CN106978428A - 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒 - Google Patents

一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒 Download PDF

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CN106978428A
CN106978428A CN201710152120.7A CN201710152120A CN106978428A CN 106978428 A CN106978428 A CN 106978428A CN 201710152120 A CN201710152120 A CN 201710152120A CN 106978428 A CN106978428 A CN 106978428A
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成秋香
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Abstract

本发明涉及一种Cas蛋白结合DNA的方法，尤其是能够方便、快速、高效地针对一个或者多个靶进行基因转录调控的方法、以及用于所述方法的试剂盒。确定基因组上的目标靶点，找到目标靶点左右区域内Cas蛋白(所述Cas蛋白为保留RNA酶活力、缺失DNA酶活力的Cas蛋白突变体)结合所需的PAM位点，针对所述PAM位点设计向导序列，确定所述Cas蛋白的同向重复序列，并构建转录所述同向重复序列与向导序列相连的crRNA序列的crRNA质粒；将Cas蛋白的编码基因与转录所述crRNA质粒在细胞内共表达，即可使Cas蛋白特异地结合到目标靶点。采用本发明的方法，可以抑制一个或多个靶标基因的转录，整个操作过程与CRISPR/dCas9相比，操作过程非常简单，且编辑效率与dCas9相当。

Description

一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及 试剂盒
技术领域
本发明涉及一种Cas蛋白结合DNA的方法，所述方法的应用以及一种试剂盒，尤其涉及一种能够方便、快速、高效地针对一个或者多个靶点进行基因转录调控的方法、以及用于所述方法的试剂盒。
背景技术
随着分子生物学的进步，科学家们的其中一项工作是致力于发展新的修饰和操作基因组的技术，其中，基于CRISPR/Cas9蛋白的基因组编辑技术显示了强大的应用前景。一般来说，在DNA水平上精确的基因组编辑或调控需要两个主要组成部分：一个DNA结合的结构域来介导特异性序列的识别和结合，一个效应结构域来实现DNA的切割或调节转录。Cas9蛋白是RNA引导的内切酶，这段RNA通过与靶标DNA碱基配对的方式结合，加之Cas9与DNA上PAM(protospacer-adjacent motif)序列相互作用，只要简单的改变引导RNA就可以改变特定的靶标位置。Cas9蛋白这个天然内切酶的活性能广泛的用于各个物种的基因组编辑，包括细菌、真菌、植物和动物。
为了实现特定基因的调控，失活的Cas9(dCas9)融合不同的效应蛋白，如转录激活子、抑制子和表观修饰子，从而实现不同的调控目的。在细菌细胞中，仅dCas9就可以通过阻碍转录机器有效的抑制转录，该技术被称为CRISPR干扰(CRISPRi)。虽然CRISPRi在原核细胞中非常高效，但是在哺乳动物细胞中，dCas9并不能有效的实现基因沉默。但在哺乳动物细胞中，如果将dCas9与一个抑制结构域(比如Kox1的KRAB结构域)进行融合，便可以成功地抑制报告基因或者内源基因的表达。除了CRISPRi，CRISPR激活(CRISPRa)也已成功实现，即在哺乳动物细胞中将dCas9融合转录激活结构域，例如VP64和p65AD，或者是在细菌中将dCas9融合ω亚单位。
除了应用于基因调控，dCas9还可以应用于基因组的表观遗传学编辑，或与荧光蛋白融合，使得可以在活细胞中直接成像来确定基因组的特定位置。另外，dCas9也被用来研究蛋白与特定位点的相互作用，并且有潜力切割RNA。最近，Doudna等人利用dCas9和一小段额外添加的DNA，实现对特定mRNA的追踪，这种Cas9被称为Rcas9(RNA-targeting Cas9)。
利用CRISPR/dCas9技术再进行基因转录调控或者基因组表观遗传学编辑的过程中，如果需要编辑多个基因(或位点)的话，则需要构建多个sgRNA表达单元，整个构建过程比较繁琐。
发明内容
针对现有技术方案无法快速、方便地使得蛋白结合到基因组上多个靶位点的问题，本发明提供了一种新的Cas蛋白结合DNA的方法和试剂盒。
本发明第一个方面提供了一种Cas蛋白结合DNA的方法，优选为一种基因转录调控的方法；更优选为针对靶标基因进行基因转录水平的调控的方法，更优选为抑制靶标基因转录的方法；尤其提供一种能够方便、快速、高效地针对一个或者多个靶点进行基因转录调控的方法。
本发明所述的Cas蛋白结合DNA的方法，步骤包括：
确定基因组上的目标靶点或者靶标基因，在所述目标靶点或者靶标基因左右两侧找到结合Cas蛋白所需的PAM位点，针对所述PAM位点设计向导序列；
确定所述Cas蛋白识别和结合所需的同向重复序列；将同向重复序列与所述向导序列连接形成crRNA序列，并构建转录所述crRNA序列的crRNA质粒；
将所述Cas蛋白的编码基因与转录所述crRNA序列的crRNA质粒在细胞内共表达，即可使Cas蛋白特异地结合到靶标位点。
其中，所述靶标基因的数量可以是一个或多个，并优选为多个。
其中，所述的目标靶点数量可以是一个或多个，并优选为多个。
其中，所述PAM位点的数量可以是一个或多个。
其中，所述Cas蛋白为保留RNA酶活力、缺失DNA酶活力的Cas蛋白突变体。
在一种优选实施例中，所述Cas蛋白可以是选自：
1)保留RNA酶活力、缺失DNA酶活力的Cpf1突变体；和/或
2)缺失DNA酶活力的其他类型Cas蛋白突变体；和/或
3)所述Cpf1突变体或Cas蛋白突变体连接一个或多个不同功能域的融合蛋白。
其中，所述其他类型Cas蛋白为具有RNA酶活性的Cas蛋白，尤其是同时具有DNA酶活性和RNA活性的Cas蛋白，如C2c1蛋白。其中，所述缺失DNA酶活力的其他类型Cas蛋白突变体为缺失DNA酶活性、但具有RNA酶活性的蛋白突变体，如C2c1蛋白突变体。
在一种更优选实施例中，
根据所述PAM位点设计一个向导序列，将向导序列与同向重复序列连接获得一个crRNA序列；或者根据多个PAM位点设计分别向导序列(即获得多个向导序列)，将多个向导序列分别与同向重复序列连接获得多个crRNA序列，将多个crRNA序列串联之后获得一个crRNA阵列；
构建所述crRNA序列或者crRNA阵列的转录质粒crRNA质粒用于转录所述一个crRNA序列、或多个crRNA序列串联的序列；然后将crRNA序列或者crRNA阵列与Cas编码基因在细胞内共表达，即可使Cas蛋白特异结合到基因组上向导序列所靶向的目标靶点或靶标基因。
