CN111849978A - 一种基于Type I-F CRISPR/Cas的染色质成像方法和染色质成像*** - Google Patents
一种基于Type I-F CRISPR/Cas的染色质成像方法和染色质成像*** Download PDFInfo
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Abstract
本发明公开了一种基于Type I‑F CRISPR/Cas***的染色质成像***。本发明首次研究显示隶属于Type I‑F CRISPR/Cas***的Cascade复合物,如PaeCascade复合物,能有效用于染色质成像;同时,本发明对Type I‑F CRISPR/Cas***进行了哺乳动物表达***优化,使得其在哺乳动物细胞内具有高效的双链DNA结合活性,并将荧光分子融合表达至优化后的Cascade亚基上,成功构建了染色质成像***。本发明使得Type I‑F CRISPR/Cas***在哺乳动物细胞的染色质成像方面的应用成为可能,并为基于Type I‑F CRISPR/Cas***的染色质成像提供了必要工具。
Description
技术领域
本发明属于分子生物学技术领域。更具体地,涉及一种基于Type I-F CRISPRCas的染色质成像方法和染色质成像***及应用。
背景技术
在真核细胞中,基因组DNA以染色质的高级形式储存于细胞核的三维空间内,因此称“三维基因组”。三维基因组结构的异常与多种人类疾病的发生密切相关。基因组的三维结构是一个动态变化,且呈现单细胞异质性的结构。因此在活细胞状态下,采用单细胞分辨率来研究基因组三维结构的动态和功能尤为重要。三维基因组领域有非常多重要科学问题等待探索。例如,一个特定基因在细胞核内的位置由什么分子决定?在哪些情况下,基因或调控元件会从细胞核的一个位置移动到另外一个位置?这种空间位置上的变化,是由哪些蛋白分子介导?这个发生位移的时间点是有规律并受某种机制控制的吗?这背后的生物学意义是什么?这一系列问题同样适用于三维基因组结构概念范畴里的任何一个方面,例如染色质环,拓扑关联结构域,染色体区室或染色体疆域等不同三维结构的形成。
近年来,活细胞DNA标记技术,作为研究基因组不可或缺的实时成像工具,受到了极大地关注。将核酸酶失活的Cas9,简称dCas9,与荧光蛋白融合,经过***改造优化后,可在人的活细胞中标记并实时追踪特异基因或特异调控元件在细胞核内的位置及动态变化。该技术可以在细胞***各个时期的多种细胞类型内进行基因组DNA特异标记,并能对目标元件进行长达十几个小时的动态观察,显示了该项技术可广泛性运用的前景。如马来西亚博特拉大学Mahdi Moradpours实验室在期刊Plant Biotechnology Journal上发表的题为“CRISPR/dCas9 Platforms in Plants:Strategies and Applications beyond GenomeEditing”的综述中记载了CRISPR/dCas9在植物活细胞染色质成像中的应用。
虽然基于CRISPR-Cas9的活细胞DNA成像技术在最近几年得到了很大的发展,但是大部分工作还是局限于标记带重复序列的DNA片段。不含重复序列的基因标记还是存在很大的挑战。技术难点主要体现在:为了富集足够强的高于背景的信号,不含重复DNA序列的基因标记需要表达26-36个sgRNA。然而不同sgRNA的活性差异很大,虽然有不少预测sgRNA活性的设计软件帮助我们筛选靶点,但是仍然无法用之准确判断sgRNA在细胞内的活性,其标记效率比较低。
在自然界中,CRISPR/Cas***拥有多种类别,和单效应蛋白的CRISPR相对,I型(Type I)CRISPR***由多效应蛋白组成,需要多亚基共同组装成为具有功能的CRISPR复合体(CRISPR-associated complex for antiviral defense,Cascade)执行功能。从亚基数量和排列等基因特征进行归类,I型CRISPR又可细分为8种亚型(I-A到I-G型,I-U型)。以目前研究最深入的大肠杆菌的I-E型CRISPR***为例,该***由5种效应蛋白和CRISPR RNA(crRNA)构成,其组装首先需要Cas6对crRNA进行加工,其后Cas5、6个Cas7、Cas8e和2个Cse2小亚基共同被招募形成具有共11个亚基的复合体。该复合体仅具有双链DNA(dsDNA)结合能力,需要与Cas3结合才能进行dsDNA切割。
和相对复杂的I-E型CRISPR相比,来自铜绿假单胞菌(Pseudomonas aeruginosa)的I-F型CRISPR复合体(PaeCascade)结合dsDNA仅需4种效应蛋白(Cas8f1、Cas5f1、Cas7f1和Cas6f)易用性存在一定的优势。研究指出PaeCascade可在体外和大肠杆菌中结合dsDNA(Chowdhury,S.,et al,Cell,2017)。在Cas3存在的情况下,PaeCascade复合体可招募Cas3在细菌内对满足PAM(protospacer abject motif)SEQ ID NO.5’-CC-3’要求的靶序列进行切割,实现靶基因编辑。
然而关于Type I-F CRISPR***是否适用于染色质成像及相应的应用价值如何,尚未有任何的研究与报道,其能否可成为新一代的染色质成像工具,仍是未知。
发明内容
本发明要解决的技术问题是克服现有Type I-F CRISPR***的应用局限,探究其是否适用于染色质成像,并开发适用于哺乳动物的基于Type I-F CRISPR/Cas***的染色质成像新工具。
本发明的目的是提供Type I-F CRISPR/Cas***在染色质成像方面的应用。
本发明另一目的是提供一种利用Type I-F CRISPR/Cas***进行染色质成像的方法。
本发明再一目的是提供一种基于Type I-F CRISPR/Cas的染色质成像***。
本发明再一目的是提供上述染色质成像***的应用。
本发明上述目的通过以下技术方案实现:
本发明通过对PaeCascade序列进行哺乳动物表达***优化,将荧光蛋白融合表达至PaeCascade上,进行了PaeCascade染色质成像***的构建、优化和活性检测。使得PaeCascade***在哺乳动物细胞的染色质成像工具开发成为可能。
因此本发明提供了一下可应用方案:
Type I-F CRISPR/Cas***在染色质成像方面或在制备染色质成像工具中的应用。所述Type I-F CRISPR/Cas***包括Cas1,Cas3,Cas8f1(又名Csy1),Cas5f1(又名Csy2),Cas7f1(又名Csy3),Cas6f(又名Csy4)等蛋白亚基,以及由正向重复序列和间隔序列组成的CRISPR阵列。
所述的Type I-F CRISPR/Cas***是来源于Pseudomonas aeruginosa UCBPP-PA14的Cascade复合物,简称为PaeCascade。
基于此,本发明提供一种利用隶属于Type I-F CRISPR/Cas***的PaeCascade复合物进行染色质成像的方法,该方法是:首先对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化,然后将目标基因的靶向序列、优化后的Cas8f1、优化后的Cas5f1、优化后的Cas7f1、优化后的Cas6f以及荧光蛋白共同呈递至靶细胞。
具体而言所述方法是:首先对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化,然后将目标基因的靶向序列、优化后的Cas8f1、优化后的Cas5f1、优化后的Cas7f1、优化后的Cas6f以任意组合***表达载体(即将crRNA、Cas8f1、Cas5f1、Cas7f1、Cas6f表达盒,或其融合蛋白,或共表达(如P2A或IRES)接头,以任意组合任意顺序,任意启动子驱动的任意载体组合),得到重组质粒组合,再将荧光蛋白***所述重组质粒组合中任一个重组质粒中,最后所得重组质粒共同转染细胞。
优化后的Cas8f1、Cas5f1、Cas7f1、Cas6f分别记为hCas8f1、hCas5f1、hCas7f1和hCas6f。
所述“以任意组合***表达载体”包括但不限于以下组合方式:
(1)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f全部***在同一个表达载体中,所得重组质粒即构成染色质成像***;
(2)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f中任意四个组分***在同一个表达载体中,余下一个组分***在另一个表达载体中,构建成由2个重组质粒构成的染色质成像***;
(3)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f中任意三个组分***在同一个表达载体中,余下两个组分***在另一个表达载体中,构建成由2个重组质粒构成的染色质成像***;
(4)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f中任意三个组分***在同一个表达载体中,余下两个组分各自分别***在另外两个表达载体中,构建成由3个重组质粒构成的染色质成像***;
(5)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f中任意两个组分***在同一个表达载体中,余下三个组分中的任意两个***在另一个表达载体中,余下一个组分***在第三个表达载体中,构建成由3个重组质粒构成的染色质成像***;
(6)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f中任意两个组分***在同一个表达载体中,余下三个组分各自分别***在另外三个表达载体中,构建成由4个重组质粒构成的染色质成像***;
(7)靶向序列、hCas8f1、hCas5f1、hCas7f1、hCas6f分别单独***5个表达载体中,构建成由5个重组质粒构成的染色质成像***。
因此,上述涉及到的融合质粒,具体是指由hCas8f1、hCas5f1、hCas7f1、hCas6f中的任一种或任几种组合***表达载体得到重组质粒,也在本发明的保护范围之内。
进一步优选地,还需将荧光蛋白分子***任意一个重组质粒中。
基于此,一种由上述融合质粒组成的基于Type I-F CRISPR/Cas的染色质成像***,也应在本发明的保护范围之内。
另外,作为一种可选择的实施方案,本发明以上述组合方式(5)为例,提供了一种利用隶属于Type I-F CRISPR/Cas***的PaeCascade复合物进行染色质成像的方法及染色质成像***,以及涉及到的优化效应蛋白及重组质粒。
具体展示如下:
一种利用隶属于Type I-F CRISPR/Cas***的PaeCascade复合物进行染色质成像的方法,包括如下步骤:
(1)对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化;
(2)选取目标基因中靶向序列,将靶向序列***表达载体中构建crRNA靶向质粒;
(3)将优化后的Cas8f1、Cas5f1、Cas7f1和Cas6f中任两个与启动子共同***表达载体构建融合质粒I;
(4)将将优化后的Cas8f1、Cas5f1、Cas7f1和Cas6f中任两个与启动子和荧光蛋白共同***表达载体构建融合质粒IV;
(5)将上述crRNA靶向质粒和融合质粒I、融合质粒IV共同转染细胞。一种优化的PaeCascade效应蛋白,包括hCas8f1、hCas5f1、hCas7f1和hCas6f。
所述hCas8f1的序列如SEQ ID NO.2所示,hCas5f1的序列如SEQ ID NO.3所示,hCas7f1的序列如SEQ ID NO.4所示,hCas6f的序列如SEQ ID NO.5所示。
一种融合质粒I,所述融合质粒I为:真核细胞表达载体-hCsy蛋白-启动子-hCsy蛋白;其中两个hCsy蛋白分别选自hCas8f1、hCas5f1、hCas7f1或hCas6f且两个hCsy蛋白不同。
即所述融合质粒I可以为如下11种:
(1)真核细胞表达载体-hCas5f1-启动子-hCas8f1;
(2)真核细胞表达载体-hCas5f1-启动子-hCas7f1;
(3)真核细胞表达载体-hCas5f1-启动子-hCas6f;
(4)真核细胞表达载体-hCas8f1-启动子-hCas5f1;
(5)真核细胞表达载体-hCas8f1-启动子-hCas7f1;
(6)真核细胞表达载体-hCas8f1-启动子-hCas6f;
(7)真核细胞表达载体-hCas7f1-启动子-hCas8f1;
(8)真核细胞表达载体-hCas7f1-启动子-hCas5f1;
(9)真核细胞表达载体-hCas7f1-启动子-hCas6f;
(10)真核细胞表达载体-hCas6f-启动子-hCas8f1;
(10)真核细胞表达载体-hCas6f-启动子-hCas5f1;
(11)真核细胞表达载体-hCas6f-启动子-hCas7f1。
其中所述真核细胞表达载体如pCDNA3.1等。
所述启动子如PGK、EF1α,CMV,CBh等一般广泛性启动子。
优选地,所述融合质粒I为pxCMV-hCas5f1-PGK-hCas8f1。
或优选地,所述融合质粒I为pxCMV-hCas6f-PGK-hCas7f1。
优选地,所述融合质粒I的N端添加核定位信号(NLS)PKKKRKV。
最优选地,所述融合质粒pxCMV-hCas5f1-PGK-hCas8f1的序列如SEQ ID NO.7所示;
或所述融合质粒pxCMV-hCas6f-PGK-hCas7f1的序列如SEQ ID NO.8所示。
再一种融合质粒IV,所述融合质粒IV为:真核细胞表达载体-hCas7f1-荧光蛋白-启动子-hCas6f。
其中所述真核细胞表达载体如pCDNA3.1等。
所述荧光蛋白如mScarlett、GFP、YFP、mCherry等。
所述启动子如PGK、EF1α,CMV,CBh等一般广泛性启动子。
优选地,所述融合质粒IV为pxCMV-hCas7f1-mScarlett-PGK-hCas6f。
优选地,所述融合质粒IV中,融合质粒IV的N端添加核定位信号(NLS)PKKKRKV。
最优选地,所述融合质粒pxCMV-hCas7f1-mScarlett-PGK-hCas6f的序列如SEQ IDNO.9所示。
再一种融合质粒V,所述融合质粒V为在适于哺乳动物的表达载体中***hCas6f-DR序列得到。
优选地,所述适于哺乳动物的表达载体包括pCDNA3.1等。
优选地,所述融合质粒V为pLenti-DR(hCas6f)-EV。其中,pLenti为骨架名称,可替换为任意真核表达载体。EV(empty vector)是指载体未连入识别任何靶向序列的序列,是可用来进行靶向序列克隆的空载体。DR(hCas6f)是指可被hCas6f识别的直接重复序列(简称DR序列)。
最优选地,所述融合质粒pLenti-DR(hCas6f)-EV的序列如SEQ ID NO.10所示。
基于上述优化的PaeCascade效应蛋白以及构建的多种融合质粒,本发明还提供基于Type I-F CRISPR/Cas的染色质成像***。具体如下:
一种基于Type I-F CRISPR/Cas的染色质成像***,包括以下三个组分:
(1)融合质粒I;
(2)融合质粒IV;
(3)融合质粒V。
另外,上述***在制备染色质成像工具方面的应用,也应在本发明保护范围之内。
