子，在靠近3'端的地方有一个终止密码子。编码序列也可称为开放阅读框。

术语“核定位序列”、“核定位信号(Nuclear Localization Signal，NLS)”是指促进蛋白质输入细胞核的氨基酸序列。核定位序列是本领域已知的并且描述于例如Plank等人的国际PCT申请，PCT/EP2000/011690，2000年11月23日提交，2001年5月31日作为WO/2001/038547公布，其内容以引用方式并入本文以用于它们对示例性核定位序列的公开。在一些实施方案中，所述NLS是优化的NLS，例如由Koblan等人，Nature Biotech.2018doi:10.1038/nbt.4172所描述。

如本文所用，术语“接头”可指共价接头(例如，共价键)、非共价接头、化学基团或连接两个分子或部分(例如，蛋白质复合物或核糖核复合体的两个组分)的分子，例如，融合蛋白的两个结构域，如多核苷酸可编程DNA结合结构域(例如，dCas9)和脱氨酶结构域(例如，腺苷脱氨酶)。接头可以连接碱基编辑器系统的不同组分或组分的不同部分。例如，在一些实施方案中，接头可以连接多核苷酸可编程核苷酸结合结构域的引导多核苷酸结合结构域和脱氨酶的催化结构域。接头可以位于两个基团、分子或其他部分之间或两侧，并通过共价键或非共价相互作用连接到每一个，从而连接这两者。在一些实施方案中，所述接头可以是多核苷酸。在一些实施方案中，所述接头可以是DNA接头。在一些实施方案中，所述接头可以是RNA接头。

在一些实施方案中，接头可以是一个氨基酸或多个氨基酸(例如，肽或蛋白质)。在一些实施方案中，所述接头的长度可为约5至100个氨基酸，例如约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、20至30、30至40、40至50、50至60、60至70、70至80、80至90或90至100个氨基酸的长度。在一些实施例中，接头的长度可为约100至150、150至200、200至250、250至300、300至350、350至400、400至450或450至500个氨基酸。也可以考虑更长或更短的接头。

如本公开所使用的，术语“切割”可以是指使核苷酸链中磷酸二酯键断裂。对于断裂的类型可以是单链断裂或双链断裂。

如本公开所使用的，术语“互补的”或“杂交的”用于指与碱基配对规则相关的“多核苷酸”和“寡核苷酸”(它们是可互换的术语，指的是核苷酸序列)。例如，序列“CAGT”与序列“GTCA”互补。互补可以是“部分的”或“全部的”。“部分”互补是指一个或多个核酸碱基根据碱基配对规则错配，核酸之间的“全部”或“完全”互补是指每个核酸碱基在碱基配对下均与另一个碱基匹配规则。核酸链之间的互补程度对核酸链之间的杂交效率和强度具有重要影响。这在扩增反应以及取决于核酸之间结合的检测方法中特别重要。

如本文所用，术语“杂交”是指使用核酸链通过碱基配对与互补链结合以形成杂交复合物的任何过程来配对互补核酸。

如本公开所使用的，术语“核酸序列”和“核苷酸序列”是指寡核苷酸或多核苷酸及其片段或部分，并且是指可以是单链或双链的基因组或合成来源的DNA或RNA，和代表有义或反义链。

如本公开所使用的，术语“序列同一性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。

示例性的，当使用序列比较算法或通过目视检查测量以最大的对应性进行比较和比对时，两个或多个序列或子序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％核苷酸的“序列同一性”或“同一性百分比”。在某些实施方案中，所述序列在任一或两个相比较的生物聚合物(例如，多核苷酸)的整个长度上基本相同。

术语“载体”是指将核酸序列引入细胞中从而产生转化细胞的手段。载体包括质粒、转座子、噬菌体、病毒、脂质体和附加体。“表达载体”是包含待在受体细胞中表达的核苷酸序列的核酸序列。表达载体可以包括额外的核酸序列以促进和/或促进引入序列的表达，例如起始、终止、增强子、启动子和分泌序列。

如本公开所使用的，术语“个体”和“受试者”可互换地使用，是指哺乳动物。哺乳动物包括但不限于驯化动物(例如，奶牛、绵羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体是人。

本文公开的方法可以在体外、离体、或体内进行，或者产品可以以体外、离体、或体内形式存在。术语“体外”是指在实验室条件或培养液中使用材料、生物物质、细胞和/或组织的实验；而术语“体内”是指使用完整多细胞有机体的实验和工序。在一些实施方案中，体内进行的方法可以在非人动物上进行。“离体”是指存在于有机体外或发生在有机体外，例如在人或动物体外的事件，例如可以在取自有机体的组织(例如整个器官)或细胞上存在或发生的事件。

如本公开所使用的，术语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，例如液体或固体填充剂、稀释剂、赋形剂、制造助剂(例如，润滑剂、滑石粉、硬脂酸镁、钙或锌或硬脂酸)或溶剂包封材料，涉及将化合物从身体的一个部位(例如，递送部位)运送或运输到另一个部位(例如，器官、组织或身体的一部分)。药学上可接受的载体是“可接受的”，意思是与制剂的其他成分相容并且对受试者的组织无害(例如，生理学相容的、无菌的、生理学的pH等)。可以充当药学上可接受的载体的材料的一些实例包括：(1)糖，例如乳糖、葡萄糖和蔗糖；(2)淀粉，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和醋酸纤维素；(4)粉末黄蓍胶；(5)麦芽；(6)明胶；(7)润滑剂，如硬脂酸镁、十二烷基硫酸钠和滑石粉；(8)赋形剂，如可可脂和栓剂蜡；(9)油，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；(10)二醇，如丙二醇；(11)多元醇，如甘油、山梨糖醇、甘露醇和聚乙二醇(PEG)；(12)酯类，如油酸乙酯和月桂酸乙酯；(13)琼脂；(14)缓冲剂，如氢氧化镁和氢氧化铝；(15)海藻酸；(16)无热原水；(17)等渗盐水；(18)林格氏液；(19)乙醇；(20)pH缓冲溶液；(21)聚酯，聚碳酸酯和/或聚酸酐；(22)增量剂(bulking agent)，如多肽和氨基酸(23)血清成分，如血清白蛋白、高密度脂蛋白(high density lipoprotein,HDL)和低密度脂蛋白(low density lipoprotein，LDL)；(22)C2-C12醇，如乙醇；和(23)药物制剂中采用的其他无毒相容物质。润湿剂、着色剂、脱模剂、包衣剂、甜味剂、调味剂、芳香剂、防腐剂和抗氧化剂也可以存在于制剂中。诸如“赋形剂”、“药学上可接受的载体”等术语在本文中可互换使用。

如本文所用，术语“有效量”是指足以引起期望的生物学反应的生物活性剂的量。例如，在一些实施方案中，碱基编辑器的有效量可以指足以诱导由碱基编辑器突变的特异性结合的靶位点的突变的碱基编辑器的量。如本领域技术人员将理解的，试剂，例如碱基编辑器融合蛋白、脱氨酶、多核苷酸等的有效量可以随各种因素而变化，例如随期望的生物学反应，例如随待编辑的特定等位基因、基因组或靶位点，随靶定的细胞或组织和使用的试剂而变化。

术语“治疗”、“处理”是指如本文所述旨在逆转、缓解疾病或病症或其一种或多种症状、延迟疾病或病症或其一种或多种症状的发作或抑制疾病或病症或其一种或多种症状进展的临床干预。如本文所用，术语“治疗”、“处理”是指如本文所述旨在逆转、缓解疾病或病症或其一种或多种症状、延迟疾病或病症或其一种或多种症状的发作或抑制疾病或病症或其一种或多种症状进展的临床干预。在一些实施方案中，可以在一种或多种症状已经得以形成之后和/或疾病已经得到诊断之后施用治疗。在其他实施方案中，可以在没有症状的情况下施用治疗，例如用于预防或延迟症状的发作或抑制疾病的发作或进展。例如，可以在症状发作之前(例如，鉴于症状的历史和/或鉴于遗传或其他易感性因素)施用治疗于易感个体。治疗也可以在症状消退后继续进行，例如以预防或延迟其复发。

<腺苷脱氨酶>

在本发明的第一方面的一些实施方案中，提供了一种腺苷脱氨酶，其包含以下序列中的一种或多种：

(i)如下SEQ ID NO:1所示的氨基酸序列；

MYNAPRFFCRSAAVSDHEFNDEYWMRHALTLAKRAREEGEVPVGAVLVLNNQVIGEGWNRAIGLHDPTAHAEIMALRQGGLVLQNYRLIDATLYVTFEPCVMCAGAMVHSRISRLVFGVRNSKRGAAGSLINVLNYPGMNHRVEITEGILAESCSAMLCDFYRWPREVFNALKKARQEEG(腺苷脱氨酶004V1)

(ii)与SEQ ID NO:1所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ ID NO:1所示的氨基酸序列的脱氨活性；

(iii)在SEQ ID NO:1所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:1所示的氨基酸序列的脱氨活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:1所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ IDNO:1所示的氨基酸序列的脱氨活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

在一些实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：

S15、D16、H17、F19、N20、D21、E22、Y23、W24、R26、H27、L29、K33、R34、A35、V41、V43、L47、L49、N59、A61、I62、A69、E72、G80、L81、V82、L83、Q84、N85、Y86、I89、D90、A91、T92、V95、F97、I112、S113、R114、L115、F117、V119、R120、N121、K123、R124、N132、V133、L134、N135、P137、G138和M139。

进一步地，在一些实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：

S15T、D16E、H17K、F19Y、N20Q、E22D、Y23F、W24F、R26K、H27R、L29I、K33R、R34K、A35S、G80A、L81N、V82A、L83I、Q84N、N85S、Y86W、I89L、D90G、A91T、T92D、I112L、S113K、R114K、N132K、V133I、L134F、N135H、P137F、G138A和M139L。

在一些具体的实施方案中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点的组合发生的取代：

(1)S15T、D16E、H17K、F19Y、N20Q(腺苷脱氨酶004V2)；

(2)E22D、Y23F、W24F、R26K、H27R、L29I(腺苷脱氨酶004V3)；

(3)K33R、R34K、A35S(腺苷脱氨酶004V4)；

(4)G80A、L81N、V82A、L83I、Q84N、N85S、Y86W(腺苷脱氨酶004V7)；

(5)I89L、D90G、A91T、T92D(腺苷脱氨酶004V8)；

(6)I112L、S113K、R114K(腺苷脱氨酶004V10)；

(7)N132K、V133I、L134F、N135H(腺苷脱氨酶004V12)；和/或

(8)P137F、G138A、M139L(腺苷脱氨酶004V13)。

本发明的第一方面的另一些实施方案中，提供了一种腺苷脱氨酶，其包含以下序列中的一种或多种：

(i)如下SEQ ID NO:2所示的氨基酸序列；

MSELNDAYWMKQALALAQKAREQGEVPVGAILVLDDEVIGQGWNRAITLHDPTAHAEIMALQQGGQIVQNYRLLNATLYVTFEPCVMCAGAMVHSRIKRLVYGVSNSKRGAAGSLLNVLNYPGMNHQIEITAGVMANECSEMLCQFYQQPREVFNAEREARRLNQPDRAD(腺苷脱氨酶005V1)