在更优选实施例中，所述一个或多个向导序列为针对启动子区域T链或/和NT链上PAM位点进行设计的向导序列、或者编码序列NT链或/和T链上PAM位点进行设计的向导序列，更优选为针对一个或多个编码序列T链上PAM位点设计的向导序列，和/或针对一个或多个编码序列NT链上PAM位点设计的向导序列，尤其优选为针对一个或多个编码序列T链上PAM位点设计的向导序列。
本发明第二个方面是提供一种用于Cas蛋白特异性结合靶标DNA的试剂盒，优选为基因转录调控的试剂盒，更优选为针对靶标基因进行基因转录水平的调控的试剂盒，更优选为抑制靶标基因转录的试剂盒。尤其提供一种能够方便、快速、高效地针对一个或者多个靶点进行基因转录调控的试剂盒。
其中，所述靶标基因的数量可以是一个或多个，并优选为多个。
本发明所述的试剂盒包括Cas蛋白编码基因、所述Cas蛋白识别和结合所需的同向重复序列、针对目标靶点设计的向导序列、以及crRNA质粒，所述crRNA质粒用于转录所述同向重复序列-向导序列连接形成的crRNA。
其中，所述试剂盒中，所述同向重复序列可以是与所述引导序列连接形成crRNA。或者，也可以是在使用时将所述同向重复序列与所述引导序列连接形成crRNA。
在所述试剂盒的一种更优选实施例中，所述引导序列为根据一个PAM位点设计的一个序列或根据多个PAM位点设计的多个向导序列；在所述试剂盒的更优选实施例中，还包括连接引物，用于连接所述同向重复序列与所述一个或多个向导序列。
在所述试剂盒的一种优选实施例中，同向重复序列-向导序列连接形成的crRNA可以是：A)根据所述PAM位点设计一个向导序列，将向导序列与同向重复序列连接获得一个crRNA序列；或者B)根据多个PAM位点分别设计向导序列，将多个向导序列分别与同向重复序列连接获得多个crRNA序列，再将多个crRNA序列串联之后获得的一个crRNA阵列。
在所述试剂盒的一种优选实施例中，所述crRNA质粒用于转录A)所述一个所述crRNA序列；或用于转录B)所述一个crRNA阵列。
其中，所述Cas蛋白为保留RNA酶活力、缺失DNA酶活力的Cas蛋白突变体。
在一种优选实施例中，所述Cas蛋白可以是选自：
1)保留RNA酶活力、缺失DNA酶活力的Cpf1突变体；和/或
2)缺失DNA酶活力的其他类型Cas蛋白突变体；和/或
3)所述Cpf1突变体或Cas蛋白突变体连接一个或多个不同功能域的融合蛋白。
其中，所述其他类型Cas蛋白为具有RNA酶活性的Cas蛋白，尤其是同时具有DNA酶活性和RNA活性的Cas蛋白，如C2c1蛋白。其中，所述缺失DNA酶活力的其他类型Cas蛋白突变体为缺失DNA酶活性、但具有RNA酶活性的蛋白突变体，如C2c1蛋白突变体。
在所述试剂盒的一种优选实施例中，所述一个或多个向导序列为针对启动子区域PAM位点设计的向导序列、或一个或多个针对编码序列NT链或/和T链上PAM位点进行设计的向导序列，更优选为针对一个或多个编码序列T链上PAM位点设计的向导序列，和/或针对一个或多个编码序列NT链上PAM位点设计的向导序列，尤其优选为针对一个或多个编码序列T链上PAM位点设计的向导序列。
在所述试剂盒的一种优选实施例中，还包括Cas蛋白编码基因序列的扩增引物。
在所述试剂盒的一种优选实施例中，还包括含有复制子、抗生素抗性基因的质粒。其中，所述复制子优选为p15A复制子。其中，所述抗生素抗性基因优选为卡那霉素(Kanamycin)抗性基因。
在所述试剂盒的更优选实施例中，还包括所述用于转录所述引导序列的crRNA质粒的扩增引物。
采用本发明的方法，能够调节一个或多个靶标基因的转录，过程非常简单，且编辑效率与dCas9相当。
附图说明
图1为本发明采用ddCpf1调节靶标基因转录原理示意图；
图2为本发明实施例1中大肠杆菌MG1655基因组lacZ基因启动子区域、编码链(即非模板链)和非编码链(即模板链)上不同向导序列的位点示意图；
图3为本发明实施例1中针对大肠杆菌lacZ基因，利用ddCpf1和不同位置向导序列的crRNA进行转录调控的实验结果；
图4为本发明实施例2中针对大肠杆菌malT、proP、degP和rseA基因，利用ddCpf1和crRNA阵列进行转录调控的实验结果。
具体实施方式
参照图1，本发明采用ddCpf1等缺失DNA酶活性的Cas(CRISPR-associated,Cas)蛋白突变体来替代dCas9，由于ddCpf1可以直接加工crRNA的前体(或者crRNA阵列)，形成一个(或多个)成熟的crRNA，从而能够引导ddCpf1结合到基因组上特定的位点，来行使基因转录调控或者基因组表观遗传学编辑的功能。
下面参照附图2-4，本发明结合具体实施例对本发明调控基因编辑的方法进行介绍。
实施例1：针对大肠杆菌lacZ基因，利用ddCpf1和不同位置的启动子区域、模板链、 非模板链crRNA进行转录调控
步骤1，构建crRNA的表达质粒
参照图2，在大肠杆菌MG1655基因组lacZ基因的编码链和非编码链上找到Cpf1的PAM(protospacer adjacent motifs)位点TTN，并根据所述位点设计相应的同向重复序列(direct repeat，DR，AsCpf1同向重复序列，SEQ ID No.38)和向导序列(guide)T1、T2、T3、NT1、NT2。
构建crRNA的表达载体pTC17014r。以pgRNA-bacteria(可参照RepurposingCRISPR as an RNA-Guided Platform for Sequence-Specific Control of GeneExpression.Qi LS等.Cell.2013,152(5):1173-83.)为模板，用引物(BsmBI-gRNA-f/BsmBI-gRNA-r2，SEQ ID No.3-4)进行PCR扩增，然后回收PCR产物，进行DpnI处理以去除原始质粒模板，然后利用上海吐露港生物科技有限公司的无缝拼接试剂盒(货号：24303-1)进行无缝拼接，然后转化DH10B，获得质粒pTC17014。为了消除pTC17014质粒上带有的BsmBI位点，设计了一对引物(BsmBI-Muf/BsmBI-Mur，SEQ ID No.5-6)，以pTC17014为模板进行扩增，使用同样的方法进行无缝拼接，获得pTC17014r质粒。
pTC17014r质粒利用BsmBI进行酶切，回收后作为载体备用。针对P、T1、T2、T3、NT1、NT2序列，分别设计引物对(见表1)；将每对引物分别进行退火，然后分别与用BsmBI酶切后的pTC17014r质粒进行连接；获得转录针对P、T1、T2、T3、NT1、NT2位点crRNA的质粒；其中，转录crRNA的启动子为J23119-SpeI(SEQ ID No.37；可参照Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression.Qi LS等.Cell.2013,152(5):1173-83.)。所有的质粒均经过测序确认。
表1，针对不同PAM位点设计的引物对
步骤2，制备ddCpf1表达质粒