另外具体应用时,选取靶向序列或crRNA设计规则是:在目标基因上游寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后32-56bp作为靶向序列。即crRNA的长度为32-56nt。
更优选地,是在目标基因上游100-200bp处寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后32-56bp作为靶向序列。即优选地crRNA的长度为32-56nt。
更优选地,是在目标基因上游100-200bp处寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后44-56bp作为靶向序列。即优选地crRNA的长度为44-56nt。
更优选地,是在目标基因上游100-200bp处寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后48-56bp作为靶向序列。即优选地crRNA的长度为48-56nt。
最优选地,是在目标基因上游100-200bp处寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后50-56bp作为靶向序列。即优选地crRNA的长度为50-56nt。
最后,作为一种可选择的具体实施方案,基于上述优化构建的染色质成像***,进行染色质成像的方法包括如下步骤:
(1)选取目标基因中靶向序列,将靶向序列***质粒pLenti-DR(hCas6f)-EV(SEQID NO.10)中构建得到crRNA靶向质粒;
(2)将上述crRNA靶向质粒、融合质粒I、融合质粒IV共同转染细胞。
优选地,步骤(1)中靶向序列选择或crRNA设计规则如上文所述。
优选地,步骤(2)中crRNA靶向质粒、融合质粒I、融合质粒IV以1:1:1摩尔比进行转染。
优选地,步骤(2)中细胞可用HEK293T细胞。
本发明具有以下有益效果:
本发明对PaeCascade***进行了哺乳动物表达***优化,使得其在哺乳动物细胞内具有双链DNA结合活性。同时将荧光蛋白融合表达至优化后的PaeCascade上,进行了PaeCascade染色质成像***的构建、优化和活性检测。
本发明提供了一种新型的PaeCascade哺乳细胞染色质成像***,使得PaeCascade***在哺乳动物细胞的染色质成像方面的应用成为可能,为I型CRISPR基因成像工具的开发成为可能,并为基于PaeCascade的染色质成像提供了必要工具。
和现有基于dCas9的成像***相比,本发明的PasCascade-mScarlett染色质成像***可同时将6个分子的mScarlett荧光蛋白信号富集于染色质特定区域,单一个效应复合体即可增强染色质成像效果。
本发明利用的PaeCascade***其crRNA识别靶序列需32个碱基,对比于已有Cas9的20个碱基配对的gRNA长度更长。由于其识别序列所需碱基更长,因此PaeCascade***对序列识别的要求更严谨,会有更强的特异性。
附图说明
图1为PaeCascade-mScarlett对HEK293T端粒位点的显示成像。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
除非另有说明,本发明的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术,这些在本领域的技能之内。参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis),《分子克隆:实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL),第2次编辑(1989);《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑,(1987));《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司):《PCR2:实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体:实验室手册》(ANTIBODIES,A LABORATORY MANUAL),以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
如下述实施例中所用的细胞培养所需培养基、转染所需optimem购买与Thermo公司。血清购买自Excell公司。RNA提取试剂RNAiso和TB Green II反转录试剂、qPCR试剂盒购买TAKARA公司。Phusion DNA聚合酶、涉及的内切酶、T4连接酶购买自Thermo公司。PCRclean up和凝胶回收试剂盒均购买自Qiagen公司。
实施例1 PaeCascade的序列优化
PaeCascade序列来源于Pseudomonas aeruginosa的I-F型CRISPR复合体。Jennifer A.Doudna团队于2011年在《Proc Natl Acad Sci》发表的一篇题为“RNA-guidedcomplex from a bacterial immune system enhances target recognition throughseed sequence interactions”的文章,其中报导PaeCascade可在体外结合dsDNA序列。其来源菌种为Pseudomonas aeruginosa。
由于PaeCascade位点4个蛋白(Cas8f1、Cas5f1、Cas7f1、Cas6f)所在序列为受原核多顺反子调控,为原核偏好密码子编码序列(SEQ ID NO.1)。因而,为促进PaeCascade在哺乳动物细胞的表达,应对其序列特征进行优化改造。改造原则有三:1)拆分出单个蛋白,每个进行哺乳动物密码子优化;2)去除意外的真核转录终止信号;3)加入真核核定位信号。
就每个序列而言,其序列优化以http://genomes.urv.es/OPTIMIZER/软件进行处理,多余真核转录终止信号以http://dnafsminer.bic.nus.edu.sg/PolyA.html进行检索并排除,最后加入真核核定位信号,对优化前后序列进行表达量差异分析,从Cas8f1、Cas5f1、Cas7f1、Cas6f原序列得到hCas8f1(SEQ ID NO.2)、hCas5f1(SEQ ID NO.3)、hCas7f1(SEQ ID NO.4)、hCas6f(SEQ ID NO.5)序列。
将mScarlett转录抑制因子融合表达至hCas7f1 C端,构建为hCas7f1-mScarlett(SEQ ID NO.6)元件。
实施例2构建重组质粒
1、构建重组质粒pxCMV-hCas5f1-PGK-hCas8f1(序列如SEQ ID NO.7所示)
通过AscI与MluI酶切位点和NotI与HindIII酶切位点分别连于改良的pxCMV质粒,将含NLS序列(PKKKRKV)的hCas8f1、hCas5f1连接并构建pxCMV-hCas5f1-PGK-hCas8f1质粒(SEQ ID NO.7)。
所用的改良pxCMV来源于px601质粒,其中在CMV启动子后加入AscI酶切位点,将PGK启动子、NotI酶切位点通过原KpnI和HindIII位点***构建。
2、构建重组质粒pxCMV-hCas6f-PGK-hCas7f1(序列如SEQ ID NO.8所示):
通过AscI与MluI酶切位点和NotI与HindIII酶切位点分别连于改良的pxCMV质粒,将含NLS序列(PKKKRKV)的hCas7f1、hCas6f连接并构建pxCMV-hCas6f-PGK-hCas7f1质粒(SEQ ID NO.8)。
所用的改良pxCMV来源于px601质粒,其中在CMV启动子后加入AscI酶切位点,将PGK启动子、NotI酶切位点通过原KpnI和HindIII位点***构建。
3、构建重组质粒pxCMV-hCas7f1-mScarlett-PGK-hCas6f(序列如SEQ ID NO.9所示):
通过AscI与MluI酶切位点和NotI与HindIII酶切位点分别连于改良的pxCMV质粒,将hCas7f1、hCas6f和荧光蛋白mScarlett连接并构建pxCMV-hCas7f1-mScarlett-PGK-hCas6f(序列如SEQ ID NO.9所示)。
所用的改良pxCMV来源于px601质粒,其中在CMV启动子后加入AscI酶切位点,将PGK启动子、NotI酶切位点通过原KpnI和HindIII位点***构建。
4、构建重组质粒pLenti-DR(hCas6f)-EV(序列如SEQ ID NO.10所示):
PaeCascade crRNA骨架(Direct Repeat,DR)序列来源于Pseudomonasaeruginosa内源CRISPR repeat序列。
其中通过BsmBI和EcoRI酶切位点将crRNA克隆序列***至Lenti gRNA puro载体中。
Direct Repeat序列:gttcactgccgtataggcagctaagaaa
crRNA克隆序列:Ggttcactgccgtataggcagctaagaaa TGAGACGGCAGCCAGGGATCCTATCCGTCTCA gttcactgccgtataggcagctaagaaaTTTTTTTg
实施例1、2中序列用于以下***的构建,序列来源为合成序列。
实施例3构建PaeCascade-mScarlett染色质成像***
PaeCascade-KRAB转录抑制***包括:
(1)pxCMV-hCas5f1-PGK-hCas8f1质粒(SEQ ID NO.7),
(2)pxCMV-hCas7f1-mScarlett-PGK-hCas6f质粒(SEQ ID NO.9),
(3)crRNA表达质粒为pLenti-DR(hCas6f)-EV(SEQ ID NO.10)。
实施例4PaeCascade-mScarlett成像指示哺乳动物内端粒位点
实验方法:在目标靶点(如端粒序列)处寻找满足PAM-SEQ ID NO.6’-CC-3’特征的后32bp作为靶向序列。通过BsmBI酶切的方法将靶向序列***至pLenti-DR(hCas6f)-EV(SEQ ID NO.10)中,构建出crRNA靶向质粒。将crRNA靶向质粒和pxCMV-hCas5f1-PGK-hCas8f1质粒(SEQ ID NO.7)、pxCMV-hCas7f1-mScarlett-PGK-hCas6f质粒(SEQ ID NO.9)以1:1:1摩尔比转染至以电转染或其他转染试剂后转染方法转染至HEK293T细胞中。转染后以荧光显微镜进行成像拍照。
本实施例以POT1为目标靶点,利用PaeCascade-mScarlett染色质成像***进行哺乳动物内端粒位点的成像指示实验。具体操作如下:
将1μg含有靶向端粒位点靶向增强子位点的crRNA靶向质粒,以及1μg质粒pxCMV-hCas5f1-PGK-hCas8f1(SEQ ID NO.7)和1μg pxCMV-hCas7f1-mScarlett-PGK-hCas6f质粒(SEQ ID NO.9)以PEI转染方式转染HEK293T细胞。转染后48h以POT1指示端粒,以荧光显微镜拍摄PaeCascade-mScarlett在端粒的成像效果。其中以非靶向序列作为空白对照。
结果如图1,PaeCascade-mScarlett可和POT1有共定位,指示细胞中端粒序。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中山大学
<120> 一种基于Type I-F CRISPR/Cas的染色质成像方法和染色质成像***
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 3881
<212> DNA
<213> PaeCascade
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atgacctctc ccctcccaac gcctacgtgg caggagcttc gccagttcat cgaatccttc 60
atccaggagc gcctccaggg caagctggac aaactccaac ccgacgaaga cgacaagcgc 120
cagacattgc tggccaccca ccggcgggag gcctggctgg ccgatgccgc ccggcgggtt 180
ggccagttgc agttggtgac ccacacgctc aagccgatcc atcccgacgc ccgcggcagc 240
aacctgcaca gcctgccgca agcacccggc caaccgggcc tcgccggttc ccatgagcta 300
ggtgacaggc tggtcagcga tgtggtgggc aatgccgcgg cgctggacgt attcaagttt 360
ctcagtctcc agtatcaggg taaaaatctt ctgaactggc tgacagaaga cagtgccgag 420
gcattacagg cgctgtccga taacgccgaa caggctcgcg aatggcggca agcgttcatc 480
ggcatcacga ccgtcaaagg cgctcccgcg tcccacagcc tggccaagca gttgtacttt 540
cccctgcccg gttccggcta ccacctgcta gcaccgctgt ttcccaccag tctggtgcat 600
cacgtccacg ctctgctccg cgaagcgcgc ttcggcgacg cggccaaggc ggcacgcgaa 660
gcgcgcagcc gccaggagtc atggccccac ggattcagcg agtaccccaa cctggcgatc 720
cagaagttcg gcggtaccaa gccgcagaac atcagtcagt tgaacaacga gcgccgtggc 780
gagaactggt tgctgccatc gcttccgccg aactggcaaa ggcagaatgt aaacgcgccg 840
atgcggcact cttcggtctt cgagcatgac ttcggaagaa ctcctgaagt atcccgccta 900
acccgtaccc tgcagcgttt tcttgccaag acagtccata acaacctcgc catccgccag 960
cggcgtgccc aattggtggc gcaaatctgc gacgaagccc tgcaatacgc cgcccgtctg 1020
cgcgaactgg agcccggctg gagcgcaacc cccgggtgcc aactgcatga cgcggagcag 1080
ctctggctcg atccgttgcg cgcacagacc gatgagacgt tcttgcagcg ccgactacga 1140
ggtgactggc ctgccgaggt cggaaatcgc ttcgccaact ggctgaaccg ggcggtcagc 1200
agcgacagtc agatactggg tagcccggaa gccgcccaat ggagccagga gctgagcaag 1260
gaactgacga tgttcaagga gatactcgaa gatgagcgtg actgatcccg aggcgctgct 1320
gttgctgcca cgcctgtcca tccagaacgc caacgccatt tccagcccct tgacctgggg 1380