(ii)与SEQ ID NO:2所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ ID NO:2所示的氨基酸序列的脱氨活性；

(iii)在SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:2所示的氨基酸序列的脱氨活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:2所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ IDNO:2所示的氨基酸序列的脱氨活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

在一些实施方案中，所述的取代为在如SEQ ID NO:2所示的氨基酸序列的以下位点中的一个或多个发生的取代：

K19、A20、R21、E22、V33、L34、D35、D36、A46、I47、T48、L49、V80、T81、F82、E83、P84、I97、K98、R99、G103、V104、S105、N106、S107、K108、R109、G110、L116、N117、V118、L119、N120、Y121、P122、G123、C144、Q145、F146、Y147、Q148、Q149、P150、R151、E152、V153、F154、N155。

在本发明中，“保留脱氨活性”的表述，例如“保留如SEQ ID NO:1所示的氨基酸序列的脱氨活性”、“保留如SEQ ID NO:2所示的氨基酸序列的脱氨活性”，可以是完全保留原始序列的腺苷脱氨酶的脱氨活性，也可以是部分保留原始序列的腺苷脱氨酶的脱氨活性，例如保留30％、40％、50％、60％、70％、80％、90％、95％、99％的脱氨活性。在另一些实施方案中，具有修改后的序列的腺苷脱氨酶，例如具有经氨基酸取代的序列的腺苷脱氨酶也可能具有高于原始序列的腺苷脱氨酶的脱氨活性。

本发明提供的腺苷脱氨酶可作用于任何多核苷酸，包括DNA、RNA和DNA-RNA杂交体。在某些实施方案中，腺苷脱氨酶可以使包含DNA的多核苷酸的靶标腺嘌呤(A)脱氨基。在某些实施方案中，腺苷脱氨酶可以使包含RNA的多核苷酸的靶标腺嘌呤(A)脱氨基。

在一些实施方案中，所述的取代为保守取代。

在本发明中，“中等严格条件”，“中-高严格条件”，“高严格条件”或“非常高严格条件”描述了核酸杂交和洗涤的条件。进行杂交反应的指导参见Current Protocols inMolecular Biology，John Wiley&Sons，N.Y.(1989)，6.3.1-6.3.6，其通过引用并入本文。在该文献中描述了含水的和非含水的方法，且可以使用任一种。例如，具体的杂交条件如下：(1)低严格性杂交条件在6×氯化钠/柠檬酸钠(SSC)中，在约45℃，然后在至少50℃，在0.2×SSC，0.1％SDS中洗涤2次(对于低严格性条件，可以将洗涤温度升高到55℃)；(2)中等严格性杂交条件在6×SSC，在约45℃，然后在60℃，在0.2×SSC，0.1％SDS中洗涤1次或多次；(3)高严格性杂交条件在6×SSC，在约45℃，然后在65℃，在0.2×SSC，0.1％SDS中洗涤1次或多次且优选；(4)非常高的严格性杂交条件是0.5M磷酸钠，7％SDS，在65℃，然后在65℃，在0.2×SSC，1％SDS中洗涤1次或多次。

<碱基编辑器融合蛋白>

本发明的第二方面提供了一种碱基编辑器融合蛋白，其包含本发明第一方面所述腺苷脱氨酶，以及核酸可编程核苷酸结合结构域。

在本发明中，核酸可编程核苷酸结合结构域当与结合的引导多核苷酸(例如，gRNA)结合时，可以特异性结合靶多核苷酸序列(即，通过结合的引导核酸的碱基和靶多核苷酸的碱基之间的互补碱基配对序列)，从而将碱基编辑器定位到需要编辑的目标核酸序列。在一些实施方案中，靶多核苷酸序列包含单链DNA或双链DNA。在一些实施方案中，所述靶多核苷酸序列包含RNA。在一些实施方案中，靶标多核苷酸序列包含DNA-RNA杂交体。应当理解，核酸可编程核苷酸结合结构域还可以包括结合RNA的核酸可编程蛋白。

在本发明的一些实施方案中，碱基编辑器中的核酸可编程核苷酸结合结构域是Cas蛋白或者AGO蛋白。所述Cas蛋白或AGO蛋白包括天然存在的Cas蛋白或AGO蛋白，以及其同源物或其修饰或工程化版本。例如，在一些实施方案中，Cas蛋白或AGO蛋白可以是包括与天然存在的Cas蛋白或AGO蛋白的氨基酸序列具有至少60％，至少65％，至少70％，至少75％，至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或至少99.5％同一性的氨基酸序列的蛋白。在另一些实施方案中，Cas蛋白还包括其切口酶或无核酸酶活性形式的蛋白。

在一些实施方案中，可以作为核酸可编程核苷酸结合结构域的Cas蛋白的非限制实例包括Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas5d、Cas5t、Cas5h、Cas5a、Cas6、Cas7、Cas8、Cas8a、Cas8b、Cas8c、Cas9(也已知为Csn1或Csx12)、Cas10、Cas10d、Cas12a/Cpfl、Cas12b/C2cl、Cas12c/C2c3、Cas12d/CasY、Cas12e/CasX、Cas12f(C2c10/Cas14)、Cas12g、Cas12h、Cas12i、Cas12j、Cas12k/C2c5、Cas12l、Cas12m、Cas12n、Cas13a(C2c2)、cas13b、Cas13c、Cas13d、Csy1、Csy2、Csy3、Csy4、Css1、Css2、Cse5e、Csc2、Csa5、Csn1、Csn2、Csm1、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr2、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、Csx1、Csx1S、Csx11、Csf1、Csf2、CsO、Csf4、Csd1、Csd2、Cst1、Cst2、Csh1、Csh2、Csa1、Csa2、Csa3、Csa4、Csa5、II类Cas效应蛋白、V型Cas效应蛋白、VI类蛋白质、CARF、DinG、其同源物或其修饰或工程化版本。其他核酸可编程核苷酸结合结构域也在本公开的范围内，尽管它们可能未在本公开中具体列出。

在本发明的一些具体的实施方案中，Cas蛋白选自Cas9家族、Cas12家族和Cas13家族，例如但不限于Cas9、Cas12a(Cpf1)、Cas12b(C2c1)、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12f(C2c10/Cas14)、Cas12g、Cas12h、Cas12i、Cas12j、Cas12k(C2c5)、Cas12l、Cas12m、Cas12n、Cas13a(C2c2)、Cas13b、Cas13c、Cas13d、其同源物或其修饰或工程化版本。在本发明的一些具体的实施方案中，Cas蛋白包括上述Cas蛋白的无核酸酶活性形式，例如dCas9、dCas12a、dCas12b、dCas12c、dCas12d、dCas12e、dCas12f、dCas12g、dCas12h、dCas12i、dCas12j、dCas12k、dCas12l、dCas12m、dCas12n、dCas13a、dcas13b、dCas13c和dCas13d。在本发明的一些具体的实施方案中，Cas蛋白还包括上述蛋白的切口酶形式，例如但不限于nCas9。

在本发明的一些优选的实施方案中，核酸可编程核苷酸结合结构域为Cas9。在本发明的一些具体的实施方案中，Cas9是来自化脓性链球菌的Cas9(SpCas9)、金黄色葡萄球菌的Cas9(SaCas9)、嗜热链球菌1的Cas9(St1Cas9)。在本发明的一些优选的实施方案中，Cas9是来自化脓性链球菌的Cas9(SpCas9)。

在本发明的一些更优选的实施方案中Cas9可以是核酸酶活性Cas9、Cas9切口酶(nCas9)或无核酸酶活性Cas9(dCas9)。

在本发明的一些进一步优选的实施方案中，核酸可编程核苷酸结合结构域为Cas9切口酶(nCas9)。在本发明的另一些进一步优选的实施方案中，核酸可编程核苷酸结合结构域包含与本文提供的Cas9切口酶(nCas9)的氨基酸序列具有至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或至少99.5％同一性的氨基酸序列。

在本发明的一些实施方案中，腺苷脱氨酶与核酸可编程核苷酸结合结构域直接融合/连接形成融合蛋白，或者通过接头融合/连接形成融合蛋白。腺苷脱氨酶与核酸可编程核苷酸的融合/连接顺序没有特别的限制，例如腺苷脱氨酶可以在碱基编辑器的N端，或者核酸可编程核苷酸结合结构域位于碱基编辑器的N端。

在腺苷脱氨酶与核酸可编程核苷酸结合结构域直接融合的实施方案中，示例性的碱基编辑器融合蛋白具有如下的结构：

NH₂-[腺苷脱氨酶]-[napDNAbp]-COOH；或

NH₂-[napDNAbp]-[腺苷脱氨酶]-COOH。

在腺苷脱氨酶与核酸可编程核苷酸结合结构域通过接头融合的实施方案中，示例性的碱基编辑器融合蛋白具有如下的结构：

NH₂-[腺苷脱氨酶]-[任选的接头]-[napDNAbp]-COOH；或

NH₂-[napDNAbp]-[任选的接头]-[腺苷脱氨酶]-COOH。

在一些实施方案中，核碱基编辑器的结构域通过包含以下氨基酸序列的接头融合：

在本发明的一些实施方案中，碱基编辑器中还包括至少一条核定位信号序列(NLS序列)，NLS序列的氨基酸序列选自：

KRTADGSEFESPKKKRKV(SEQ ID NO:13)、

KRPAATKKAGQAKKKK(SEQ ID NO:14)、

KKTELQTTNAENKTKKL(SEQ ID NO:15)、

KRGINDRNFWRGENGRKTR(SEQ ID NO:16)、

RKSGKIAAIVVKRPRK(SEQ ID NO:17)、

PKKKRKV(SEQ ID NO:18)、

MDSLLMNRRKFLYQFKNVRWAKGRRETY(SEQ ID NO:19)、

MKRTADGSEFESPKKKRKV(SEQ ID NO:20)、或

SGGSKRTADGSEFEPKKKRKV(SEQ ID NO:21)。

在本发明的一些实施方案中，核定位信号序列可以位于碱基编辑器的N端、C端、两端，或者位于腺苷脱氨酶与核酸可编程核苷酸结合结构域之间。在本发明的一些实施方案中，核定位信号序列可以直接融合于碱基编辑器中，或者通过接头融合在碱基编辑器中。包含核定位信号序列的碱基编辑器的示例性结构如下：