合成AsCpf1基因(SEQ ID No.1)并克隆到含有p15A复制子和卡纳霉素抗性基因的质粒上，获得pXX55-AsCpf1。AsCpf1的启动子为强启动子J23100(SEQ ID No.2)。设计引物(AsCpf1-E993A-F/AsCpf1-E993A-R，SEQ ID No.7-8)进行PCR扩增。得到的PCR产物用DpnI去除模板，直接转化到DH10B中，并测序验证。正确的克隆将AsCpf1蛋白(SEQ ID No.9)的第993位谷氨酸(GAG编码)替换成了丙氨酸(GCC编码)，从而使Cpf1缺失DNA酶的活力，但是保留其RNA酶的活力。获得的质粒命名为pXX55-ddCpf1，可以在大肠杆菌中表达ddCpf1蛋白(SEQ ID No.10)。
步骤3，在大肠杆菌中共表达ddCpf1和crRNA
将pXX55-ddCpf1与表达针对P位点、以及T1、T2、T3、NT1、NT2位点crRNA的质粒，以及crRNA对照质粒pTC17014r，分别共转化到大肠杆菌MG1655中，并在添加了卡纳霉素和氨苄青霉素的LB平板上进行筛选。培养条件是37℃培养箱，过夜培养，直至平板上长出单克隆。
挑选单克隆接种至添加了卡纳霉素和氨苄青霉素的LB液体中，置于37℃摇床中过夜培养；以1％的接种量，将过夜培养的菌液接种至添加了卡纳霉素和氨苄青霉素的新鲜LB液体中，37℃振荡培养至OD600为1.0；添加终浓度为0.5mM IPTG来诱导lacZ基因的表达；诱导1h之后，收集3ml菌体，用Zymo Research的RNA提取试剂盒(货号：R2014)来抽提总RNA。取1μg的总RNA，用TaKaRa的反转录试剂盒(货号：RR047A)进行反转录，用TaKaRa的荧光定量检测试剂盒(货号：RR820A)进行定量检测相应基因的表达水平。荧光定量PCR仪为StepOnePlus Real-Time PCR System(Thermo Fisher Scientific)。以大肠杆菌MG1655的gapA基因为内参基因。实验操作均按照试剂盒或仪器的说明进行。
参照图3，验结果表明，转入表达ddCpf1和表达针对P、T1、T2和T3位点crRNA质粒的菌株，lacZ的转录水平有大幅度的降低；相比于对照组(转入了ddCpf1的表达质粒和crRNA对照质粒pTC17014r)，转录水平降低了300倍左右，抑制效果非常显著。相比而言，转入了表达ddCpf1和表达针对NT1、NT2位点crRNA质粒的菌株，lacZ的转录水平有一定幅度的降低，但是降低的幅度相对较小。因此，使用ddCpf1进行靶标基因抑制时，首先选择针对启动子区域和模板链(T链)上的位点设计crRNA的表达质粒。
实施例2：针对大肠杆菌多个基因，利用ddCpf1和crRNA阵列进行转录调控
除了步骤1之外，与实施例1相同。
本实施例中，步骤1为在malT、proP、degP和rseA基因的模板链上分别选择一条ddCpf1的向导序列，详见表2。
表2，针对不同基因设计的向导序列
基因 向导序列
malT cacagtgaagtgattaactatgc SEQ ID No.21
proP ttgcttacgcattaggtaaagtt SEQ ID No.22
degP gcgttatctccgctctctgcaac SEQ ID No.23
rseA atggatggcgaaacgctggatag SEQ ID No.24
针对四个向导序列分别设计PCR引物malTcrRNA-TF/malTcrRNA-TR(malT基因向导序列)、proPcrRNA-TF/proPcrRNA-TR(proP基因向导序列)、degPcrRNA-TF/degPcrRNA-TR(degP基因向导序列)和rseAcrRNA-TF/rseAcrRNA-TR(rseA基因向导序列)；分别进行退火，获得的4个片段分别与pTC17014r质粒用BsmBI酶切处理后的载体进行连接，获得表达针对malT、proP、degP和rseA基因的crRNA表达质粒pTC160309、pTC17050、pTC17041、pTC17043。
表3，针对不同基因向导序列设计的引物对
设计引物(详见表4)，构建crRNA阵列1(malT-proP-degP-rseA)，方法如下：将crRNA-array1f/crRNA-array1r、crRNA-array2f/crRNA-array2r和crRNA-array3f/crRNA-array3r分别退火；获得的3个片段与pTC17014r质粒用BsmBI酶切处理后的载体进行连接，获得crRNA阵列1的表达质粒pTC17055。在该阵列中，4个guide的串联顺序为malT-proP-degP-rseA。
表4，构建crRNA阵列1所设计的引物
引物名称 引物序列
crRNA-array1f agatcacagtgaagtgattaactatgcaatttctactcttgtagatttgctt
crRNA-array1r gcgtaagcaaatctacaagagtagaaattgcatagttaatcacttcactgtg
crRNA-array2f acgcattaggtaaagttaatttctactcttgtagatgcgttatctccgctc
crRNA-array2r cagagagcggagataacgcatctacaagagtagaaattaactttacctaat
crRNA-array3f tctgcaacaatttctactcttgtagatatggatggcgaaacgctggatag
crRNA-array3r aaaactatccagcgtttcgccatccatatctacaagagtagaaattgttg
设计引物(详见表5)，构建crRNA阵列2(proP-degP-rseA-malT)，方法如下：将crRNA-array4f/crRNA-array4r、crRNA-array5f/crRNA-array5r和crRNA-array6f/crRNA-array6r分别退火；获得的3个片段与pTC17014r质粒用BsmBI酶切处理后的载体进行连接，获得crRNA阵列2的表达质粒pTC17056。在该阵列中，4个guide的串联顺序为proP-degP-rseA-malT。
表5，构建crRNA阵列2所设计的引物
引物名称 引物序列
crRNA-array4f agatttgcttacgcattaggtaaagttaatttctactcttgtagatgcgtta
crRNA-array4r gagataacgcatctacaagagtagaaattaactttacctaatgcgtaagcaa
crRNA-array5f tctccgctctctgcaacaatttctactcttgtagatatggatggcgaaacgc
crRNA-array5r tccagcgtttcgccatccatatctacaagagtagaaattgttgcagagagcg
crRNA-array6f tggatagaatttctactcttgtagatcacagtgaagtgattaactatgc
crRNA-array6r aaaagcatagttaatcacttcactgtgatctacaagagtagaaattcta