tttcccttcg cccggcgctt tcaccggatt cgtccatgcc ttgcagcgtc gagtcggaat 1440
ctccctcgat atcgaactgg acggtgtcgg catcgtctgt caccgcttcg aagcgcagat 1500
atcgcaaccg gccggcaagc gcaccaaggt cttcaacctg acccgcaatc cgctgaaccg 1560
cgacggctcc accgcagcca tcgtcgaaga aggtcgcgcc catctggagg tcagtctgct 1620
gctcggagtg catggcgatg gtctggacga tcaccccgca caggaaatcg ccaggcaggt 1680
acaggagcag gctggcgcca tgcgcctcgc cggtggcagc atcctgccct ggtgcaatga 1740
gcgcttcccg gctccgaacg ccgaactgct gatgctgggt ggcagcgacg aacagcggcg 1800
caagaaccag cgccggctta cccgccgcct gctccccggc ttcgccctgg tgagtcgcga 1860
ggcgttgttg caacaacatc tggaaacctt gcgcaccacg ctccccgaag ccaccacgct 1920
cgatgcactg ctcgatcttt gtcgaatcaa cttcgagcct cctgcaacct cgtccgaaga 1980
agaagcatcg ccgcccgatg cagcctggca agtgcgcgac aagcccggct ggctggtgcc 2040
gatcccggct ggctacaacg ccctgtcacc cctgtacctc cccggcgaag tgcgaaatgc 2100
ccgtgataga gaaacaccgc tgcgcttcgt cgaaaacctc ttcggtctgg gcgaatggct 2160
cagcccccat cgcgtcgccg cgttatcgga cctgctttgg tatcaccacg ccgagcctga 2220
caaagggctc taccgctgga gcacaccccg tttcgtcgaa cacgccatcg cataaggaaa 2280
tcagcatgtc caagccaata ctgagcaccg cttccgtcct cgccttcgaa cgtaagctcg 2340
acccttccga cgcccttatg agcgctggcg cctgggcgca acgcgacgcc tcgcaggaat 2400
ggccggccgt gaccgtgcgc gagaagtccg tacgcggcac catctccaac cgcctcaaga 2460
ccaaggaccg tgacccggcc aagctggacg cctcgatcca gtcgcccaac ctgcagacgg 2520
tggacgtggc caacctgccg agcgacgccg acaccctcaa ggtccgcttc accctccgtg 2580
tgctcggcgg cgccggaaca ccgtctgcct gcaacgacgc ggcctaccgt gacaagctgc 2640
tgcaaacggt cgccacctac gtgaacgatc agggcttcgc cgagctggct cgtcgttatg 2700
cgcacaacct ggccaacgcc cgcttcctgt ggcgcaaccg ggtgggcgcg gaagcggtgg 2760
aagtccgtat caaccatatc cgccagggcg aggtggctcg cgcctggcgc ttcgacgccc 2820
tggccatcgg cttgcgcgac ttcaaggccg acgccgaact ggacgcgctc gccgaactga 2880
tcgccagcgg tctctcaggc agtgggcatg tcctgctcga agtggtcgcc ttcgcccgta 2940
tcggcgacgg ccaggaagtc ttcccctccc aggaactgat cctcgacaaa ggcgacaaga 3000
aaggccagaa gagcaagacc ctgtacagcg tacgcgatgc cgcggccatc cactcgcaga 3060
agatcggcaa tgccctgcgc accatcgata cgtggtatcc cgacgaagat ggcctcggcc 3120
ccatcgccgt ggagccctac ggctccgtca catcccaggg caaagcctat cgccagccca 3180
agcagaagct ggacttctat acgctgctcg acaactgggt actgcgcgac gaggcgcccg 3240
ccgtggagca acagcattat gtgatcgcca acctgatccg tggcggcgtg ttcggtgaag 3300
ccgaagagaa gtaagccatg gaccactacc tcgacattcg cttgcgaccg gacccggaat 3360
ttcccccggc gcaactcatg agcgtgctct tcggcaagct ccaccaggcc ctggtggcac 3420
agggcgggga caggatcggc gtgagcttcc ccgacctcga cgaaagccgc tcccggctgg 3480
gcgagcgcct gcgcattcat gcctcggcgg acgaccttcg tgccctgctc gcccggccct 3540
ggctggaagg gttgcgggac catctgcaat tcggagaacc ggcagtcgtg cctcacccca 3600
caccgtaccg tcaggtcagt cgggttcagg cgaaaagcaa tccggaacgc ctgcggcggc 3660
ggctcatgcg ccggcacgat ctgagtgagg aggaggctcg gaaacgcatt cccgatacgg 3720
tcgcgagagc cttggacctg cccttcgtca cgctacgcag ccagagcacc ggacagcact 3780
tccgtctctt catccgccac gggccgttgc aggtgacggc agaggaagga ggattcacct 3840
gttacgggtt gagcaaagga ggtttcgttc cctggttctg a 3881
<210> 2
<211> 1401
<212> DNA
<213> hCas8f1
<400> 2
atgcccaaga agaagaggaa agtcacctct cccctcccaa cgcctacgtg gcaggagctt 60
cgccagttca tcgaatcctt catccaggag cgcctccagg gcaagctgga caaactccaa 120
cccgacgaag acgacaagcg ccagacattg ctggccaccc accggcggga ggcctggctg 180
gccgatgccg cccggcgggt tggccagttg cagttggtga cccacacgct caagccgatc 240
catcccgacg cccgcggcag caacctgcac agcctgccgc aagcacccgg ccaaccgggc 300
ctcgccggtt cccatgagct aggtgacagg ctggtcagcg atgtggtggg caatgccgcg 360
gcgctggacg tattcaagtt tctcagtctc cagtatcagg gtaaaaatct tctgaactgg 420
ctgacagaag acagtgccga ggcattacag gcgctgtccg ataacgccga acaggctcgc 480
gaatggcggc aagcgttcat cggcatcacg accgtcaaag gcgctcccgc gtcccacagc 540
ctggccaagc agttgtactt tcccctgccc ggttccggct accacctgct agcaccgctg 600
tttcccacca gtctggtgca tcacgtccac gctctgctcc gcgaagcgcg cttcggcgac 660
gcggccaagg cggcacgcga agcgcgcagc cgccaggagt catggcccca cggattcagc 720
gagtacccca acctggcgat ccagaagttc ggcggtacca agccgcagaa catcagtcag 780
ttgaacaacg agcgccgtgg cgagaactgg ttgctgccat cgcttccgcc gaactggcaa 840
aggcagaatg taaacgcgcc gatgcggcac tcttcggtct tcgagcatga cttcggaaga 900
actcctgaag tatcccgcct aacccgtacc ctgcagcgtt ttcttgccaa gacagtccat 960
aacaacctcg ccatccgcca gcggcgtgcc caattggtgg cgcaaatctg cgacgaagcc 1020
ctgcaatacg ccgcccgtct gcgcgaactg gagcccggct ggagcgcaac ccccgggtgc 1080
caactgcatg acgcggagca gctctggctc gatccgttgc gcgcacagac cgatgagacg 1140
ttcttgcagc gccgactacg aggtgactgg cctgccgagg tcggaaatcg cttcgccaac 1200
tggctgaacc gggcggtcag cagcgacagt cagatactgg gtagcccgga agccgcccaa 1260
tggagccagg agctgagcaa ggaactgacg atgttcaagg agatactcga agatgagcgt 1320
gaccaagctt gcgactataa ggaccacgac ggagactaca aggatcatga tattgattac 1380
aaagacgatg acgataagta a 1401
<210> 3
<211> 1092
<212> DNA
<213> hCas5f1
<400> 3
atgcctaaga agaagagaaa agtgagcgtg actgatcccg aggcgctgct gttgctgcca 60
cgcctgtcca tccagaacgc caacgccatt tccagcccct tgacctgggg tttcccttcg 120
cccggcgctt tcaccggatt cgtccatgcc ttgcagcgtc gagtcggaat ctccctcgat 180
atcgaactgg acggtgtcgg catcgtctgt caccgcttcg aagcgcagat atcgcaaccg 240
gccggcaagc gcaccaaggt cttcaacctg acccgcaatc cgctgaaccg cgacggctcc 300
accgcagcca tcgtcgaaga aggtcgcgcc catctggagg tcagtctgct gctcggagtg 360
catggcgatg gtctggacga tcaccccgca caggaaatcg ccaggcaggt acaggagcag 420
gctggcgcca tgcgcctcgc cggtggcagc atcctgccct ggtgcaatga gcgcttcccg 480
gctccgaacg ccgaactgct gatgctgggt ggcagcgacg aacagcggcg caagaaccag 540
cgccggctta cccgccgcct gctccccggc ttcgccctgg tgagtcgcga ggcgttgttg 600
caacaacatc tggaaacctt gcgcaccacg ctccccgaag ccaccacgct cgatgcactg 660
ctcgatcttt gtcgaatcaa cttcgagcct cctgcaacct cgtccgaaga agaagcatcg 720
ccgcccgatg cagcctggca agtgcgcgac aagcccggct ggctggtgcc gatcccggct 780
ggctacaacg ccctgtcacc cctgtacctc cccggcgaag tgcgaaatgc ccgtgataga 840
gaaacaccgc tgcgcttcgt cgaaaacctc ttcggtctgg gcgaatggct cagcccccat 900
cgcgtcgccg cgttatcgga cctgctttgg tatcaccacg ccgagcctga caaagggctc 960
taccgctgga gcacaccccg tttcgtcgaa cacgccatcg caggatccta cccatacgat 1020
gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata cgatgttcca 1080
gattacgctt aa 1092
<210> 4
<211> 1134
<212> DNA
<213> hCas7f1
<400> 4
atgccgaaga agaagaggaa agtttccaat gcatccaagc caatactgag caccgcttcc 60
gtcctcgcct tcgaacgtaa gctcgaccct tccgacgccc ttatgagcgc tggcgcctgg 120
gcgcaacgcg acgcctcgca ggaatggccg gccgtgaccg tgcgcgagaa gtccgtacgc 180
ggcaccatct ccaaccgcct caagaccaag gaccgtgacc cggccaagct ggacgcctcg 240
atccagtcgc ccaacctgca gacggtggac gtggccaacc tgccgagcga cgccgacacc 300
ctcaaggtcc gcttcaccct ccgtgtgctc ggcggcgccg gaacaccgtc tgcctgcaac 360
gacgcggcct accgtgacaa gctgctgcaa acggtcgcca cctacgtgaa cgatcagggc 420
ttcgccgagc tggctcgtcg ttatgcgcac aacctggcca acgcccgctt cctgtggcgc 480
aaccgggtgg gcgcggaagc ggtggaagtc cgtatcaacc atatccgcca gggcgaggtg 540
gctcgcgcct ggcgcttcga cgccctggcc atcggcttgc gcgacttcaa ggccgacgcc 600