NH₂-[NLS]-[腺苷脱氨酶]-[napDNAbp]-COOH；

NH₂-[腺苷脱氨酶]-[NLS]-[napDNAbp]-COOH；

NH₂-[腺苷脱氨酶]-[napDNAbp]-[NLS]-COOH；

NH₂-[NLS]-[napDNAbp]-[腺苷脱氨酶]-COOH；

NH₂-[napDNAbp]-[NLS]-[腺苷脱氨酶]-COOH；

NH₂-[napDNAbp]-[腺苷脱氨酶]-[NLS]-COOH；

NH₂-[NLS]-[腺苷脱氨酶]-[任选的接头]-[napDNAbp]-COOH；

NH₂-[腺苷脱氨酶]-[任选的接头]-[NLS]-[任选的接头]-[napDNAbp]-COOH；

NH₂-[腺苷脱氨酶]-[任选的接头]-[napDNAbp]-[NLS]-COOH；

NH₂-[NLS]-[napDNAbp]-[任选的接头]-[腺苷脱氨酶]-COOH；

NH₂-[napDNAbp]-[任选的接头]-[NLS]-[任选的接头]-[腺苷脱氨酶]-COOH；

NH₂-[napDNAbp]-[任选的接头]-[腺苷脱氨酶]-[NLS]-COOH；

NH₂-[NLS]-[腺苷脱氨酶]-[任选的接头]-[napDNAbp]-[NLS]-COOH。

在本发明的一些优选的实施方案中，碱基编辑器具有如下的结构：

NH₂-[NLS]-[腺苷脱氨酶]-[任选的接头]-[napDNAbp]-[NLS]-COOH。

在本发明的一些进一步优选的实施方案中，所述碱基编辑器包含以下序列中的一种或多种：

(i)如SEQ ID NO:22所示的氨基酸序列；

(ii)与SEQ ID NO:22所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ ID NO:22所示的氨基酸序列的结合多核苷酸和碱基编辑活性；

(iii)在SEQ ID NO:22所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:22所示的氨基酸序列的结合多核苷酸和碱基编辑活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:22所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ IDNO:22所示的氨基酸序列的结合多核苷酸和碱基编辑活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

004V1-nCas9的氨基酸序列(SEQ ID NO:22)：

在本发明中，基于腺苷脱氨酶004V1进行氨基酸取代获得腺苷脱氨酶004V2、腺苷脱氨酶004V3、腺苷脱氨酶004V4、腺苷脱氨酶004V7、腺苷脱氨酶004V8、腺苷脱氨酶004V10、腺苷脱氨酶004V12、腺苷脱氨酶004V13，具体如下：

名称	基于腺苷脱氨酶004V1(SEQ ID NO:1)存在的取代
		腺苷脱氨酶004V2	S15T、D16E、H17K、F19Y、N20Q
腺苷脱氨酶004V3	E22D、Y23F、W24F、R26K、H27R、L29I
		腺苷脱氨酶004V4	K33R、R34K、A35S
腺苷脱氨酶004V7	G80A、L81N、V82A、L83I、Q84N、N85S、Y86W
		腺苷脱氨酶004V8	I89L、D90G、A91T、T92D
腺苷脱氨酶004V10	I112L、S113K、R114K
		腺苷脱氨酶004V12	N132K、V133I、L134F、N135H
腺苷脱氨酶004V13	P137F、G138A、M139L

上述腺苷脱氨酶各自所构成的碱基编辑器序列如下：

004V2-nCas9的氨基酸序列如下所示(SEQ ID NO:46)：

004V3-nCas9的氨基酸序列如下所示(SEQ ID NO:47)：

004V4-nCas9的氨基酸序列如下所示(SEQ ID NO:48)：

004V7-nCas9的氨基酸序列如下所示(SEQ ID NO:49)：

004V8-nCas9的氨基酸序列如下所示(SEQ ID NO:50)：

004V10-nCas9的氨基酸序列如下所示(SEQ ID NO:51)：

004V12-nCas9的氨基酸序列如下所示(SEQ ID NO:52)：

004V13-nCas9的氨基酸序列如下所示(SEQ ID NO:53)：

其中，粗体序列表示源自nCas9的序列；斜体表示接头序列；双下划线序列表示核定位序列；单下划线序列是004V1-004V4、004V7-004V8、004V10、004V12或004V13脱氨酶序列；C端的星号代表终止密码子位置。

在本发明的一些进一步优选的实施方案中，所述碱基编辑器包含以下序列中的一种或多种：

(i)如下SEQ ID NO:23所示的氨基酸序列；

(ii)与SEQ ID NO:23所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；

(iii)在SEQ ID NO:23所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:23所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ IDNO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

005V1-nCas9的氨基酸序列(SEQ ID NO:23)：

其中，粗体序列表示源自nCas9的序列；斜体表示接头序列；双下划线序列表示核定位序列；单下划线序列是005V1脱氨酶序列；C端的星号代表终止密码子位置。

在本发明中，“保留如SEQ ID NO:22所示的氨基酸序列的结合多核苷酸和碱基编辑活性”、“保留如SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性”可以是完全保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的碱基编辑器的结合多核苷酸和碱基编辑活性，也可以是部分保留其活性。在另一些实施方案中，具有修改序列的碱基编辑器也可以具有高于如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的碱基编辑器的结合多核苷酸和碱基编辑活性。

应当理解，本公开的碱基编辑器融合蛋白可以包含一个或多个附加特征。例如，在一些实施方案中，融合蛋白可包含抑制剂、细胞质定位序列、输出序列，例如核输出序列或其他定位序列，以及可用于溶解、纯化或检测融合的标签。本文提供的合适的标签包括但不限于生物素羧化酶载体蛋白(BCCP)标签、myc标签、钙调蛋白标签、FLAG标签、血凝素(HA)标签、多组氨酸标签，也称为组氨酸标签或His-标签、麦芽糖结合蛋白(MBP)-标签、nus-标签、谷胱甘肽-S-转移酶(GST)-标签、绿色荧光蛋白(GFP)-标签、硫氧还蛋白-标签、S-标签、Softags(例如，Softag 1、Softag 3)、链标签、生物素连接酶标签、Flash标签、V5标签和SBP标签。其他合适的序列对本领域技术人员来说是显而易见的。在一些实施方案中，融合蛋白包含一个或多个His标签。

<多核苷酸>

本发明的第三方面提供了一种多核苷酸，其编码本发明第一方面所述的腺苷脱氨酶或者编码本发明第二方面所述的碱基编辑器融合蛋白。

<表达载体>

本发明的第四方面提供了一种载体，其包含本发明第三方面所述的多核苷酸。在本发明的一些实施方案中，所述载体是哺乳动物表达载体。在一些实施方案中，所述表达载体选自腺相关病毒、逆转录病毒载体、腺病毒载体、慢病毒载体、仙台病毒载体和疱疹病毒载体中的一种或多种。在一些实施方案中，所述载体包含启动子。

<细胞>

本发明的第五方面提供了一种细胞，其包含本发明第一方面所述的腺苷脱氨酶、本发明第二方面所述的碱基编辑器融合蛋白、本发明第三方面所述的多核苷酸和本发明第四方面所述的载体中的一种或多种。在本发明的一些实施方案中，所述细胞是原核细胞、真核细胞，进一步地，可以是细菌细胞、植物细胞、昆虫细胞、人类细胞或哺乳动物细胞。

<碱基编辑器系统>

本发明的第六方面提供了一种碱基编辑器系统。在一些实施方案中，碱基编辑器系统包含本发明第一方面所述的腺苷脱氨酶、核酸可编程核苷酸结合结构域、以及引导多核苷酸。

在另一些实施方案中，碱基编辑器系统包含本发明第二方面所述的碱基编辑器融合蛋白，以及引导多核苷酸。

在本发明的一些实施方案中，引导多核苷酸为引导RNA(gRNA)，其是一种短的合成RNA，由Cas结合所需的骨架序列和用户定义的约20个核苷酸间隔序列组成，该间隔序列定义了要修改的基因组靶标。因此，技术人员可以改变Cas蛋白的基因组靶标特异性部分取决于gRNA靶向序列与基因组其余部分相比对基因组靶标的特异性。

在本发明的更具体的一些实施方案中，引导多核苷酸为sgRNA，其由Cas结合所需的骨架序列和用户定义的约20个核苷酸间隔序列组成。

针对不同来源或种类的Cas蛋白，可以选择不同的骨架序列。在本发明的一些更具体的具体实施方案中，其中，结合Cas9蛋白的结构域(SpCas9)，即sgRNA的骨架序列为(SEQID NO:24)：

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC

<药物组合物、试剂盒、递送系统、用途及方法>

本发明第七方面提供了一种药物组合物，其包含本发明第一方面所述的腺苷脱氨酶、本发明第二方面所述的碱基编辑器融合蛋白、本发明第三方面所述的多核苷酸、本发明第四方面所述的载体、本发明第五方面所述的细胞和本发明第六方面所述的碱基编辑器系统中的一种或多种，以及药学上可接受的载体。

在一些实施方案中，所述的药学上可接受的载体可以是递送载体，例如脂质、阳离子脂质或其他具有药物递送功能的聚合物。

本发明第八方面提供了一种试剂盒，具体为一种疾病治疗试剂盒，其包含本发明第一方面所述的腺苷脱氨酶、本发明第二方面所述的碱基编辑器融合蛋白、本发明第三方面所述的多核苷酸、本发明第四方面所述的载体、本发明第五方面所述的细胞、本发明第六方面所述的碱基编辑器系统和本发明第七方面所述的药物组合物中的一种或多种。

本发明的第九方面提供了一种递送系统，其包含本发明第一方面所述的腺苷脱氨酶、本发明第二方面所述的碱基编辑器融合蛋白、本发明第三方面所述的多核苷酸、本发明第四方面所述的载体、本发明第五方面所述的细胞和本发明第六方面所述的碱基编辑器系统中的一种或多种，以及递送介质。

在一些实施方案中，所述递送介质可以为纳米颗粒、脂质体、外泌体、微囊泡或基因枪、细胞穿膜肽等。

本发明第十方面提供了本发明第一方面所述的腺苷脱氨酶在制备碱基编辑器或剪辑编辑器系统中的用途。

本发明第十一方面提供了本发明第一方面所述的腺苷脱氨酶、本发明第二方面所述的碱基编辑器融合蛋白、本发明第三方面所述的多核苷酸、本发明第四方面所述的载体、本发明第五方面所述的细胞、本发明第六方面所述的碱基编辑器系统或本发明第七方面所述的药物组合物、本发明第九方面所述的递送系统在制备用于治疗与点突变相关或由点突变引起的疾病的药物中的用途。

在一些实施方案中，所述药物可以将所述点突变进行校正。在一些实施方案中，所述点突变为G至A和/或C至T。

在一些实施方案中，与点突变相关或由点突变引起的疾病包括高胆固醇血症。在另一些实施方案中，所述疾病非限制性的实例包括梅尔-戈林综合征(Meier-Gorlinsyndrome)；塞克尔综合征(Seckel syndrome)；乔伯特综合征(Joubert syndrome)；莱伯氏先天性黑蒙症(Leber congenital amaurosis)；夏科-马里-图思病(Charcot-Marie-Toothdisease)，2型；夏科-马里-图思病，2型；乌谢尔综合征(Usher syndrome)，2C型；脊髓小脑性共济失调；长QT综合征2；西奥格林-拉尔逊氏综合征(-Larsson syndrome)；遗传性果糖尿病；遗传性果糖尿病；神经母细胞瘤；神经母细胞瘤；卡尔曼综合征(Kallmannsyndrome)1；卡尔曼综合征；异染性脑白质营养不良；雷特综合征(Rettsyndrome)；肌萎缩性侧索硬化症10型；李-佛美尼综合征(Li-Fraumeni syndrome)。