参照图4，当把ddCpf1的表达质粒和靶向malT-proP-degP-rseA的crRNA阵列1表达质粒一起转入大肠杆菌时，相比于对照组(转入了ddCpf1的表达质粒和crRNA对照质粒pTC17014r)，4个基因的转录水平都被大幅度抑制；并且，其抑制水平与单转靶向单个基因的crRNA时的抑制水平相当。
类似地，当把ddCpf1的表达质粒和靶向proP-degP-rseA-malT的crRNA阵列2表达质粒一起转入大肠杆菌时，相比于对照组(转入了ddCpf1的表达质粒和crRNA对照质粒pTC17014r)，4个基因的转录水平也被大幅度抑制；并且，其抑制水平与单转靶向单个基因的crRNA时的抑制水平相当。
以上结果说明，本发明利用构建crRNA阵列的方法可以来方便地调控多个靶标基因的转录水平；而且，针对各个靶标基因的guide序列的排列顺序不会显著影响对各个靶标基因的抑制效率。
本发明上述实施例中，进行荧光定量PCR检测基因转录水平的引物如下：
表6，荧光定量PCR检测基因转录水平的引物
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
SEQUENCE LISTING
<110> 上海吐露港生物科技有限公司
<120> 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒
<130> CN
<160> 60
<170> PatentIn version 3.3
<210> 1
<211> 3924
<212> DNA
<213> Artificial
<220>
<223> AsCpf1基因
<400> 1
atgacacagt tcgagggctt taccaacctg tatcaggtga gcaagacact gaggtttgag 60
ctgatcccac agggcaagac cctgaagcac atccaggagc agggcttcat cgaggaggac 120
aaggccagaa atgatcacta caaggagctg aagcccatca tcgataggat ctacaagacc 180
tatgccgacc agtgcctgca gctggtgcag ctggattggg agaacctgag cgccgccatc 240
gactcctatc gcaaggagaa gacagaggag acccggaacg ccctgatcga ggagcaggcc 300
acatatagaa atgccatcca cgactacttc atcggcagga cagacaacct gaccgatgcc 360
atcaataagc gccacgccga gatctacaag ggcctgttca aggccgagct gtttaatggc 420
aaggtgctga agcagctggg caccgtgacc acaaccgagc acgagaacgc cctgctgcgc 480
agcttcgata agtttacaac ctacttctcc ggcttttatg agaaccggaa gaacgtgttc 540
agcgccgagg atatcagcac agccatccca cacagaatcg tgcaggacaa cttccccaag 600
tttaaggaga attgtcacat cttcacaaga ctgatcaccg ccgtgcccag cctgagggag 660
cactttgaga acgtgaagaa ggccatcggc atcttcgtga gcacctccat cgaggaggtg 720
ttttccttcc ctttttataa ccagctgctg acacagaccc agatcgacct gtacaatcag 780
ctgctgggag gaatctctag ggaggcagga accgagaaga tcaagggcct gaacgaggtg 840
ctgaatctgg ccatccagaa gaatgatgag acagcccaca tcatcgcctc cctgccacac 900
cgcttcatcc ccctgtttaa gcagatcctg tccgatcgga acaccctgtc tttcatcctg 960
gaggagttta agagcgacga ggaagtgatc cagtccttct gcaagtacaa gacactgctg 1020
cgcaacgaga atgtgctgga gaccgccgag gccctgttta acgagctgaa cagcatcgac 1080
ctgacacaca tcttcatcag ccacaagaag ctggagacca tcagcagcgc cctgtgcgac 1140
cactgggata cactgcggaa tgccctgtac gagcggagaa tctccgagct gacaggcaag 1200
atcaccaagt ctgccaagga gaaggtgcag agaagcctga agcacgagga tatcaacctg 1260
caggagatca tctctgccgc aggcaaggag ctgagcgagg ccttcaagca gaagaccagc 1320
gagatcctgt cccacgcaca cgccgccctg gatcagccac tgcctacaac cctgaagaag 1380
caggaggaga aggagatcct gaagtctcag ctggacagcc tgctgggcct gtaccacctg 1440
ctggactggt ttgccgtgga tgagtccaac gaggtggacc ccgagttctc tgccaggctg 1500
accggcatca agctggagat ggagccttct ctgagcttct acaacaaggc ccgcaattat 1560
gccaccaaga agccctactc cgtggagaag ttcaagctga actttcagat gcctacactg 1620
gcctctggct gggacgtgaa taaggagaag aacaatggcg ccatcctgtt tgtgaagaac 1680
ggcctgtact atctgggcat catgccaaag cagaagggca ggtataaggc cctgagcttc 1740
gagcccacag agaagacctc cgagggcttt gataagatgt actatgacta cttccctgat 1800
gccgccaaga tgatcccaaa gtgcagcacc cagctgaagg ccgtgacagc ccactttcag 1860
acccacacaa cccccatcct gctgtccaac aatttcatcg agcctctgga gatcacaaag 1920
gagatctacg acctgaacaa tcctgagaag gagccaaaga agtttcagac agcctatgcc 1980
aagaagaccg gcgatcagaa gggatacagg gaggccctgt gcaagtggat cgacttcaca 2040