gaactggacg cgctcgccga actgatcgcc agcggtctct caggcagtgg gcatgtcctg 660
ctcgaagtgg tcgccttcgc ccgtatcggc gacggccagg aagtcttccc ctcccaggaa 720
ctgatcctcg acaaaggcga caagaaaggc cagaagagca agaccctgta cagcgtacgc 780
gatgccgcgg ccatccactc gcagaagatc ggcaatgccc tgcgcaccat cgatacgtgg 840
tatcccgacg aagatggcct cggccccatc gccgtggagc cctacggctc cgtcacatcc 900
cagggcaaag cctatcgcca gcccaagcag aagctggact tctatacgct gctcgacaac 960
tgggtactgc gcgacgaggc gcccgccgtg gagcaacagc attatgtgat cgccaacctg 1020
atccgtggcg gcgtgttcgg tgaagccgaa gagaagcaag cttgcgacta taaggaccac 1080
gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa gtaa 1134
<210> 5
<211> 726
<212> DNA
<213> hCas6f
<400> 5
atgccaaaga agaagcggaa agtcgaccac tatctggaca tcagactgag gcccgatcct 60
gagttccctc ccgcccagct gatgagcgtg ctgtttggca agctgcatca ggctctggtc 120
gcccaaggcg gagacagaat cggcgtgtcc ttccccgacc tggacgagtc ccggagtcgc 180
ctgggcgagc ggctgagaat ccacgccagc gcagacgatc tgcgcgccct gctggcccgg 240
ccttggctgg agggcctgcg ggatcatctg cagtttggcg agcccgccgt ggtgccacac 300
ccaacaccct accgccaggt gagccgcgtg caggccaagt caaatcccga gagactgcgg 360
cggaggctga tgaggcgaca tgatctgagc gaggaggagg ccagaaagag aatccccgac 420
acagtggcca gagccctgga tctgccattt gtgaccctgc ggagccagag cactggccag 480
catttcagac tgttcatcag acacgggccc ctgcaggtga cagccgagga gggcggattt 540
acatgctatg gcctgtctaa aggcggcttc gtgccctggt tccaagcttg cgactataag 600
gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgactataag 660
gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga cgataaggat 720
aagtaa 726
<210> 6
<211> 1761
<212> DNA
<213> hCas7f1-mScarlett
<400> 6
atgccgaaga agaagaggaa agtttccaat gcatccaagc caatactgag caccgcttcc 60
gtcctcgcct tcgaacgtaa gctcgaccct tccgacgccc ttatgagcgc tggcgcctgg 120
gcgcaacgcg acgcctcgca ggaatggccg gccgtgaccg tgcgcgagaa gtccgtacgc 180
ggcaccatct ccaaccgcct caagaccaag gaccgtgacc cggccaagct ggacgcctcg 240
atccagtcgc ccaacctgca gacggtggac gtggccaacc tgccgagcga cgccgacacc 300
ctcaaggtcc gcttcaccct ccgtgtgctc ggcggcgccg gaacaccgtc tgcctgcaac 360
gacgcggcct accgtgacaa gctgctgcaa acggtcgcca cctacgtgaa cgatcagggc 420
ttcgccgagc tggctcgtcg ttatgcgcac aacctggcca acgcccgctt cctgtggcgc 480
aaccgggtgg gcgcggaagc ggtggaagtc cgtatcaacc atatccgcca gggcgaggtg 540
gctcgcgcct ggcgcttcga cgccctggcc atcggcttgc gcgacttcaa ggccgacgcc 600
gaactggacg cgctcgccga actgatcgcc agcggtctct caggcagtgg gcatgtcctg 660
ctcgaagtgg tcgccttcgc ccgtatcggc gacggccagg aagtcttccc ctcccaggaa 720
ctgatcctcg acaaaggcga caagaaaggc cagaagagca agaccctgta cagcgtacgc 780
gatgccgcgg ccatccactc gcagaagatc ggcaatgccc tgcgcaccat cgatacgtgg 840
tatcccgacg aagatggcct cggccccatc gccgtggagc cctacggctc cgtcacatcc 900
cagggcaaag cctatcgcca gcccaagcag aagctggact tctatacgct gctcgacaac 960
tgggtactgc gcgacgaggc gcccgccgtg gagcaacagc attatgtgat cgccaacctg 1020
atccgtggcg gcgtgttcgg tgaagccgaa gagaagacgc gtatggtgag caagggcgag 1080
gcagtgatca aggagttcat gcggttcaag gtgcacatgg agggctccat gaacggccac 1140
gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag 1200
ctgaaggtga ccaagggtgg ccccctgccc ttctcctggg acatcctgtc ccctcagttc 1260
atgtacggct ccagggcctt catcaagcac cccgccgaca tccccgacta ctataagcag 1320
tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg 1380
accgtgaccc aggacacctc cctggaggac ggcaccctga tctacaaggt gaagctccgc 1440
ggcaccaact tccctcctga cggccccgta atgcagaaga agacaatggg ctgggaagcg 1500
tccaccgagc ggttgtaccc cgaggacggc gtgctgaagg gcgacattaa gatggccctg 1560
cgcctgaagg acggcggccg ctacctggcg gacttcaaga ccacctacaa ggccaagaag 1620
cccgtgcaga tgcccggcgc ctacaacgtc gaccgcaagt tggacatcac ctcccacaac 1680
gaggactaca ccgtggtgga acagtacgaa cgctccgagg gccgccactc caccggcggc 1740
atggacgagc tgtacaagta a 1761
<210> 7
<211> 6918
<212> DNA
<213> pxCMV-hCas5f1-PGK-hCas8f1质粒
<400> 7
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggcgcgcc accatgccta agaagaagag 780
aaaagtgagc gtgactgatc ccgaggcgct gctgttgctg ccacgcctgt ccatccagaa 840
cgccaacgcc atttccagcc ccttgacctg gggtttccct tcgcccggcg ctttcaccgg 900
attcgtccat gccttgcagc gtcgagtcgg aatctccctc gatatcgaac tggacggtgt 960
cggcatcgtc tgtcaccgct tcgaagcgca gatatcgcaa ccggccggca agcgcaccaa 1020
ggtcttcaac ctgacccgca atccgctgaa ccgcgacggc tccaccgcag ccatcgtcga 1080
agaaggtcgc gcccatctgg aggtcagtct gctgctcgga gtgcatggcg atggtctgga 1140
cgatcacccc gcacaggaaa tcgccaggca ggtacaggag caggctggcg ccatgcgcct 1200
cgccggtggc agcatcctgc cctggtgcaa tgagcgcttc ccggctccga acgccgaact 1260
gctgatgctg ggtggcagcg acgaacagcg gcgcaagaac cagcgccggc ttacccgccg 1320
cctgctcccc ggcttcgccc tggtgagtcg cgaggcgttg ttgcaacaac atctggaaac 1380
cttgcgcacc acgctccccg aagccaccac gctcgatgca ctgctcgatc tttgtcgaat 1440
caacttcgag cctcctgcaa cctcgtccga agaagaagca tcgccgcccg atgcagcctg 1500
gcaagtgcgc gacaagcccg gctggctggt gccgatcccg gctggctaca acgccctgtc 1560
acccctgtac ctccccggcg aagtgcgaaa tgcccgtgat agagaaacac cgctgcgctt 1620
cgtcgaaaac ctcttcggtc tgggcgaatg gctcagcccc catcgcgtcg ccgcgttatc 1680
ggacctgctt tggtatcacc acgccgagcc tgacaaaggg ctctaccgct ggagcacacc 1740
ccgtttcgtc gaacacgcca tcgcaggatc ctacccatac gatgttccag attacgctta 1800
cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttaagaatt 1860
cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc 1920
ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 1980
aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 2040
gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggaggtaccg 2100
ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg 2160
cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg 2220
ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag 2280
gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg 2340
tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg 2400
gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg 2460
tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc 2520
cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc 2580
ctcatctccg ggcctttcga cctgcatccc gcggccgcgc caccatgccc aagaagaaga 2640
ggaaagtcac ctctcccctc ccaacgccta cgtggcagga gcttcgccag ttcatcgaat 2700
ccttcatcca ggagcgcctc cagggcaagc tggacaaact ccaacccgac gaagacgaca 2760
agcgccagac attgctggcc acccaccggc gggaggcctg gctggccgat gccgcccggc 2820
gggttggcca gttgcagttg gtgacccaca cgctcaagcc gatccatccc gacgcccgcg 2880
gcagcaacct gcacagcctg ccgcaagcac ccggccaacc gggcctcgcc ggttcccatg 2940
agctaggtga caggctggtc agcgatgtgg tgggcaatgc cgcggcgctg gacgtattca 3000
agtttctcag tctccagtat cagggtaaaa atcttctgaa ctggctgaca gaagacagtg 3060
ccgaggcatt acaggcgctg tccgataacg ccgaacaggc tcgcgaatgg cggcaagcgt 3120
tcatcggcat cacgaccgtc aaaggcgctc ccgcgtccca cagcctggcc aagcagttgt 3180
actttcccct gcccggttcc ggctaccacc tgctagcacc gctgtttccc accagtctgg 3240
tgcatcacgt ccacgctctg ctccgcgaag cgcgcttcgg cgacgcggcc aaggcggcac 3300
gcgaagcgcg cagccgccag gagtcatggc cccacggatt cagcgagtac cccaacctgg 3360