本发明第十二方面提供了核酸的碱基编辑方法，其包括将核酸与本发明第六方面所述的碱基编辑器系统相接触的步骤。

在一些实施方案中，核酸是DNA。进一步地，核酸是双链DNA。

在一些实施方案中，所述核酸包含与疾病相关的靶序列。

在一些实施方案中，所述靶序列包含与疾病相关的点突变。

在一些具体的实施方案中，所述靶序列包含G至A或C至T的与疾病或病症相关的点突变，并且其中突变A碱基的脱氨化导致与疾病或病症不相关的序列。

在一些实施方案中，所述靶序列编码蛋白质，并且其中所述点突变在密码子中，并且导致与野生型密码子相比由该突变密码子编码的氨基酸变化。

在一些实施方案中，所述靶序列位于剪接位点处，并且其中所述点突变导致与野生型转录物相比mRNA转录物的剪接变化。

在一些实施方案中，所述靶序列位于基因的启动子处，并且其中所述点突变导致基因表达增加。

在一些实施方案中，所述靶序列位于基因的启动子处，并且其中所述点突变导致基因表达减少。

在一些实施方案中，核酸位于生物体的基因组内。

在一些实施方案中，所述生物体为原核生物或真核生物或脊椎动物或哺乳动物。

在一些实施方案中，所述突变A碱基的脱氨化导致该突变密码子所编码的氨基酸变化，或导致编码野生型氨基酸的密码子，或导致mRNA转录物变化，或导致野生型mRNA转录物，或导致基因表达增加，或导致基因表达减少。

在一些实施方案中，所述接触在体外实施。

在一些实施方案中，所述接触在受试者体内实施。

在一些实施方案中，所述受试者已被诊断患有疾病或病症。

在一些实施方案中，所述疾病或病症与前蛋白转化酶枯草溶菌素9(PCSK9)基因的点突变相关。

在一些实施方案中，所述疾病包括高胆固醇血症。在另一些实施方案中，所述疾病包括非限制性的实例包括梅尔-戈林综合征(Meier-Gorlin syndrome)；塞克尔综合征(Seckel syndrome)；乔伯特综合征(Joubert syndrome)；莱伯氏先天性黑蒙症(Lebercongenital amaurosis)；夏科-马里-图思病(Charcot-Marie-Tooth disease)，2型；夏科-马里-图思病，2型；乌谢尔综合征(Usher syndrome)，2C型；脊髓小脑性共济失调；长QT综合征；西奥格林-拉尔逊氏综合征(-Larsson syndrome)；遗传性果糖尿病；遗传性果糖尿病；神经母细胞瘤；神经母细胞瘤；卡尔曼综合征(Kallmannsyndrome)；异染性脑白质营养不良；雷特综合征(Rettsyndrome)；肌萎缩性侧索硬化症10型；李-佛美尼综合征(Li-Fraumeni syndrome)。

本发明第十三方面提供了用于治疗患有与点突变相关或由点突变引起的疾病的方法，在一些实施方案中，提供的方法包括向患有此类疾病的受试者施用有效量的校正点突变或将失活性突变引入到疾病相关基因中的本发明第二方面所述的碱基编辑器融合蛋白、本发明第六方面所述的碱基编辑器系统、本发明第七方面所述的药物组合物、本发明第八方面所述的试剂盒、本发明第九方面所述的递送系统。

以下结合附图，通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

在以下实施例中，使用David R.Liu团队所进化出的高效碱基编辑器ABE8e(Richter MF,Zhao KT,Eton E,Lapinaite A,Newby GA,Thuronyi BW,Wilson C,KoblanLW,Zeng J,Bauer DE,Doudna JA,Liu DR.Phage-assisted evolution of an adeninebase editor with improved Cas domain compatibility and activity.NatBiotechnol.2020Jul；38(7):883-891.doi:10.1038/s41587-020-0453-z.Epub 2020Mar16.Erratum in:Nat Biotechnol.2020May 20；:PMID:32433547；PMCID:PMC7357821.)作为对比，与包含本发明提供的腺苷脱氨酶的碱基编辑器进行比较，ABE8e是基于对ABE7.10(Gaudelli NM,Komor AC,Rees HA,Packer MS,Badran AH,Bryson DI,LiuDR.Programmable base editing of A·T to G·C in genomic DNA without DNAcleavage.Nature.2017Nov 23；551(7681):464-471.doi:10.1038/nature24644.Epub2017Oct 25.Erratum in:Nature.2018May 2；:PMID:29160308；PMCID:PMC5726555.)的脱氨酶成分进行优化后得到的；根据前述团队所得出的实验结果，相对于ABE7.10的活性(以一级脱氨常数(first-order kinetics.deamination rate constants,kapp)表示)提高590倍。

具体地，在以下实施例中，ABE8e的氨基酸序列如下(SEQ ID NO:25)：

其中，粗体序列表示源自nCas9的序列；斜体表示接头序列；双下划线序列表示核定位序列；单下划线序列是ecTadA*脱氨酶序列；C端末尾*表示终止密码子位置。

相应地，ABE8e核苷酸序列如下(SEQ ID NO:26)：

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtctctgaggtggagttttcccacgagtactggatgagacatgccctgaccctggccaagagggcacgggatgagagggaggtgcctgtgggagccgtgctggtgctgaacaatagagtgatcggcgagggctggaacagagccatcggcctgcacgacccaacagcccatgccgaaattatggccctgagacagggcggcctggtcatgcagaactacagactgattgacgccaccctgtacgtgacattcgagccttgcgtgatgtgcgccggcgccatgatccactctaggatcggccgcgtggtgtttggcgtgaggaactcaaaaagaggcgccgcaggctccctgatgaacgtgctgaactaccccggcatgaatcaccgcgtcgaaattaccgagggaatcctggcagatgaatgtgccgccctgctgtgcgatttctatcggatgcctagacaggtgttcaatgctcagaagaaggcccagagctccatcaactccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtctaa

在以下实施例中，ABE8e以及包含本发明提供的腺苷脱氨酶的碱基编辑器中所使用的napDNAbp均为nCas9，其氨基酸序列如下(SEQ ID NO:27)：

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

nCas9核苷酸序列如下(SEQ ID NO:28)：

gacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgac

在以下实施例中，包含本发明提供的包含腺苷脱氨酶和nCas9的碱基编辑器结构如下：

NH₂-[NLS]-[腺苷脱氨酶]-接头-[napDNAbp]-[NLS]-COOH。

在以下实施例中，所使用的其他序列见下表：

序号	名称	序列
			SEQ ID NO:29	位点1-sgRNA	gaacacaaagcatagactgc
SEQ ID NO:30	位点17-sgRNA	acaaagaggaagagagacg
			SEQ ID NO:31	位点18-sgRNA	acacacacacttagaatctg
SEQ ID NO:32	PCSK9-sgRNA	cccgcaccttggcgcagcgg
			SEQ ID NO:33	位点1-正向引物	cctcagcattcagccactaa
SEQ ID NO:34	位点1-反向引物	agaggcccattaacgtttgg
			SEQ ID NO:35	位点17-正向引物	ctggctgaccaagagtgaag
SEQ ID NO:36	位点17-反向引物	aatgagcctctggtggagat
			SEQ ID NO:37	位点18-正向引物	gagaggctgccaagctaaat
SEQ ID NO:38	位点18-反向引物	tggagctcaagatcacgttg
			SEQ ID NO:39	PCSK9-正向引物	gctagccttgcgttccg
SEQ ID NO:40	PCSK9-反向引物	gtccccaagatcgtgccaa

实施例

实施例1

本实施例采用腺苷脱氨酶004V1，将其与nCas9融合表达构成一种新的编辑窗口更窄的腺嘌呤碱基编辑器004V1-nCas9。

在本实施例中，腺嘌呤碱基编辑器004V1-nCas9构建策略为：将ABE8e中的腺苷脱氨酶替换为004V1，从而得到一种新的腺苷碱基编辑器004V1-nCas9。sgRNA及碱基编辑器的具体构建如下。

1、sgRNA表达载体(sgRNA质粒)构建

根据目标序列设计sgRNA并合成寡核苷酸(oligos)，所用到的sgRNA序列如SEQ IDNO:29～31所示。在每个sgRNA的上游序列5’端加CACC序列，下游序列的5’端加AAAC序列，因此用于合成的每个sgRNA上游序列形式为：5’-CACCXXXXXXXXXXXXXXXXXXXX(20nt)-3’，下游序列形式为：5’-AAACXXXXXXXXXXXXXXXXXXXX(20nt)-3’。经合成后，上、下游序列通过预设程序(95℃，5min；95℃-85℃以-2℃/s；85℃-25℃以-0.1℃/s；保持在4℃)进行退火，将退火产物连接到经过BsmBI(NEB：R0739L)线性化的PHK09载体(质粒图谱如图1A所示，实验室自有，其已包含sgRNA的骨架序列)上，PHK09载体的序列如下所示(SEQ ID NO:41)

TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGAGCTGCAAGCTTAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCAGAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACGGACGTCTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTAAGCTTGGCGTAACTAGATCTTGAGACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCACTTTGGCGCCGGCTCGAGGGGGCCCGGGTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACGTACGGCCACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCCGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGAGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAACGCGTTAAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCGTCGACTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTACGTATAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGGACGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG

其中，sgRNA质粒构建中所使用的体系如下：

PHK09载体的线性化体系如下：PHK09载体3μg；缓冲液(NEB：R0539L)6μL；BsmBI 2μL；ddH₂O补齐到60μL，37℃酶切过夜。

sgRNA退火产物与线性化载体连接体系如下：T4连接酶缓冲液(NEB：M0202L)1μL，线性化载体20ng，经退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH₂O补齐到10μL，16℃连接过夜。

将连接的载体转化大肠杆菌DH5a感受态细胞(唯地生物，DL1001)。具体流程如下：DH5α感受态细胞从-80℃拿出，迅速插入冰中，5分钟后待菌块融化，加入连接产物并用手拨打离心管底轻轻混匀，冰中静置25分钟。42℃水浴热激45秒，迅速放回冰中并静置2分钟。向离心管中加入700μl不含抗生素的无菌LB培养基，混匀后37℃，200rpm复苏60分钟。5000rpm离心一分钟收菌，留取100μl左右上清轻轻吹打重悬菌块并涂布到Amp抗生素的LB培养基上。将平板倒置放于37℃培养箱过夜培养。挑取单菌落，经过测序确认后对阳性克隆摇菌并提取质粒(TIANGEN：DP120-01)后测定浓度，-20℃冰箱中保存备用。