cgggattttc tgtccaagta taccaagaca acctctatcg atctgtctag cctgaggcca 2100
tcctctcagt ataaggacct gggcgagtac tatgccgagc tgaatcccct gctgtaccac 2160
atcagcttcc agcgcatcgc cgagaaggag atcatggatg ccgtggagac cggcaagctg 2220
tacctgttcc agatctataa caaggacttt gccaagggcc accacggcaa gcctaatctg 2280
cacacactgt attggaccgg cctgttttct ccagagaacc tggccaagac aagcatcaag 2340
ctgaatggcc aggccgagct gttctacaga cctaagtcca ggatgaagcg catggcccac 2400
aggctgggcg agaagatgct gaacaagaag ctgaaggatc agaagacacc aatccccgac 2460
accctgtacc aggagctgta cgactatgtg aatcaccgcc tgtcccacga cctgtctgat 2520
gaggcccggg ccctgctgcc aaacgtgatc accaaggagg tgtctcacga gatcatcaag 2580
gataggcgct ttaccagcga caagttcttt ttccacgtgc ctatcacact gaactatcag 2640
gccgccaatt ccccatctaa gttcaaccag cgggtgaatg cctacctgaa ggagcacccc 2700
gagaccccta tcatcggcat cgatcggggc gagagaaacc tgatctatat cacagtgatc 2760
gactccaccg gcaagatcct ggagcagaga tctctgaata ccatccagca gtttgattac 2820
cagaagaagc tggacaaccg ggagaaggag agagtggcag caaggcaggc ctggtctgtg 2880
gtgggcacaa tcaaggatct gaagcagggc tatctgagcc aggtcatcca cgagatcgtg 2940
gacctgatga tccactacca ggccgtggtg gtgctggaga acctgaattt cggctttaag 3000
agcaagagga ccggaatcgc agagaaggcc gtgtaccagc agttcgagaa gatgctgatc 3060
gataagctga attgcctggt gctgaaggac tatccagcag agaaagtggg aggcgtgctg 3120
aacccatacc agctgacaga ccagttcacc tcctttgcca agatgggcac ccagtctggc 3180
ttcctgtttt acgtgcctgc cccatataca tctaagatcg atcccctgac cggcttcgtg 3240
gaccccttcg tgtggaagac catcaagaat cacgagagca gaaagcactt cctggagggc 3300
ttcgactttc tgcactacga tgtgaagaca ggcgacttca tcctgcactt taagatgaac 3360
cgcaatctgt ccttccagag gggactgcca ggctttatgc ctgcatggga tatcgtgttc 3420
gagaagaacg agacacagtt tgacgccaag ggcacccctt tcatcgccgg caagcgcatc 3480
gtgccagtga tcgagaatca ccggtttacc ggccggtaca gagacctgta tcctgccaac 3540
gagctgatcg ccctgctgga ggagaagggc atcgtgttca gagatggctc caacatcctg 3600
ccaaagctgc tggagaatga cgattctcac gccatcgaca caatggtggc cctgatcaga 3660
agcgtgctgc agatgaggaa ctccaatgcc gccacaggcg aggactatat caacagcccc 3720
gtgagagatc tgaatggcgt gtgcttcgac tccaggtttc agaacccaga gtggcctatg 3780
gacgccgatg ccaatggcgc ctaccacatc gccctgaagg gccagctgct gctgaatcac 3840
ctgaaggaga gcaaggatct gaagctgcag aacggcatct ccaatcagga ctggctggcc 3900
tacatccagg agctgaggaa ctag 3924
<210> 2
<211> 35
<212> DNA
<213> Unknown
<220>
<223> J23100启动子序列
<400> 2
ttgacggcta gctcagtcct aggtacagtg ctagc 35
<210> 3
<211> 44
<212> DNA
<213> Artificial
<220>
<223> BsmBI-gRNA-f引物
<400> 3
gagacgacct gcccgtctcg tttttttgaa gcttgggccc gaac 44
<210> 4
<211> 59
<212> DNA
<213> Artificial
<220>
<223> BsmBI-gRNA-r2引物
<400> 4
gacgggcagg tcgtctcgat ctacaagagt agaaattact agtattatac ctaggactg 59
<210> 5
<211> 23
<212> DNA
<213> Artificial
<220>
<223> BsmBI-Muf引物
<400> 5
caaggtctca ttttcgccaa aag 23
<210> 6
<211> 22
<212> DNA
<213> Artificial
<220>
<223> BsmBI-mur引物
<400> 6
ggcgaaaatg agaccttgat cg 22
<210> 7
<211> 32
<212> DNA
<213> Artificial
<220>
<223> AsCpf1-E993A-F引物
<400> 7
tcaggttggc cagcaccacc acggcctggt ag 32
<210> 8
<211> 33
<212> DNA
<213> Artificial
<220>
<223> AsCpf1-E993A-R引物
<400> 8