cgatccagaa gttcggcggt accaagccgc agaacatcag tcagttgaac aacgagcgcc 3420
gtggcgagaa ctggttgctg ccatcgcttc cgccgaactg gcaaaggcag aatgtaaacg 3480
cgccgatgcg gcactcttcg gtcttcgagc atgacttcgg aagaactcct gaagtatccc 3540
gcctaacccg taccctgcag cgttttcttg ccaagacagt ccataacaac ctcgccatcc 3600
gccagcggcg tgcccaattg gtggcgcaaa tctgcgacga agccctgcaa tacgccgccc 3660
gtctgcgcga actggagccc ggctggagcg caacccccgg gtgccaactg catgacgcgg 3720
agcagctctg gctcgatccg ttgcgcgcac agaccgatga gacgttcttg cagcgccgac 3780
tacgaggtga ctggcctgcc gaggtcggaa atcgcttcgc caactggctg aaccgggcgg 3840
tcagcagcga cagtcagata ctgggtagcc cggaagccgc ccaatggagc caggagctga 3900
gcaaggaact gacgatgttc aaggagatac tcgaagatga gcgtgaccaa gcttgcgact 3960
ataaggacca cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata 4020
agtaagggcc cgtgtaggct agtcgagcag acatgataag atacattgat gagtttggac 4080
aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg 4140
ctttatttgt aaccattata agctgcaata aacaagttgc aggaacccct agtgatggag 4200
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 4260
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag 4320
gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt 4380
caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 4440
cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc 4500
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 4560
tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg 4620
gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 4680
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct 4740
attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga 4800
tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca 4860
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 4920
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 4980
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 5040
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 5100
agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 5160
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 5220
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 5280
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 5340
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 5400
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 5460
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 5520
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 5580
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 5640
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 5700
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 5760
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 5820
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 5880
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 5940
ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc 6000
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 6060
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 6120
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 6180
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 6240
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 6300
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 6360
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 6420
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 6480
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 6540
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 6600
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 6660
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 6720
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 6780
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 6840
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 6900
ggccttttgc tcacatgt 6918
<210> 8
<211> 6210
<212> DNA
<213> pxCMV-hCas6f-PGK-hCas7f1质粒
<400> 8
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggcgcgcc accatggacc actatctgga 780
catcagactg aggcccgatc ctgagttccc tcccgcccag ctgatgagcg tgctgtttgg 840
caagctgcat caggctctgg tcgcccaagg cggagacaga atcggcgtgt ccttccccga 900
cctggacgag tcccggagtc gcctgggcga gcggctgaga atccacgcca gcgcagacga 960
tctgcgcgcc ctgctggccc ggccttggct ggagggcctg cgggatcatc tgcagtttgg 1020
cgagcccgcc gtggtgccac acccaacacc ctaccgccag gtgagccgcg tgcaggccaa 1080
gtcaaatccc gagagactgc ggcggaggct gatgaggcga catgatctga gcgaggagga 1140
ggccagaaag agaatccccg acacagtggc cagagccctg gatctgccat ttgtgaccct 1200
gcggagccag agcactggcc agcatttcag actgttcatc agacacgggc ccctgcaggt 1260
gacagccgag gagggcggat ttacatgcta tggcctgtct aaaggcggct tcgtgccctg 1320
gttcggatcc tacccatacg atgttccaga ttacgcttac ccatacgatg ttccagatta 1380
cgcttaccca tacgatgttc cagattacgc ttaagaattc ctagagctcg ctgatcagcc 1440
tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 1500
accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 1560
tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1620
gattgggaag agaatagcag gcatgctggg gaggtaccgg gtaggggagg cgcttttccc 1680
aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 1740
cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 1800
tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 1860
gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 1920
ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 1980
ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 2040
gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 2100
gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac 2160
ctgcatcccg cggccgcgcc accatgccga agaagaagag gaaagtttcc aatgcatcca 2220
agccaatact gagcaccgct tccgtcctcg ccttcgaacg taagctcgac ccttccgacg 2280
cccttatgag cgctggcgcc tgggcgcaac gcgacgcctc gcaggaatgg ccggccgtga 2340
ccgtgcgcga gaagtccgta cgcggcacca tctccaaccg cctcaagacc aaggaccgtg 2400
acccggccaa gctggacgcc tcgatccagt cgcccaacct gcagacggtg gacgtggcca 2460
acctgccgag cgacgccgac accctcaagg tccgcttcac cctccgtgtg ctcggcggcg 2520
ccggaacacc gtctgcctgc aacgacgcgg cctaccgtga caagctgctg caaacggtcg 2580
ccacctacgt gaacgatcag ggcttcgccg agctggctcg tcgttatgcg cacaacctgg 2640
ccaacgcccg cttcctgtgg cgcaaccggg tgggcgcgga agcggtggaa gtccgtatca 2700
accatatccg ccagggcgag gtggctcgcg cctggcgctt cgacgccctg gccatcggct 2760
tgcgcgactt caaggccgac gccgaactgg acgcgctcgc cgaactgatc gccagcggtc 2820
tctcaggcag tgggcatgtc ctgctcgaag tggtcgcctt cgcccgtatc ggcgacggcc 2880
aggaagtctt cccctcccag gaactgatcc tcgacaaagg cgacaagaaa ggccagaaga 2940
gcaagaccct gtacagcgta cgcgatgccg cggccatcca ctcgcagaag atcggcaatg 3000
ccctgcgcac catcgatacg tggtatcccg acgaagatgg cctcggcccc atcgccgtgg 3060
agccctacgg ctccgtcaca tcccagggca aagcctatcg ccagcccaag cagaagctgg 3120
acttctatac gctgctcgac aactgggtac tgcgcgacga ggcgcccgcc gtggagcaac 3180
agcattatgt gatcgccaac ctgatccgtg gcggcgtgtt cggtgaagcc gaagagaagc 3240
aagcttgcga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag 3300
acgatgacga taagtaaggg cccgtgtagg ctagtcgagc agacatgata agatacattg 3360
atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt 3420
gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt gcaggaaccc 3480
ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 3540
ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 3600
agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 3660
caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 3720
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt 3780
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3840
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3900
atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 3960
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact 4020
ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa 4080
aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 4140
ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4200
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4260
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4320
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 4380
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4440
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4500
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4560
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4620
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 4680
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 4740
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 4800
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4860
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4920
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4980
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 5040
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5100
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5160
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5220
ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg 5280
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5340
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5400
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5460
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5520
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5580
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5640
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5700
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5760
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5820
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5880
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5940
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 6000
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6060
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6120
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6180
tggccttttg ctggcctttt gctcacatgt 6210
<210> 9
<211> 6846
<212> DNA
<213> pxCMV-hCas7f1-mScarlett-PGK-hCas6f质粒
<400> 9
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 180
agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 240
ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 300
tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 360
atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 420
ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 480
gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 540
ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 600
tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 660
aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 720
tctatataag cagagctctc tggctaacta ccggcgcgcc accatgccga agaagaagag 780
gaaagtttcc aatgcatcca agccaatact gagcaccgct tccgtcctcg ccttcgaacg 840
taagctcgac ccttccgacg cccttatgag cgctggcgcc tgggcgcaac gcgacgcctc 900
gcaggaatgg ccggccgtga ccgtgcgcga gaagtccgta cgcggcacca tctccaaccg 960
cctcaagacc aaggaccgtg acccggccaa gctggacgcc tcgatccagt cgcccaacct 1020
gcagacggtg gacgtggcca acctgccgag cgacgccgac accctcaagg tccgcttcac 1080
cctccgtgtg ctcggcggcg ccggaacacc gtctgcctgc aacgacgcgg cctaccgtga 1140
caagctgctg caaacggtcg ccacctacgt gaacgatcag ggcttcgccg agctggctcg 1200
tcgttatgcg cacaacctgg ccaacgcccg cttcctgtgg cgcaaccggg tgggcgcgga 1260
agcggtggaa gtccgtatca accatatccg ccagggcgag gtggctcgcg cctggcgctt 1320
cgacgccctg gccatcggct tgcgcgactt caaggccgac gccgaactgg acgcgctcgc 1380
cgaactgatc gccagcggtc tctcaggcag tgggcatgtc ctgctcgaag tggtcgcctt 1440
cgcccgtatc ggcgacggcc aggaagtctt cccctcccag gaactgatcc tcgacaaagg 1500
cgacaagaaa ggccagaaga gcaagaccct gtacagcgta cgcgatgccg cggccatcca 1560
ctcgcagaag atcggcaatg ccctgcgcac catcgatacg tggtatcccg acgaagatgg 1620
cctcggcccc atcgccgtgg agccctacgg ctccgtcaca tcccagggca aagcctatcg 1680
ccagcccaag cagaagctgg acttctatac gctgctcgac aactgggtac tgcgcgacga 1740
ggcgcccgcc gtggagcaac agcattatgt gatcgccaac ctgatccgtg gcggcgtgtt 1800
cggtgaagcc gaagagaaga cgcgtatggt gagcaagggc gaggcagtga tcaaggagtt 1860
catgcggttc aaggtgcaca tggagggctc catgaacggc cacgagttcg agatcgaggg 1920
cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg 1980
tggccccctg cccttctcct gggacatcct gtcccctcag ttcatgtacg gctccagggc 2040
cttcatcaag caccccgccg acatccccga ctactataag cagtccttcc ccgagggctt 2100
caagtgggag cgcgtgatga acttcgagga cggcggcgcc gtgaccgtga cccaggacac 2160
ctccctggag gacggcaccc tgatctacaa ggtgaagctc cgcggcacca acttccctcc 2220
tgacggcccc gtaatgcaga agaagacaat gggctgggaa gcgtccaccg agcggttgta 2280
ccccgaggac ggcgtgctga agggcgacat taagatggcc ctgcgcctga aggacggcgg 2340
ccgctacctg gcggacttca agaccaccta caaggccaag aagcccgtgc agatgcccgg 2400
cgcctacaac gtcgaccgca agttggacat cacctcccac aacgaggact acaccgtggt 2460
ggaacagtac gaacgctccg agggccgcca ctccaccggc ggcatggacg agctgtacaa 2520
gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 2580
gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 2640
tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 2700
ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 2760
gaggtaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatgcg ctttagcagc 2820
cccgctgggc acttggcgct acacaagtgg cctctggcct cgcacacatt ccacatccac 2880
cggtaggcgc caaccggctc cgttctttgg tggccccttc gcgccacctt ctactcctcc 2940
cctagtcagg aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga 3000
agtagcacgt ctcactagtc tcgtgcagat ggacagcacc gctgagcaat ggaagcgggt 3060
aggcctttgg ggcagcggcc aatagcagct ttgctccttc gctttctggg ctcagaggct 3120
gggaaggggt gggtccgggg gcgggctcag gggcgggctc aggggcgggg cgggcgcccg 3180
aaggtcctcc ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt 3240
ctcctcttcc tcatctccgg gcctttcgac ctgcatcccg cggccgcgcc accatgccaa 3300
agaagaagcg gaaagtcgac cactatctgg acatcagact gaggcccgat cctgagttcc 3360
ctcccgccca gctgatgagc gtgctgtttg gcaagctgca tcaggctctg gtcgcccaag 3420
gcggagacag aatcggcgtg tccttccccg acctggacga gtcccggagt cgcctgggcg 3480
agcggctgag aatccacgcc agcgcagacg atctgcgcgc cctgctggcc cggccttggc 3540