2、腺嘌呤碱基编辑器004V1-nCas9表达载体(p004V1-nCas9质粒)的构建

本实施例制备了腺嘌呤碱基编辑器表达载体p004V1-nCas9。004V1的核苷酸序列如SEQ ID NO:42所示。

004V1的核苷酸序列(SEQ ID NO:42)：

atgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggc

上述004V1脱氨酶的核苷酸序列已根据人密码子使用的偏好性进行密码子优化，委托生工生物工程(上海)股份有限公司完成540bp的004V1基因人工合成工作并将合成的基因替换ABE8e序列的第63-560位核苷酸。

p004V1-nCas9质粒图谱如图1B所示。

其中，004V1-nCas9的氨基酸序列如SEQ ID NO:22所示。

004V1-nCas9的核苷酸序列如下所示(SEQ ID NO:43)：

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

3、细胞培养与转染

将HEK293T细胞(购自ATCC)接种于添加了10％FBS(v/v)的DMEM培养基中(Gibco,11965092)，其中含1％Penicillin Streptomycin(v/v)(Gibco,15140122)，在含有5％CO₂的37℃细胞培养箱中进行培养。用于转染的细胞，前一天接种于24孔细胞培养板中进行培养，第二天观察细胞，当细胞生长到细胞密度为80％左右时进行转染。24孔板每孔转染的质粒用量分别是p004V1-nCas9质粒0.4μg，sgRNA质粒0.4μg。将质粒混合后用25μl的减血清培养基(源培生物,L530KJ)培养基稀释，再加入2μl p3000试剂，吹打混匀作为试剂A，静置5分钟。同时，将2μl的Lipofectamine 3000转染试剂(Thermo,11668019)用25μl的减血清培养基稀释并混匀，作为试剂B，静置5分钟。将上述试剂A与试剂B混合并吹打均匀，静置20分钟。静置结束后将混合试剂逐滴加入待转染的24孔板细胞中，放回37℃培养箱培养。转染6小时后将培养基换为含有10％FBS的DMEM培养基。转染48小时后收集细胞进行编辑效率的检测。

4、本实施例的腺嘌呤碱基编辑器004V1-nCas9在内源基因位点编辑效率检测

将“3、细胞培养与转染”中所述细胞进行基因组抽提(TIANGEN，DP304-03)。根据实验需求设计引物，所用到的鉴定引物序列如SEQ ID NO:33～38所示。以基因组为模板，对靶点附近序列进行PCR扩增，扩增的PCR产物用于高通量深度测序(金唯智生物科技有限公司)或Sanger测序(铂尚生物技术(上海)有限公司)进行编辑效率的鉴定。用于目标位点序列扩增的体系如下：2×Taq Master Mix(Vazyme，P112-03)25μL；Primer-F(10pmol/μL)1μL；Primer-R(10pmol/μL)1μL；模板1μL；ddH₂O补齐到50μL。

基因编辑效果试验过程如下：

将p004V1-nCas9质粒分别与不同位点的sgRNA质粒共转到HEK293T细胞(ATCC购买)中，相比于将ABE8e质粒(addgene,Plasmid#138489)，发现在位点1和位点17处，p004V1-nCas9质粒的编辑效率与ABE8e相似(图2，图3)，而在位点18处p004V1-nCas9质粒的编辑效率明显优于ABE8e(图4)。

至于编辑窗口，位点1、位点17、位点18处p004V1-nCas9质粒的编辑窗口都小于ABE8e(参见图2～图4)。

基因编辑效率计算方法参见Kluesner MG,Nedveck DA,Lahr WS,Garbe JR,Abrahante JE,Webber BR,Moriarity BS.EditR:A Method to Quantify Base Editingfrom Sanger Sequencing.CRISPR J.2018Jun；1(3):239-250.doi:10.1089/crispr.2018.0014.PMID:31021262；PMCID:PMC6694769。本实施例相关结果如图2-4所示。

实施例2

本实施例中，将实施例1中获得的腺嘌呤碱基编辑器表达载体p004V1-nCas9应用到疾病治疗中。

前蛋白转化酶枯草溶菌素9(PCSK9)是kexin样前蛋白转化酶枯草溶菌素家族的第9个成员，由692个氨基酸残基组成。作为低密度脂蛋白受体(Low-Density LipoproteinReceptor,LDLR)的负调节剂，过量PCSK9与肝细胞表面LDLR结合后可加速其降解，导致肝细胞对低密度脂蛋白-胆固醇(Low-Density Lipoprotein Cholesterol,LDL-C)的摄取下降，进而增加外周循环的LDL-C水平，最终使血液中胆固醇水平升高。

1、sgRNA表达载体(sgRNA质粒)构建

本实施例中靶向PCSK9的sgRNA质粒的构建方式如实施例1中所述。所用到的sgRNA序列如SEQ ID NO:32所示。

2、细胞培养与转染

本实施例中HEK293T细胞的培养与转染方法与实施例1相同。

3、优化后的碱基编辑工具在PCSK9位点编辑效率检测

本实施例中编辑效率检测方法同实施例1中所述，所用到的鉴定引物序列如SEQID NO:39～40所示。

本实施例将腺嘌呤碱基编辑器表达载体p004V1-nCas9与靶向PCSK9的sgRNA质粒共转到HEK293T细胞中，如图5所示，相比于ABE8e腺嘌呤碱基编辑器，p004V1-nCas9的编辑效率优于ABE8e。说明p004V1-nCas9可以靶向治疗PCSK9高表达导致的高胆固醇血症。

实施例3

本实施例采用腺苷脱氨酶004V2、腺苷脱氨酶004V3、腺苷脱氨酶004V4、腺苷脱氨酶004V7、腺苷脱氨酶004V8、腺苷脱氨酶004V10、腺苷脱氨酶004V12、腺苷脱氨酶004V13采用与实施例1或实施例2相同的方法构建腺嘌呤碱基编辑器表达载体、sgRNA表达载体，并进行细胞培养及转染，检测编辑效率。

各腺苷脱氨酶的核苷酸序列如下：

004V2-nCas9的核苷酸序列(SEQ ID NO:54)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgacagagaaggaatatcaggatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V3-nCas9的核苷酸序列(SEQ ID NO:55)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgattttttcatgaagcgcgcaattacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatac gtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggaca ttctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaac atcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V4-nCas9的核苷酸序列(SEQ ID NO:56)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccagaaagtctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V7-nCas9的核苷酸序列(SEQ ID NO: 57)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcgccaatgccattaacagctggcggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatac gtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactggg accctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcac cgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V8-nCas9的核苷酸序列(SEQ ID NO: 58)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgctgggaacagacctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V10-nCas9的核苷酸序列(SEQ ID NO: 59)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagattgaagaaactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V12-nCas9的核苷酸序列(SEQ ID NO: 60)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaagatcttccactaccccggcatgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctg ggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggtt caacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

004V13-nCas9的核苷酸序列(SEQ ID NO:61)

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgtacaatgcccctagattcttttgtagatctgctgccgtgtctgatcatgagtttaacgatgagtactggatgagacacgccctgacactggccaagagggctcgggaggaaggagaggtgccagtgggagctgtgctggtgctgaataatcaggtcattggagaaggatggaatagagccatcggcctgcacgaccccaccgcccacgccgagatcatggccctgagacagggcggcctggtgctgcagaactaccggctgatcgacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcatagcagaatcagcagactggtgttcggcgtgagaaactcaaagagaggcgccgccggcagcctgatcaacgtgctgaactacttcgccctgaaccacagagtggagatcaccgagggcatcctggccgagagctgcagcgccatgctgtgcgacttctataggtggcctagggaggtgttcaatgccctgaagaaggccagacaggaggagggctccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

本实施例具体结果如图6所示。其中，如图6所示，相比于ABE8e，004V2-nCas9、004V3-nCas9、004V4-nCas9、004V7-nCas9、004V8-nCas9、004V10-nCas9、004V12-nCas9、004V13-nCas9在位点1的编辑窗口明显变窄(+5，+7)，其中以004v12-nCas9的编辑窗口最窄，仅对位置+5处具有编辑活性，并且碱基编辑效率为46％，可以实现高效精准编辑。在位点18，004V1-nCas9的不同突变体的编辑效率都明显优于ABE8e。对于PCSK9位点，004V1-nCas9和004V3-nCas9的编辑效率也优于ABE8e。

实施例4

本实施例采用腺苷脱氨酶005V1，将其与nCas9融合表达构成一种新的编辑窗口更窄的腺嘌呤碱基编辑器005V1-nCas9。

在本实施例中，腺嘌呤碱基编辑器005V1-nCas9构建策略为：将ABE8e中的腺苷脱氨酶替换为005V1，从而得到一种新的腺苷碱基编辑器005V1-nCas9。

1、腺嘌呤碱基编辑器005V1-nCas9表达载体(p005V1-nCas9质粒)的构建

本实施例制备了腺嘌呤碱基编辑器表达载体p005V1-nCas9。005V1的核苷酸序列如SEQ ID NO:44所示。

005V1的核苷酸序列(SEQ ID NO:44)

ATGAGTGAGCTGAATGATGCTTACTGGATGAAACAGGCACTCGCTTTAGCTCAGAAGGCCCGGGAACAGGGAGAAGTTCCAGTGGGCGCTATTCTGGTGCTGGATGATGAAGTGATAGGACAGGGATGGAATAGAGCCATCACCCTGCACGACCCCACCGCCCACGCCGAGATCATGGCCCTGCAGCAGGGCGGCCAGATCGTGCAGAACTACCGGCTGCTGAACGCCACCCTGTACGTGACCTTCGAGCCCTGCGTGATGTGCGCCGGCGCCATGGTGCACAGCAGGATCAAGAGACTGGTCTACGGCGTGAGCAACTCAAAAAGAGGCGCCGCCGGCAGCCTGCTGAACGTGCTGAACTACCCCGGCATGAACCACCAGATCGAGATCACCGCCGGCGTGATGGCCAACGAGTGCAGCGAGATGCTGTGCCAGTTCTATCAGCAGCCTAGGGAAGTGTTCAATGCTGAGCGTGAGGCTAGGCGGCTGAACCAACCTGATAGAGCTGAC

上述005V1脱氨酶的核苷酸序列已根据人密码子使用的偏好性进行密码子优化，委托生工生物工程(上海)股份有限公司完成510bp的005V1基因人工合成工作并将合成的基因替换ABE8e序列的第63-560位核苷酸。p005V1-nCas9质粒图谱如图7所示。

其中，005V1-nCas9的氨基酸序列如SEQ ID NO:23所示。

005V1-nCas9的核苷酸序列如下所示(SEQ ID NO:45)：

atgaaacggacagccgacggaagcgagttcgagtcaccaaagaagaagcggaaagtcatgagtgagctgaatgatgcttactggatgaaacaggcactcgctttagctcagaaggcccgggaacagggagaagttccagtgggcgctattctggtgctggatgatgaagtgataggacagggatggaatagagccatcaccctgcacgaccccaccgcccacgccgagatcatggccctgcagcagggcggccagatcgtgcagaactaccggctgctgaacgccaccctgtacgtgaccttcgagccctgcgtgatgtgcgccggcgccatggtgcacagcaggatcaagagactggtctacggcgtgagcaactcaaaaagaggcgccgccggcagcctgctgaacgtgctgaactaccccggcatgaaccaccagatcgagatcaccgccggcgtgatggccaacgagtgcagcgagatgctgtgccagttctatcagcagcctagggaagtgttcaatgctgagcgtgaggctaggcggctgaaccaacctgatagagctgactccggaggatctagcggaggctcctctggctctgagacacctggcacaagcgagagcgcaacacctgaaagcagcgggggcagcagcggggggtcagacaagaagtacagcatcggcctggccatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggtgactctggcggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtc

2、细胞培养与转染

本实施例中HEK293T细胞的培养与转染的方法同实施例1。

3、本实施例的腺嘌呤碱基编辑器005V1-nCas9在位点1编辑效率检测

本实施例中编辑效率检测方法如实施例1中所述，所用到的鉴定引物序列如SEQID NO:33～34所示。相关结果如图8所示。在位点1，ABE8e的编辑窗口为+3，+5，+7，+8，而005V1-nCas9的编辑窗口仅为+5。而且在+5处的编辑效率，005V1-nCas9与ABE8e相近，证明005V1-nCas9可以实现高效精准编辑。