ggtgctggcc aacctgaatt tcggctttaa gag 33
<210> 9
<211> 1352
<212> PRT
<213> Unknown
<220>
<223> AsCpf1氨基酸序列
<400> 9
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys
1295 1300 1305
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1310 1315 1320
Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp
1325 1330 1335
Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1340 1345 1350
<210> 10
<211> 1352
<212> PRT
<213> Unknown
<220>
<223> ddAsCpf1氨基酸序列
<400> 10
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Ala Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn Lys
1295 1300 1305
Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys
1310 1315 1320
Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Tyr Pro Tyr Asp
1325 1330 1335
Val Pro Asp Tyr Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1340 1345 1350
<210> 11
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T1F引物
<400> 11
agatcaacgt cgtgactggg aaaaccc 27
<210> 12
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T1R引物
<400> 12
aaaagggttt tcccagtcac gacgttg 27
<210> 13
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T2F引物
<400> 13
agatgccagc tggcgtaata gcgaaga 27
<210> 14
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T2R引物
<400> 14
aaaatcttcg ctattacgcc agctggc 27
<210> 15
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T3F引物
<400> 15
agatatgttg atgaaagctg gctacag 27
<210> 16
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-T3R引物
<400> 16
aaaactgtag ccagctttca tcaacat 27
<210> 17
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-NT1F引物
<400> 17
agatccagtc acgacgttgt aaaacga 27
<210> 18
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-NT1R引物
<400> 18
aaaatcgttt tacaacgtcg tgactgg 27
<210> 19
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-NT3F引物
<400> 19
agatagggga cgacgacagt atcggcc 27
<210> 20
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-NT3R引物
<400> 20
aaaaggccga tactgtcgtc gtcccct 27
<210> 21
<211> 23
<212> DNA
<213> Artificial
<220>
<223> malT向导序列
<400> 21
cacagtgaag tgattaacta tgc 23
<210> 22
<211> 23
<212> DNA
<213> Artificial
<220>
<223> proP向导序列
<400> 22
ttgcttacgc attaggtaaa gtt 23
<210> 23
<211> 23
<212> DNA
<213> Artificial
<220>
<223> degP向导序列
<400> 23
gcgttatctc cgctctctgc aac 23
<210> 24
<211> 23
<212> DNA
<213> Artificial
<220>
<223> rseA向导序列
<400> 24
atggatggcg aaacgctgga tag 23
<210> 25
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array1f引物
<400> 25
agatcacagt gaagtgatta actatgcaat ttctactctt gtagatttgc tt 52
<210> 26
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array1r引物
<400> 26
gcgtaagcaa atctacaaga gtagaaattg catagttaat cacttcactg tg 52
<210> 27
<211> 51
<212> DNA
<213> Artificial
<220>
<223> crRNA-array2f引物
<400> 27
acgcattagg taaagttaat ttctactctt gtagatgcgt tatctccgct c 51
<210> 28
<211> 51
<212> DNA
<213> Artificial
<220>
<223> crRNA-array2r引物
<400> 28
cagagagcgg agataacgca tctacaagag tagaaattaa ctttacctaa t 51
<210> 29
<211> 50
<212> DNA
<213> Artificial
<220>
<223> crRNA-array3f引物
<400> 29
tctgcaacaa tttctactct tgtagatatg gatggcgaaa cgctggatag 50
<210> 30