tggagggcct gcgggatcat ctgcagtttg gcgagcccgc cgtggtgcca cacccaacac 3600
cctaccgcca ggtgagccgc gtgcaggcca agtcaaatcc cgagagactg cggcggaggc 3660
tgatgaggcg acatgatctg agcgaggagg aggccagaaa gagaatcccc gacacagtgg 3720
ccagagccct ggatctgcca tttgtgaccc tgcggagcca gagcactggc cagcatttca 3780
gactgttcat cagacacggg cccctgcagg tgacagccga ggagggcgga tttacatgct 3840
atggcctgtc taaaggcggc ttcgtgccct ggttccaagc ttgcgactat aaggaccacg 3900
acggagacta caaggatcat gatattgatt acaaagacga tgacgataag taagggcccg 3960
tgtaggctag tcgagcagac atgataagat acattgatga gtttggacaa accacaacta 4020
gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 4080
ccattataag ctgcaataaa caagttgcag gaacccctag tgatggagtt ggccactccc 4140
tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc 4200
tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg 4260
cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat 4320
agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4380
ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg 4440
ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4500
ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg 4560
ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4620
gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt 4680
tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat 4740
ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa 4800
tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 4860
cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 4920
gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 4980
tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 5040
gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 5100
atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 5160
agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 5220
ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 5280
gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 5340
gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 5400
tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 5460
acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 5520
aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 5580
cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 5640
gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 5700
cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 5760
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 5820
tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 5880
gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 5940
tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 6000
gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 6060
ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 6120
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 6180
agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 6240
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 6300
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 6360
agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 6420
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 6480
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 6540
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 6600
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 6660
gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 6720
ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 6780
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 6840
acatgt 6846
<210> 10
<211> 7350
<212> DNA
<213> pLenti-DR(hCas6f)-EV质粒
<400> 10
ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa 60
catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac 120
gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa 180
ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc 240
tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 300
cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 360
ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 420
cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 480
ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 540
tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 600
gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 660
taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 720
tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 780
gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 840
ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 900
gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 960
gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 1020
gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 1080
gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 1140
ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 1200
agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 1260
caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 1320
ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 1380
aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1440
aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1500
gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1560
aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1620
atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1680
tttcagaccc acctcccaac cccgagggga cccagagagg gcctatttcc catgattcct 1740
tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa tttgactgta 1800
aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 1860
gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 1920
ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggttc actgccgtat 1980
aggcagctaa gaaatgagac ggcagccagg gatcctatcc gtctcagttc actgccgtat 2040
aggcagctaa gaaaactttt tttgaattct agatcttgag acaaatggca gtattcatcc 2100
acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca 2160
taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt 2220
ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag tggctccggt 2280
gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc 2340
ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 2400
tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 2460
gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc 2520
ctagcggcag cggcgccacc aacttcagcc tgctgaagca ggccggcgac gtggaggaga 2580
accccggccc catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc 2640
ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg 2700
tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg 2760
tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgcggccgtg gcggtctgga 2820
ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg 2880
agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc 2940
ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca 3000
agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc 3060
ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca 3120
ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc 3180
ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat 3240
tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc 3300
ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct 3360
ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca 3420
ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt 3480
ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 3540
cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga 3600
aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt 3660
ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc 3720
cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt 3780
gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag 3840
ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga 3900
tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc 3960
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 4020
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 4080
agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt 4140
ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa 4200
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 4260
ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc 4320
ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 4380
catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta 4440
ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata 4500
gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 4560
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 4620
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg 4680
acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 4740
ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 4800
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 4860
gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 4920
catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 4980
gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 5040
aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5100
acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt 5160
gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 5220
acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 5280
tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 5340
agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 5400
cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 5460
aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 5520
gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 5580
agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 5640
aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 5700
gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 5760
ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 5820
aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 5880
aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 5940
tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 6000
agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 6060
ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 6120
ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 6180
ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 6240
acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 6300
agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 6360
ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 6420
cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 6480
gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 6540
cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 6600
gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 6660
caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 6720
aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 6780
tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 6840
gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 6900
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 6960
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 7020
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 7080
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 7140
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 7200
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 7260
acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca 7320
ctaaagggaa caaaagctgg agctgcaagc 7350
Claims (21)
1.Type I-F CRISPR/Cas***在染色质成像方面或在制备染色质成像工具中的应用。
2.一种利用隶属于Type I-F CRISPR/Cas***的Cascade复合物进行染色质成像的方法,以PaeCascade复合物为例,其特征在于,首先对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化,然后将目标基因的靶向序列、优化后的Cas8f1、优化后的Cas5f1、优化后的Cas7f1、优化后的Cas6f以及荧光蛋白共同呈递至靶细胞。
3.根据权利要求2所述方法,其特征在于,首先对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化,然后将目标基因的靶向序列、优化后的Cas8f1、优化后的Cas5f1、优化后的Cas7f1、优化后的Cas6f以任意组合***表达载体,得到重组质粒组合,再将荧光蛋白***所述重组质粒组合中任一个重组质粒中,最后所得重组质粒共同转染细胞。
4.一种融合质粒,其特征在于,是由hCas8f1、hCas5f1、hCas7f1、hCas6f中的任一种或任几种组合***表达载体得到重组质粒。
5.根据权利要求4所述融合质粒,其特征在于,荧光蛋白***任意一个重组质粒中。
6.一种基于Type I-F CRISPR/Cas的染色质成像***,其特征在于,包含权利要求4或5所述融合质粒。
7.一种利用隶属于Type I-F CRISPR/Cas***的PaeCascade复合物进行染色质成像的方法,其特征在于,包括如下步骤:
(1)对PaeCascade复合物的Cas8f1、Cas5f1、Cas7f1和Cas6f分别进行适应编辑对象的优化;
(2)选取目标基因中靶向序列,将靶向序列***表达载体中构建crRNA靶向质粒;
(3)将优化后的Cas8f1、Cas5f1、Cas7f1和Cas6f中任两个与启动子共同***表达载体构建融合质粒I;
(4)将将优化后的Cas8f1、Cas5f1、Cas7f1和Cas6f中任两个与启动子和荧光蛋白共同***表达载体构建融合质粒IV;
(5)将上述crRNA靶向质粒和融合质粒I、融合质粒IV共同转染细胞。
8.优化的PaeCascade效应蛋白,包括hCas8f1、hCas5f1、hCas7f1和hCas6f,其特征在于,hCas8f1的序列如SEQ ID NO.2所示,hCas5f1的序列如SEQ ID NO.3所示,hCas7f1的序列如SEQ ID NO.4所示,hCas6f的序列如SEQ ID NO.5所示。
9.一种融合质粒I,其特征在于,所述融合质粒I为:真核细胞表达载体-hCsy蛋白-启动子-hCsy蛋白;其中两个hCsy蛋白分别选自hCas8f1、hCas5f1、hCas7f1或hCas6f且两个hCsy蛋白不同。
10.根据权利要求9所述融合质粒I,其特征在于,所述融合质粒I为pxCMV-hCas5f1-PGK-hCas8f1,或所述融合质粒I为pxCMV-hCas6f-PGK-hCas7f1。
11.根据权利要求10所述融合质粒I,其特征在于,融合质粒I的N端添加核定位信号PKKKRKV。
12.根据权利要求11所述融合质粒I,其特征在于,所述融合质粒pxCMV-hCas5f1-PGK-hCas8f1的序列如SEQ ID NO.7所示;所述融合质粒pxCMV-hCas6f-PGK-hCas7f1的序列如SEQ ID NO.8所示。
13.一种融合质粒IV,其特征在于,所述融合质粒IV为:真核细胞表达载体-hCas7f1-荧光蛋白-启动子-hCas6f。
14.根据权利要求13所述融合质粒IV,其特征在于,所述融合质粒IV为pxCMV-hCas7f1-mScarlett-PGK-hCas6f。
15.根据权利要求14所述融合质粒IV,其特征在于,融合质粒IV的N端添加核定位信号PKKKRKV。
16.根据权利要求15所述融合质粒IV,其特征在于,所述融合质粒pxCMV-hCas7f1-mScarlett-PGK-hCas6f的序列如SEQ ID NO.9所示。
17.一种融合质粒V,其特征在于,所述融合质粒V为在适于哺乳动物的表达载体中***hCas6f-DR序列得到。
18.根据权利要求17所述融合质粒V,其特征在于,所述融合质粒V为pLenti-DR(hCas6f)-EV。
19.根据权利要求18所述融合质粒V,其特征在于,所述融合质粒pLenti-DR(hCas6f)-EV的序列如SEQ ID NO.10所示。
20.一种基于Type I-F CRISPR/Cas的染色质成像***,其特征在于,包括以下三个组分:
(1)权利要求9-12任一所述融合质粒I;
(2)权利要求13-16任一所述融合质粒IV;
(3)权利要求17-19任一所述融合质粒V。
21.权利要求20所述***在制备染色质成像工具方面的应用。
Priority Applications (1)
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CN115806922A (zh) * | 2022-09-19 | 2023-03-17 | 湖北大学 | 运动发酵单胞菌的基因工程菌株及其应用 |
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CN115806922A (zh) * | 2022-09-19 | 2023-03-17 | 湖北大学 | 运动发酵单胞菌的基因工程菌株及其应用 |
CN115806922B (zh) * | 2022-09-19 | 2023-08-11 | 湖北大学 | 运动发酵单胞菌的基因工程菌株及其应用 |
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