以上示例性实施方式所呈现的描述仅用以说明本发明的技术方案，并不想要成为毫无遗漏的，也不想要把本发明限制为所描述的精确形式。显然，本领域的普通技术人员根据上述教导做出很多改变和变化都是可能的。选择示例性实施方式并进行描述是为了解释本发明的特定原理及其实际应用，从而使得本领域的其它技术人员便于理解、实现并利用本发明的各种示例性实施方式及其各种选择形式和修改形式。本发明的保护范围意在由所附权利要求书及其等效形式所限定。

序列表

<110> 尧唐（上海）生物科技有限公司

<120> 腺苷脱氨酶、碱基编辑器融合蛋白、碱基编辑器系统及用途

<130> 6D19-2233060IP

<160> 61

<170> PatentIn version 3.5

<210> 1

<211> 180

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<213> 人工序列

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<223> 腺苷脱氨酶004V1

<400> 1

Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala Val Ser Asp

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His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala

20 25 30

Lys Arg Ala Arg Glu Glu Gly Glu Val Pro Val Gly Ala Val Leu Val

35 40 45

Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu

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His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly

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

85 90 95

Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His Ser Arg Ile

100 105 110

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

115 120 125

Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg Val Glu

130 135 140

Ile Thr Glu Gly Ile Leu Ala Glu Ser Cys Ser Ala Met Leu Cys Asp

145 150 155 160

Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys Lys Ala Arg

165 170 175

Gln Glu Glu Gly

180

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<223> 腺苷脱氨酶005V1

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Met Ser Glu Leu Asn Asp Ala Tyr Trp Met Lys Gln Ala Leu Ala Leu

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

20 25 30

Val Leu Asp Asp Glu Val Ile Gly Gln Gly Trp Asn Arg Ala Ile Thr

35 40 45

Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Gln Gln Gly

50 55 60

Gly Gln Ile Val Gln Asn Tyr Arg Leu Leu Asn Ala Thr Leu Tyr Val

65 70 75 80

Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His Ser Arg

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

100 105 110

Gly Ser Leu Leu Asn Val Leu Asn Tyr Pro Gly Met Asn His Gln Ile

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Glu Ile Thr Ala Gly Val Met Ala Asn Glu Cys Ser Glu Met Leu Cys

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Gln Phe Tyr Gln Gln Pro Arg Glu Val Phe Asn Ala Glu Arg Glu Ala

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Arg Arg Leu Asn Gln Pro Asp Arg Ala Asp

165 170

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Gly Gly Gly Gly Ser

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Ser Gly Gly Ser

1

<210> 5

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Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala

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Thr Pro Glu Ser Ser Gly Gly Ser

20

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Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

1 5 10 15

Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

<210> 7

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Gly Gly Ser Gly Gly Ser Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser

1 5 10 15

Thr Glu Glu Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly

20 25 30

Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly

35 40 45

Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly

50 55 60

Ser Ala Pro Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly

65 70 75 80

Thr Ser Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala

85 90 95

Thr Ser Gly Gly Ser Gly Gly Ser

100

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Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

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Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

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Ser Glu Ser Ala Thr Pro Glu Ser

20

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Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

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Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

Ser Gly Gly Ser Ser Gly Gly Ser

35 40

<210> 11

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Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

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Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

20 25 30

Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr

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Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser

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Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu Glu Gly Thr Ser

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Glu Ser Ala Thr Pro Glu Ser Gly Pro Gly Thr Ser Thr Glu Pro Ser

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Glu Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Ser Thr Glu

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Glu Gly Thr Ser Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser

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Thr Glu Pro Ser Glu Gly Ser Ala Pro Gly Thr Ser Glu Ser Ala Thr

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Pro Glu Ser Gly Pro Gly Ser Glu Pro Ala Thr Ser

85 90

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Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

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Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

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Lys Lys Thr Glu Leu Gln Thr Thr Asn Ala Glu Asn Lys Thr Lys Lys

1 5 10 15

Leu

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

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Lys Thr Arg

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Arg Lys Ser Gly Lys Ile Ala Ala Ile Val Val Lys Arg Pro Arg Lys

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Pro Lys Lys Lys Arg Lys Val

1 5

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

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

20 25

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Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

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Arg Lys Val

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Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys

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Lys Lys Arg Lys Val

20

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<223> 004V1-nCas9的氨基酸序列

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Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

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Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

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Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

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Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

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Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

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Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

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

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Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

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

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Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 23

<211> 1609

<212> PRT

<213> 人工序列

<220>

<223> 005V1-nCas9的氨基酸序列

<400> 23

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Ser Glu Leu Asn Asp Ala Tyr Trp Met Lys Gln Ala