<211> 50
<212> DNA
<213> Artificial
<220>
<223> crRNA-array3r引物
<400> 30
aaaactatcc agcgtttcgc catccatatc tacaagagta gaaattgttg 50
<210> 31
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array4f引物
<400> 31
agatttgctt acgcattagg taaagttaat ttctactctt gtagatgcgt ta 52
<210> 32
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array4r引物
<400> 32
gagataacgc atctacaaga gtagaaatta actttaccta atgcgtaagc aa 52
<210> 33
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array5f引物
<400> 33
tctccgctct ctgcaacaat ttctactctt gtagatatgg atggcgaaac gc 52
<210> 34
<211> 52
<212> DNA
<213> Artificial
<220>
<223> crRNA-array5r引物
<400> 34
tccagcgttt cgccatccat atctacaaga gtagaaattg ttgcagagag cg 52
<210> 35
<211> 49
<212> DNA
<213> Artificial
<220>
<223> crRNA-array6f引物
<400> 35
tggatagaat ttctactctt gtagatcaca gtgaagtgat taactatgc 49
<210> 36
<211> 49
<212> DNA
<213> Artificial
<220>
<223> crRNA-array6r引物
<400> 36
aaaagcatag ttaatcactt cactgtgatc tacaagagta gaaattcta 49
<210> 37
<211> 50
<212> DNA
<213> Unknown
<220>
<223> J23119-SpeI启动子序列
<400> 37
gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt 50
<210> 38
<211> 19
<212> DNA
<213> Artificial
<220>
<223> AsCpf1同向重复序列
<400> 38
aatttctact cttgtagat 19
<210> 39
<211> 27
<212> DNA
<213> Artificial
<220>
<223> malTcrRNA-TF引物
<400> 39
agatcacagt gaagtgatta actatgc 27
<210> 40
<211> 28
<212> DNA
<213> Artificial
<220>
<223> malTcrRNA-TR引物
<400> 40
aaaaagcata gttaatcact tcactgtg 28
<210> 41
<211> 27
<212> DNA
<213> Artificial
<220>
<223> proPcrRNA-TF引物
<400> 41
agatttgctt acgcattagg taaagtt 27
<210> 42
<211> 27
<212> DNA
<213> Artificial
<220>
<223> proPcrRNA-TR引物
<400> 42
aaaaaacttt acctaatgcg taagcaa 27
<210> 43
<211> 27
<212> DNA
<213> Artificial
<220>
<223> degPcrRNA-TF引物
<400> 43
agatgcgtta tctccgctct ctgcaac 27
<210> 44
<211> 27
<212> DNA
<213> Artificial
<220>
<223> degPcrRNA-TR引物
<400> 44
aaaagttgca gagagcggag ataacgc 27
<210> 45
<211> 27
<212> DNA
<213> Artificial
<220>
<223> rseAcrRNA-TF引物
<400> 45
agatatggat ggcgaaacgc tggatag 27
<210> 46
<211> 27
<212> DNA
<213> Artificial
<220>
<223> rseAcrRNA-TR引物
<400> 46
aaaactatcc agcgtttcgc catccat 27
<210> 47
<211> 23
<212> DNA
<213> Artificial
<220>
<223> gapA-qF引物
<400> 47
caacgacctg ttagacgctg att 23
<210> 48
<211> 22
<212> DNA
<213> Artificial
<220>
<223> gapA-qR引物
<400> 48
acgttcagcg gtaacacgga tt 22
<210> 49
<211> 22
<212> DNA
<213> Artificial
<220>
<223> lacZ-qF引物
<400> 49
gacgtctcgt tgctgcataa ac 22
<210> 50
<211> 21
<212> DNA
<213> Artificial
<220>
<223> lacZ-qR引物
<400> 50
tcgccgcaca tctgaacttc a 21
<210> 51
<211> 21
<212> DNA
<213> Artificial
<220>
<223> malT-qF引物
<400> 51
ctctaacgcc aacaaagtcc g 21
<210> 52
<211> 20
<212> DNA
<213> Artificial
<220>
<223> malT-qR引物
<400> 52
tagccgtatg acgcaaccag 20
<210> 53
<211> 20
<212> DNA
<213> Artificial
<220>
<223> proP-qF引物
<400> 53
cgatgttccc gacgcatatc 20
<210> 54
<211> 22
<212> DNA
<213> Artificial
<220>
<223> proP-qR引物
<400> 54
catcaggtaa taggcaggca tc 22
<210> 55
<211> 20
<212> DNA
<213> Artificial
<220>
<223> degP-qF引物
<400> 55
aggtgatgcc ttcagtggtc 20
<210> 56