20 25 30

Leu Ala Leu Ala Gln Lys Ala Arg Glu Gln Gly Glu Val Pro Val Gly

35 40 45

Ala Ile Leu Val Leu Asp Asp Glu Val Ile Gly Gln Gly Trp Asn Arg

50 55 60

Ala Ile Thr Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu

65 70 75 80

Gln Gln Gly Gly Gln Ile Val Gln Asn Tyr Arg Leu Leu Asn Ala Thr

85 90 95

Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val

100 105 110

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

115 120 125

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

130 135 140

His Gln Ile Glu Ile Thr Ala Gly Val Met Ala Asn Glu Cys Ser Glu

145 150 155 160

Met Leu Cys Gln Phe Tyr Gln Gln Pro Arg Glu Val Phe Asn Ala Glu

165 170 175

Arg Glu Ala Arg Arg Leu Asn Gln Pro Asp Arg Ala Asp Ser Gly Gly

180 185 190

Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser

195 200 205

Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys

210 215 220

Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu

290 295 300

Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg

305 310 315 320

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

325 330 335

Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala

500 505 510

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

515 520 525

Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His

625 630 635 640

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

645 650 655

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

660 665 670

Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys

675 680 685

Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys

690 695 700

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

705 710 715 720

Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile

785 790 795 800

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

805 810 815

Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn

820 825 830

Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp

835 840 845

Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp

850 855 860

Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly

865 870 875 880

Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro

965 970 975

Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys

980 985 990

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

995 1000 1005

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

1010 1015 1020

Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met

1250 1255 1260

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

1265 1270 1275

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

1280 1285 1290

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

1295 1300 1305

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly

1310 1315 1320

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

1325 1330 1335

Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1340 1345 1350

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

1355 1360 1365

Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu

1370 1375 1380

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

1385 1390 1395

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

1400 1405 1410

Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn

1415 1420 1425

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

1430 1435 1440

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

1445 1450 1455

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

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1535 1540 1545

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

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys

1595 1600 1605

Val

<210> 24

<211> 76

<212> DNA

<213> 人工序列

<220>

<223> sgRNA的骨架序列

<400> 24

gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60

ggcaccgagt cggtgc 76

<210> 25

<211> 1605

<212> PRT

<213> 人工序列

<220>

<223> ABE8e的氨基酸序列

<400> 25

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His

20 25 30

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

35 40 45

Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn

50 55 60

Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala

65 70 75 80

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

85 90 95

Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met

100 105 110

Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys

115 120 125

Arg Gly Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met

130 135 140

Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala

145 150 155 160

Ala Leu Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala

165 170 175

Gln Lys Lys Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly

180 185 190

Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu

195 200 205

Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr

275 280 285

Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn

290 295 300

Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg

625 630 635 640

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

645 650 655

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

660 665 670

Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr

675 680 685

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

690 695 700

Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu

820 825 830

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

835 840 845

Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met

850 855 860

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

865 870 875 880

Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu

885 890 895

Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu

900 905 910

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

915 920 925

Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala

930 935 940

Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val

945 950 955 960

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

965 970 975

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

980 985 990

Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile

1055 1060 1065

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

1070 1075 1080

Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr

1085 1090 1095

Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala

1235 1240 1245

Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys

1250 1255 1260

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

1265 1270 1275

Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly

1280 1285 1290

Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro

1340 1345 1350

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

1355 1360 1365

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

1370 1375 1380

Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu

1385 1390 1395

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

1400 1405 1410

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

1415 1420 1425

Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala

1430 1435 1440

Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr

1445 1450 1455

Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp

1535 1540 1545

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

1550 1555 1560

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

1565 1570 1575

Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp

1580 1585 1590

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

1595 1600 1605

<210> 26

<211> 4818

<212> DNA

<213> 人工序列

<220>

<223> ABE8e的核苷酸序列

<400> 26

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtctct 60

gaggtggagt tttcccacga gtactggatg agacatgccc tgaccctggc caagagggca 120

cgggatgaga gggaggtgcc tgtgggagcc gtgctggtgc tgaacaatag agtgatcggc 180

gagggctgga acagagccat cggcctgcac gacccaacag cccatgccga aattatggcc 240

ctgagacagg gcggcctggt catgcagaac tacagactga ttgacgccac cctgtacgtg 300

acattcgagc cttgcgtgat gtgcgccggc gccatgatcc actctaggat cggccgcgtg 360

gtgtttggcg tgaggaactc aaaaagaggc gccgcaggct ccctgatgaa cgtgctgaac 420

taccccggca tgaatcaccg cgtcgaaatt accgagggaa tcctggcaga tgaatgtgcc 480

gccctgctgt gcgatttcta tcggatgcct agacaggtgt tcaatgctca gaagaaggcc 540

cagagctcca tcaactccgg aggatctagc ggaggctcct ctggctctga gacacctggc 600

acaagcgaga gcgcaacacc tgaaagcagc gggggcagca gcggggggtc agacaagaag 660

tacagcatcg gcctggccat cggcaccaac tctgtgggct gggccgtgat caccgacgag 720

tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 780

aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 840

aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 900

atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 960

ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 1020

gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 1080

agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 1140

cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 1200

ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 1260

ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 1320

ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 1380

agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 1440

cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 1500

cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 1560

atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 1620

tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 1680

gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 1740

ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 1800

ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 1860

ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 1920

cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 1980

accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 2040

atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 2100

ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 2160

gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 2220

accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 2280

aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 2340

aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 2400

gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 2460

gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 2520

ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 2580

gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 2640

aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 2700

tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 2760

aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 2820

gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 2880

gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 2940

agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 3000

gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 3060

cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 3120

gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 3180

agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 3240

ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 3300

cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 3360

agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 3420

cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 3480

gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 3540

gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 3600

cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 3660

gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 3720

agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 3780

tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 3840

acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 3900

aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 3960

ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 4020

gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 4080

gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 4140

gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 4200

aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 4260

gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 4320

gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 4380

ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 4440

tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 4500

gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 4560

caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 4620

aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 4680

gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 4740

ctgggaggtg actctggcgg ctcaaaaaga accgccgacg gcagcgaatt cgagcccaag 4800

aagaagagga aagtctaa 4818

<210> 27

<211> 1367

<212> PRT

<213> 人工序列

<220>

<223> nCas9氨基酸序列

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

115 120 125

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

130 135 140

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

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

165 170 175

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

180 185 190

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

405 410 415

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

420 425 430

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

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

450 455 460

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

610 615 620

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

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

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

865 870 875 880

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

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

1070 1075 1080

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

1085 1090 1095

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

1100 1105 1110

Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys

1115 1120 1125

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

1130 1135 1140

Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn

1220 1225 1230

Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

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

1265 1270 1275

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

1280 1285 1290

Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1295 1300 1305

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe

1310 1315 1320

Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr

1325 1330 1335

Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly

1340 1345 1350

Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1355 1360 1365

<210> 28

<211> 4101

<212> DNA

<213> 人工序列

<220>

<223> nCas9核苷酸序列

<400> 28

gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggtga c 4101

<210> 29

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点1-sgRNA

<400> 29

gaacacaaag catagactgc 20

<210> 30

<211> 19

<212> DNA

<213> 人工序列

<220>

<223> 位点17-sgRNA

<400> 30

acaaagagga agagagacg 19

<210> 31

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点18-sgRNA

<400> 31

acacacacac ttagaatctg 20

<210> 32

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> PCSK9-sgRNA

<400> 32

cccgcacctt ggcgcagcgg 20

<210> 33

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点1-正向引物

<400> 33

cctcagcatt cagccactaa 20

<210> 34

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点1-反向引物

<400> 34

agaggcccat taacgtttgg 20

<210> 35

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点17-正向引物

<400> 35

ctggctgacc aagagtgaag 20

<210> 36

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点17-反向引物

<400> 36

aatgagcctc tggtggagat 20

<210> 37

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点18-正向引物

<400> 37

gagaggctgc caagctaaat 20

<210> 38

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> 位点18-反向引物

<400> 38

tggagctcaa gatcacgttg 20

<210> 39

<211> 17

<212> DNA

<213> 人工序列

<220>

<223> PCSK9-正向引物

<400> 39

gctagccttg cgttccg 17

<210> 40

<211> 19

<212> DNA

<213> 人工序列

<220>

<223> PCSK9-反向引物

<400> 40

gtccccaaga tcgtgccaa 19

<210> 41

<211> 8318

<212> DNA

<213> 人工序列

<220>

<223> PHK09载体序列

<400> 41

tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 60

gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 120

agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 180

agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg 240

tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc 300

aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt 360

cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac 420

aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct 480

tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt 540

gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc 600

agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 660

gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 720

gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga 780

cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc 840

gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg 900

aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc 960

gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat 1020

agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc 1080

agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga 1140

acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat 1200

aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac 1260

cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga 1320

gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca 1380

aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc 1440

ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac 1500

aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg 1560

aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa 1620

tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg 1680

gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg 1740

aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa 1800

gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat 1860

tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt 1920

ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg 1980

ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc 2040

acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct tcatatttgc 2100

atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga 2160

tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa 2220

aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct 2280

tggctttata tatcttgtgg aaaggacgaa acaccggaga cggacgtctc tgttttagag 2340

ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 2400

tcggtgcttt tttaagcttg gcgtaactag atcttgagac aaatggcagt attcatccac 2460

aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 2520

atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 2580

cgggtttatt acagggacag cagagatcca ctttggcgcc ggctcgaggg ggcccgggtg 2640

caaagatgga taaagtttta aacagagagg aatctttgca gctaatggac cttctaggtc 2700

ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca 2760

gtccccgaga agttgggggg aggggtcggc aattgatccg gtgcctagag aaggtggcgc 2820

ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga 2880

gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc 2940

agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac gggttatggc 3000

ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg tgattcttga tcccgagctt 3060

cgggttggaa gtgggtggga gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt 3120

gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt 3180

cgcgcctgtc tcgctgcttt cgataagtct ctagccattt aaaatttttg atgacctgct 3240

gcgacgcttt ttttctggca agatagtctt gtaaatgcgg gccaagatct gcacactggt 3300

atttcggttt ttggggccgc gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg 3360

gcgaggcggg gcctgcgagc gcggccaccg agaatcggac gggggtagtc tcaagctggc 3420

cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg 3480

ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca 3540

gggagctcaa aatggaggac gcggcgctcg ggagagcggg cgggtgagtc acccacacaa 3600

aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg actccacgga gtaccgggcg 3660

ccgtccaggc acctcgatta gttctcgagc ttttggagta cgtcgtcttt aggttggggg 3720

gaggggtttt atgcgatgga gtttccccac actgagtggg tggagactga agttaggcca 3780

gcttggcact tgatgtaatt ctccttggaa tttgcccttt ttgagtttgg atcttggttc 3840

attctcaagc ctcagacagt ggttcaaagt ttttttcttc catttcaggt gtcgtgacgt 3900

acggccacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc 3960

agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc 4020

gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc 4080

gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc 4140

acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag 4200

ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg 4260

cccaaggagc ccgcgtggtt cctggccacc gtcggagtct cgcccgacca ccagggcaag 4320

ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc 4380

gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc 4440

gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc 4500

ggtgcctgaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg 4560

actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 4620

ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 4680

ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 4740

gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 4800

gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 4860

cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa 4920

tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 4980

ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 5040

gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 5100

gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc 5160

actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc 5220

tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg 5280

gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag 5340

tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag 5400

tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat 5460

aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg 5520

gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 5580

ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc 5640

gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 5700

tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 5760

ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt 5820

gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 5880

ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc 5940

gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac 6000

gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6060

acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6120

ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6180

gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 6240

tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6300

ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 6360

gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 6420

gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc 6480

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 6540

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 6600

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 6660

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 6720

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 6780

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 6840

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 6900

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 6960

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 7020

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 7080

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 7140

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 7200

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 7260

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 7320

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 7380

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 7440

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 7500

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 7560

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 7620

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 7680

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 7740

gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 7800

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 7860

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 7920

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 7980

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 8040

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 8100

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 8160

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 8220

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 8280

cccctgattc tgtggataac cgtattaccg cctttgag 8318

<210> 42

<211> 540

<212> DNA

<213> 人工序列

<220>

<223> 004V1的核苷酸序列

<400> 42

atgtacaatg cccctagatt cttttgtaga tctgctgccg tgtctgatca tgagtttaac 60

gatgagtact ggatgagaca cgccctgaca ctggccaaga gggctcggga ggaaggagag 120

gtgccagtgg gagctgtgct ggtgctgaat aatcaggtca ttggagaagg atggaataga 180

gccatcggcc tgcacgaccc caccgcccac gccgagatca tggccctgag acagggcggc 240

ctggtgctgc agaactaccg gctgatcgac gccaccctgt acgtgacctt cgagccctgc 300

gtgatgtgcg ccggcgccat ggtgcatagc agaatcagca gactggtgtt cggcgtgaga 360

aactcaaaga gaggcgccgc cggcagcctg atcaacgtgc tgaactaccc cggcatgaac 420

cacagagtgg agatcaccga gggcatcctg gccgagagct gcagcgccat gctgtgcgac 480

ttctataggt ggcctaggga ggtgttcaat gccctgaaga aggccagaca ggaggagggc 540

<210> 43

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V1-nCas9的核苷酸序列

<400> 43

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 44

<211> 510

<212> DNA

<213> 人工序列

<220>

<223> 005V1的核苷酸序列

<400> 44

atgagtgagc tgaatgatgc ttactggatg aaacaggcac tcgctttagc tcagaaggcc 60

cgggaacagg gagaagttcc agtgggcgct attctggtgc tggatgatga agtgatagga 120

cagggatgga atagagccat caccctgcac gaccccaccg cccacgccga gatcatggcc 180

ctgcagcagg gcggccagat cgtgcagaac taccggctgc tgaacgccac cctgtacgtg 240

accttcgagc cctgcgtgat gtgcgccggc gccatggtgc acagcaggat caagagactg 300

gtctacggcg tgagcaactc aaaaagaggc gccgccggca gcctgctgaa cgtgctgaac 360

taccccggca tgaaccacca gatcgagatc accgccggcg tgatggccaa cgagtgcagc 420

gagatgctgt gccagttcta tcagcagcct agggaagtgt tcaatgctga gcgtgaggct 480

aggcggctga accaacctga tagagctgac 510

<210> 45

<211> 4827

<212> DNA

<213> 人工序列

<220>

<223> 005V1-nCas9的核苷酸序列

<400> 45

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

agtgagctga atgatgctta ctggatgaaa caggcactcg ctttagctca gaaggcccgg 120

gaacagggag aagttccagt gggcgctatt ctggtgctgg atgatgaagt gataggacag 180

ggatggaata gagccatcac cctgcacgac cccaccgccc acgccgagat catggccctg 240

cagcagggcg gccagatcgt gcagaactac cggctgctga acgccaccct gtacgtgacc 300

ttcgagccct gcgtgatgtg cgccggcgcc atggtgcaca gcaggatcaa gagactggtc 360

tacggcgtga gcaactcaaa aagaggcgcc gccggcagcc tgctgaacgt gctgaactac 420

cccggcatga accaccagat cgagatcacc gccggcgtga tggccaacga gtgcagcgag 480

atgctgtgcc agttctatca gcagcctagg gaagtgttca atgctgagcg tgaggctagg 540

cggctgaacc aacctgatag agctgactcc ggaggatcta gcggaggctc ctctggctct 600

gagacacctg gcacaagcga gagcgcaaca cctgaaagca gcgggggcag cagcgggggg 660

tcagacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg 720

atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 780

cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 840

gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 900

tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 960

ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 1020

aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 1080

aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 1140

atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 1200

gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 1260

atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 1320

cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 1380

ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 1440

gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 1500

cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 1560

ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 1620

atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 1680

cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1740

ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1800

gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1860

aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac 1920

gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1980

gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 2040

agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgaggaa 2100

gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 2160

aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 2220

tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 2280

agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 2340

gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 2400

tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 2460

atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 2520

ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 2580

cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 2640

aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 2700

gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat ccacgacgac 2760

agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2820

cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2880

gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2940

atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 3000

atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 3060

gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 3120

gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 3180

atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 3240

gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 3300

aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 3360

accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 3420

ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 3480

actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 3540

aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 3600

taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3660

taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3720

atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3780

aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3840

cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3900

gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3960

cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 4020

gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 4080

tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 4140

aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 4200

tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 4260

tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg 4320

cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 4380

cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 4440

cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 4500

atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 4560

cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 4620

cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 4680

gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4740

gacctgtctc agctgggagg tgactctggc ggctcaaaaa gaaccgccga cggcagcgaa 4800

ttcgagccca agaagaagag gaaagtc 4827

<210> 46

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V2-nCas9的氨基酸序列

<400> 46

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

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

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 47

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V3-nCas9的氨基酸序列

<400> 47

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Asp Phe Phe Met Lys Arg Ala Ile

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 48

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V4-nCas9的氨基酸序列

<400> 48

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 49

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V7-nCas9的氨基酸序列

<400> 49

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Ala Asn Ala Ile Asn Ser Trp Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 50

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V8-nCas9的氨基酸序列

<400> 50

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Leu Gly Thr Asp Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 51

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V10-nCas9的氨基酸序列

<400> 51

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 52

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V12-nCas9的氨基酸序列

<400> 52

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Lys Ile Phe His Tyr Pro Gly Met Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 53

<211> 1619

<212> PRT

<213> 人工序列

<220>

<223> 004V13-nCas9的氨基酸序列

<400> 53

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Met Tyr Asn Ala Pro Arg Phe Phe Cys Arg Ser Ala Ala