<211> 20
<212> DNA
<213> Artificial
<220>
<223> degP-qR引物
<400> 56
gagctctgga acggagaacc 20
<210> 57
<211> 20
<212> DNA
<213> Artificial
<220>
<223> rseA-qF引物
<400> 57
tacgtcaacc ggcgacattg 20
<210> 58
<211> 20
<212> DNA
<213> Artificial
<220>
<223> rseA-qR引物
<400> 58
acgcccattt gggtaagctg 20
<210> 59
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-PF引物
<400> 59
agatcacttt atgcttccgg ctcgtat 27
<210> 60
<211> 27
<212> DNA
<213> Artificial
<220>
<223> lacZcrRNA-PR引物
<400> 60
aaaaatacga gccggaagca taaagtg 27

Claims (10)

1.一种Cas蛋白特异性结合靶标DNA的方法，其特征在于，步骤包括：
确定基因组上的目标靶点或者靶标基因，在所述目标靶点或者靶标基因左右两侧找到结合Cas蛋白所需的PAM位点，针对所述PAM位点设计向导序列；
确定所述Cas蛋白识别和结合所需的同向重复序列；将同向重复序列与所述向导序列连接形成crRNA序列，并构建转录所述crRNA序列的crRNA质粒；
将所述Cas蛋白的编码基因与转录所述crRNA序列的crRNA质粒在细胞内共表达，即可使Cas蛋白特异地结合到靶标位点。
2.根据权利要求1所述的方法，其特征在于，所述的目标靶点的数量为一个或多个；所述靶标基因的数量为一个或多个。
3.根据权利要求1所述的方法，其特征在于，所述Cas蛋白为：
1)保留RNA酶活力、缺失DNA酶活力的Cpf1突变体；和/或
2)缺失DNA酶活力的其他类型Cas蛋白突变体；和/或
3)所述Cpf1突变体或Cas蛋白突变体连接一个或多个不同功能域的融合蛋白。
4.根据权利要求3所述的方法，其特征在于，所述其他类型的Cas蛋白为具有RNA酶活性的Cas蛋白。
5.根据权利要求1或2所述的方法，其特征在于，
根据所述PAM位点设计一个向导序列，将向导序列与同向重复序列连接获得一个crRNA序列；或者根据多个PAM位点分别设计向导序列，将多个向导序列分别与同向重复序列连接获得多个crRNA序列，将多个crRNA序列串联之后获得一个crRNA阵列；
构建所述crRNA序列或者crRNA阵列的转录质粒crRNA质粒用于转录所述一个crRNA序列、或多个crRNA序列串联的序列；然后将crRNA序列或者crRNA阵列与Cas编码基因在细胞内共表达，即可使Cas蛋白特异结合到基因组上向导序列所靶向的目标靶点或靶标基因。
6.根据权利要求5所述的方法，其特征在于，所述一个或多个向导序列为针对一个或多个启动子区域内的PAM位点设计的向导序列，和/或针对一个或多个编码序列T链上的PAM位点设计的向导序列，和/或针对一个或多个编码序列NT链上的PAM位点设计的向导序列。
7.根据权利要求1所述的方法，其特征在于，所述方法为调控基因转录、或抑制基因转录的方法。
8.一种用于Cas蛋白特异性结合靶标DNA的试剂盒，其特征在于，包括Cas蛋白编码基因、所述Cas蛋白识别和结合所需的同向重复序列、针对目标靶点设计的向导序列、以及crRNA质粒，所述crRNA质粒用于转录所述同向重复序列-向导序列连接形成的crRNA。
9.根据权利要求8所述的试剂盒，其特征在于，所述Cas蛋白为：
1)保留RNA酶活力、缺失DNA酶活力的Cpf1突变体；和/或
2)缺失DNA酶活力的其他类型Cas蛋白突变体；和/或
3)所述Cpf1突变体或Cas蛋白突变体连接一个或多个不同功能域的融合蛋白。
10.根据权利要求8所述的试剂盒，其特征在于，所述其他类型的Cas蛋白突变体为具有RNA酶活性的Cas蛋白。
CN201710152120.7A 2017-03-15 2017-03-15 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒 Pending CN106978428A (zh)

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CN107488649A (zh) * 2017-08-25 2017-12-19 南方医科大学 一种Cpf1和p300核心结构域的融合蛋白、相应的DNA靶向激活系统和应用
CN107602707A (zh) * 2017-10-17 2018-01-19 湖北大学 一种特异性调节枯草芽孢杆菌外源基因表达的dcas9‑ω融合蛋白及其应用
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
WO2019134630A1 (zh) * 2018-01-03 2019-07-11 苏州克睿基因生物科技有限公司 一种Cas蛋白体系分离DNA的方法
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
WO2019237383A1 (zh) * 2018-06-16 2019-12-19 深圳市博奥康生物科技有限公司 用于人 tnfsf18 基因编辑的修饰载体、其制备方法及应用
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
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US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
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US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2023011659A1 (zh) * 2021-08-06 2023-02-09 华东理工大学 基于CRISPRi和CRISPRa的转录调控系统、其建立方法及应用
CN113897397A (zh) * 2021-09-30 2022-01-07 中南大学 一种基于DNAzyme调控基因编辑的方法
CN113897397B (zh) * 2021-09-30 2024-04-02 中南大学 一种基于DNAzyme调控基因编辑的方法
CN114507654A (zh) * 2022-04-20 2022-05-17 山东舜丰生物科技有限公司 新型Cas酶和系统以及应用
CN114507654B (zh) * 2022-04-20 2022-07-08 山东舜丰生物科技有限公司 Cas酶和系统以及应用

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