20 25 30

Val Ser Asp His Glu Phe Asn Asp Glu Tyr Trp Met Arg His Ala Leu

35 40 45

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

50 55 60

Val Leu Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ala

65 70 75 80

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

85 90 95

Gln Gly Gly Leu Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

100 105 110

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Val His

115 120 125

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

130 135 140

Ala Ala Gly Ser Leu Ile Asn Val Leu Asn Tyr Phe Ala Leu Asn His

145 150 155 160

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

165 170 175

Leu Cys Asp Phe Tyr Arg Trp Pro Arg Glu Val Phe Asn Ala Leu Lys

180 185 190

Lys Ala Arg Gln Glu Glu Gly Ser Gly Gly Ser Ser Gly Gly Ser Ser

195 200 205

Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser

210 215 220

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

225 230 235 240

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

245 250 255

Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser

260 265 270

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

275 280 285

Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln

645 650 655

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

660 665 670

Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly

675 680 685

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

690 695 700

Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg

805 810 815

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

820 825 830

Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp

835 840 845

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

850 855 860

Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln

865 870 875 880

Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu

965 970 975

Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu

980 985 990

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

995 1000 1005

Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp

1070 1075 1080

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

1085 1090 1095

Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

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

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr

1265 1270 1275

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

1280 1285 1290

Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg

1295 1300 1305

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

1310 1315 1320

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

1325 1330 1335

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

1340 1345 1350

Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr

1355 1360 1365

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

1370 1375 1380

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

1385 1390 1395

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu

1400 1405 1410

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

1415 1420 1425

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

1430 1435 1440

Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu

1445 1450 1455

Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu

1460 1465 1470

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

1475 1480 1485

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

1490 1495 1500

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

1505 1510 1515

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

1520 1525 1530

Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu

1535 1540 1545

Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg

1550 1555 1560

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

1565 1570 1575

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

1580 1585 1590

Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly

1595 1600 1605

Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val

1610 1615

<210> 54

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V2-nCas9的核苷酸序列

<400> 54

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtga cagagaagga atatcaggat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 55

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V3-nCas9的核苷酸序列

<400> 55

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gattttttca tgaagcgcgc aattacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 56

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V4-nCas9的核苷酸序列

<400> 56

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccagaaagt ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 57

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V7-nCas9的核苷酸序列

<400> 57

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcgccaat 300

gccattaaca gctggcggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 58

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V8-nCas9的核苷酸序列

<400> 58

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gctgggaaca gacctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 59

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V10-nCas9的核苷酸序列

<400> 59

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga ttgaagaaac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 60

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V12-nCas9的核苷酸序列

<400> 60

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aagatcttcc actaccccgg catgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

<210> 61

<211> 4857

<212> DNA

<213> 人工序列

<220>

<223> 004V13-nCas9的核苷酸序列

<400> 61

atgaaacgga cagccgacgg aagcgagttc gagtcaccaa agaagaagcg gaaagtcatg 60

tacaatgccc ctagattctt ttgtagatct gctgccgtgt ctgatcatga gtttaacgat 120

gagtactgga tgagacacgc cctgacactg gccaagaggg ctcgggagga aggagaggtg 180

ccagtgggag ctgtgctggt gctgaataat caggtcattg gagaaggatg gaatagagcc 240

atcggcctgc acgaccccac cgcccacgcc gagatcatgg ccctgagaca gggcggcctg 300

gtgctgcaga actaccggct gatcgacgcc accctgtacg tgaccttcga gccctgcgtg 360

atgtgcgccg gcgccatggt gcatagcaga atcagcagac tggtgttcgg cgtgagaaac 420

tcaaagagag gcgccgccgg cagcctgatc aacgtgctga actacttcgc cctgaaccac 480

agagtggaga tcaccgaggg catcctggcc gagagctgca gcgccatgct gtgcgacttc 540

tataggtggc ctagggaggt gttcaatgcc ctgaagaagg ccagacagga ggagggctcc 600

ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 660

cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 720

atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 780

aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 840

ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 900

agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 960

gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1020

aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 1080

aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 1140

ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 1200

gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 1260

acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 1320

atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 1380

ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 1440

aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 1500

tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 1560

ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 1620

gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 1680

gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 1740

ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 1800

gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 1860

gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 1920

ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 1980

ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 2040

tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 2100

gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 2160

ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 2220

cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 2280

accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 2340

ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 2400

aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 2460

ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 2520

gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 2580

atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 2640

ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 2700

cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 2760

agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 2820

gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 2880

cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 2940

atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3000

cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 3060

ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 3120

ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 3180

aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 3240

tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 3300

ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 3360

ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 3420

ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 3480

gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 3540

cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 3600

cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 3660

gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 3720

ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 3780

aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 3840

accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 3900

gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 3960

caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4020

ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 4080

tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 4140

aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 4200

agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 4260

aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 4320

aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc 4380

aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 4440

gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 4500

gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 4560

ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 4620

cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc 4680

atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag 4740

agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 4800

ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtc 4857

Claims (17)

1.一种腺苷脱氨酶，其包含以下序列中的一种或多种：

(i)如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列；

(ii)与SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ IDNO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性；

(iii)在SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:1或SEQID NO:2所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列的脱氨活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

2.根据权利要求1所述的腺苷脱氨酶，其中，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：

S15、D16、H17、F19、N20、D21、E22、Y23、W24、R26、H27、L29、K33、R34、A35、V41、V43、L47、L49、N59、A61、I62、A69、E72、G80、L81、V82、L83、Q84、N85、Y86、I89、D90、A91、T92、V95、F97、I112、S113、R114、L115、F117、V119、R120、N121、K123、R124、N132、V133、L134、N135、P137、G138和M139；和/或，

所述的取代为在如SEQ ID NO:2所示的氨基酸序列的以下位点中的一个或多个发生的取代：

K19、A20、R21、E22、V33、L34、D35、D36、A46、I47、T48、L49、V80、T81、F82、E83、P84、I97、K98、R99、G103、V104、S105、N106、S107、K108、R109、G110、L116、N117、V118、L119、N120、Y121、P122、G123、C144、Q145、F146、Y147、Q148、Q149、P150、R151、E152、V153、F154、N155。

3.根据权利要求2所述的腺苷脱氨酶，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点中的一个或多个发生的取代：S15T、D16E、H17K、F19Y、N20Q、E22D、Y23F、W24F、R26K、H27R、L29I、K33R、R34K、A35S、G80A、L81N、V82A、L83I、Q84N、N85S、Y86W、I89L、D90G、A91T、T92D、I112L、S113K、R114K、N132K、V133I、L134F、N135H、P137F、G138A和M139L；

优选地，所述的取代为在如SEQ ID NO:1所示的氨基酸序列的以下位点的组合发生的取代：

(1)S15T、D16E、H17K、F19Y、N20Q；

(2)E22D、Y23F、W24F、R26K、H27R、L29I；

(3)K33R、R34K、A35S；

(4)G80A、L81N、V82A、L83I、Q84N、N85S、Y86W；

(5)I89L、D90G、A91T、T92D；

(6)I112L、S113K、R114K；

(7)N132K、V133I、L134F、N135H；和/或

(8)P137F、G138A、M139L。

4.一种碱基编辑器融合蛋白，其包含如权利要求1～3中任一项所述的腺苷脱氨酶，以及核酸可编程核苷酸结合结构域。

5.根据权利要求4所述的碱基编辑器融合蛋白，其中，所述核酸可编程核苷酸结合结构域为Cas蛋白或AGO蛋白。

6.根据权利要求4或5所述的碱基编辑器融合蛋白，其中，所述碱基编辑器融合蛋白中还包括至少一条核定位信号序列；

可选地，所述碱基编辑器融合蛋白中还包括接头；

任选地，所述接头包含如SEQ ID NO:3～12所示的序列中的一种或多种。

7.根据权利要求4～6中任一项所述的碱基编辑器融合蛋白，其中，所述碱基编辑器融合蛋白包含以下序列中的一种或多种：

(i)如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列；

(ii)与SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列具有至少80％、82％、85％、87％、90％、92％、95％、96％、97％、98％或99％同一性的氨基酸序列，并且其保留如SEQ IDNO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；

(iii)在SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列，并且其保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性；或者，

(iv)由核苷酸序列编码的氨基酸序列，所述核苷酸序列与编码如SEQ ID NO:22或SEQID NO:23所示的氨基酸序列的多核苷酸序列在严格条件下杂交，并且所述氨基酸序列保留如SEQ ID NO:22或SEQ ID NO:23所示的氨基酸序列的结合多核苷酸和碱基编辑活性，所述严格条件是中等严格条件，中-高严格条件，高严格条件或非常高严格条件。

8.根据权利要求7所述的碱基编辑器融合蛋白，其中，所述的碱基编辑器融合蛋白包含SEQ ID NO:46～53所示序列中的任一种。

9.一种多核苷酸，其编码如权利要求1～3中任一项所述的腺苷脱氨酶或者编码如权利要求4～8中任一项所述的碱基编辑器融合蛋白。

10.一种载体，其包含如权利要求9所述的多核苷酸。

11.一种细胞，其包含如权利要求1～3中任一项所述的腺苷脱氨酶、如权利要求4～8中任一项所述的碱基编辑器融合蛋白、如权利要求9所述的多核苷酸和如权利要求10所述的载体中的一种或多种。

12.一种碱基编辑器系统，其包含如权利要求1～3中任一项所述的腺苷脱氨酶、核酸可编程核苷酸结合结构域、以及引导多核苷酸；

或者，其包含如权利要求4～8中任一项所述的碱基编辑器融合蛋白，以及引导多核苷酸。

13.一种药物组合物，其包含如权利要求1～3中任一项所述的腺苷脱氨酶、如权利要求4～8中任一项所述的碱基编辑器融合蛋白、如权利要求9所述的多核苷酸、如权利要求10所述的载体、如权利要求11所述的细胞和如权利要求12所述的碱基编辑器系统中的一种或多种，以及药学上可接受的载体。

14.一种试剂盒，其包含如权利要求1～3中任一项所述的腺苷脱氨酶、如权利要求4～8中任一项所述的碱基编辑器融合蛋白、如权利要求9所述的多核苷酸、如权利要求10所述的载体、如权利要求11所述的细胞和如权利要求12所述的碱基编辑器系统中的一种或多种。

15.一种递送系统，其包含如权利要求1～3中任一项所述的腺苷脱氨酶、如权利要求4～8中任一项所述的碱基编辑器融合蛋白、如权利要求9所述的多核苷酸、如权利要求10所述的载体、如权利要求11所述的细胞和如权利要求12所述的碱基编辑器系统中的一种或多种，以及递送介质。

16.一种核酸的碱基编辑方法，其包括将待被编辑碱基的核酸与如权利要求12所述的碱基编辑器系统相接触的步骤。

17.如权利要求1～3中任一项所述的腺苷脱氨酶、如权利要求4～8中任一项所述的碱基编辑器融合蛋白、如权利要求9所述的多核苷酸、如权利要求10所述的载体、如权利要求11所述的细胞、如权利要求12所述的碱基编辑器系统、如权利要求13所述的药物组合物或如权利要求15所述的递送系统在制备用于治疗与点突变相关或由点突变引起的疾病的药物中的用途。

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