CN113563476A - 遗传和表观遗传调节蛋白至特定基因组基因座的rna引导的靶向 - Google Patents

遗传和表观遗传调节蛋白至特定基因组基因座的rna引导的靶向 Download PDF

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CN113563476A
CN113563476A CN202110709688.0A CN202110709688A CN113563476A CN 113563476 A CN113563476 A CN 113563476A CN 202110709688 A CN202110709688 A CN 202110709688A CN 113563476 A CN113563476 A CN 113563476A
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J.K.乔昂格
M.梅德
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Abstract

用于将异源功能性结构域诸如转录激活物RNA引导靶向特定基因组基因座的方法和构建体。本发明涉及用于将基因和表观基因调控蛋白，例如转录及货物，组蛋白修饰酶，DNA甲基化修饰物，RNA引导靶向特定的基因座的方法和构建物。本发明至少部分基于融合蛋白的开发，包括融合到Cas9核酸酶的异源功能性结构域(例如，转录激活结构域)，所述Cas9核酸酶通过突变具有其核酸酶活性失活(也称为“dCas9”)。

Description

遗传和表观遗传调节蛋白至特定基因组基因座的RNA引导的 靶向
本申请是申请日为2014年3月14日，申请号为201480026276.5，发明名称为“遗传和表观遗传调节蛋白至特定基因组基因座的RNA引导的靶向”的专利申请的分案申请。
优先权的声明
本申请要求2013年3月15日提交的美国专利申请系列第61/799,647号、2013年6月21日提交的美国专利申请系列第61/838,178号、2013年6月21日提交的美国专利申请系列第61/838,148号和2013年12月26日提交的美国专利申请系列第61/921,007号的权益。前述专利申请系列号的完整内容在此通过引用并入。
联邦资助的研究或开发
本发明是在由美国国家卫生研究院授予的基金第DP1GM105378号和由国防部的国防高等研究计划署(DARPA)授予的W911NF-11-2-0056下借助政府资助进行的。政府具有本发明的某些权利。
技术领域
本发明涉及用于将遗传和表观遗传调节蛋白，例如，转录激活物、组蛋白修饰酶、DNA甲基化修饰剂RNA引导靶向至特定基因组基因座的方法和组合物。
背景
被称为CRISPR/Cas系统的成簇规律间隔短回文重复(CRISPR)和CRISPR相关(cas)基因被不同细菌和古细菌(archaea)用来介导抵抗病毒和其它外来核酸的防御。这些系统使用小RNA来以序列特异性方式检测和沉默外来核酸。
已描述了3个类型的CRISPR/Cas系统(Makarova等，Nat.Rev.Microbiol.9,467(2011)；Makarova等，Biol.Direct 1,7(2006)；Makarova等，Biol.Direct 6,38(2011))。最近的工作已显示II型CRISPR/Cas系统可被工程化来通过使用与DNA靶位点具有互补性的单一“引导RNA”和Cas9核酸酶在体外将靶向双链DNA断裂导向特定序列(Jinek等，Science2012；337:816–821)。该可靶向的基于Cas9的系统也在培养的人细胞中(Mali等，Science.2013年2月15日；339(6121):823-6；Cong等，Science.2013年2月15日；339(6121):819-23)以及在斑马鱼中在体内起作用(Hwang和Fu等，Nat Biotechnol.2013年3月；31(3):227-9)以诱导至内源基因内的靶向改变。
概述
本发明至少部分基于融合蛋白的开发，所述融合蛋白包括与其核酸酶活性已通过突变灭活的Cas9核酸酶(也称为“dCas9”)融合的异源功能性结构域(例如，转录激活结构域)。虽然公布的研究已使用引导RNA将具有催化活性但失活的Cas9核酸酶蛋白靶向特定基因组基因座，但还没有工作改造该系统来适用于招募另外的效应子结构域。该工作还提供了导致靶基因的表达水平的升高(而非降低)的RNA引导的过程的首次例证。
另外，本公开还提供了多重gRNA可用于将多种dCas9-VP64融合物带至单个启动子，从而导致转录的协同激活的首次例证。
因此，在第一方面，本发明提供包含连接于异源功能性结构域(HFD)的无催化活性的CRISPR相关9(dCas9)蛋白的融合蛋白，所述异源功能性结构域修饰基因表达、组蛋白或DNA，例如转录激活结构域、转录阻遏物(例如，沉默子诸如异染色质蛋白1(HP1)，例如HP1α或HP1β，或转录抑制结构域，例如，Krueppel相关盒(KRAB)结构域、ERF抑制结构域(ERD)或mSin3A相互作用结构域(SID))、修饰DNA的甲基化状态的酶(例如，DNA甲基转移酶(DNMT)或10-11易位(TET)蛋白，例如，也称为Tet甲基胞嘧啶双加氧酶1的TET1)或修饰组蛋白亚单位的酶(例如，组蛋白乙酰转移酶(HAT)、组蛋白脱乙酰酶(HDAC)或组蛋白脱甲基酶)。在一些实施方案中，所述异源功能性结构域是转录激活结构域，例如，来自VP64或NF-κB p65的转录激活结构域；催化DNA脱甲基化的酶，例如，TET；或组蛋白修饰(例如，LSD1、组蛋白甲基转移酶、HDAC或HAT)或转录沉默结构域，例如，来自异染色质蛋白1(HP1)，例如，HP1α或HP1β；或生物系链，例如CRISPR/Cas亚型Ypest蛋白4(Csy4)、MS2或λN蛋白。
在一些实施方案中，无催化活性的Cas9蛋白来自化脓性链球菌(S.pyogenes)。
在一些实施方案中，无催化活性的Cas9蛋白在D10、E762、H983或D986；以及在H840或N863，例如在D10和H840处包含突变，例如D10A或D10N和H840A或H840N或H840Y。
在一些实施方案中，所述异源功能性结构域通过任选的间插接头连接于无催化活性的Cas9蛋白的N末端或C末端，其中所述接头不干扰融合蛋白的活性。
在一些实施方案中，所述融合蛋白在N末端、C末端或无催化活性的CRISPR相关9(Cas9)蛋白与异源功能性结构域之间中包括核定位序列和一个或多个表位标签(例如，c-myc、6His或FLAG标签)的任一者或两者，任选地具有一个或多个间插接头。
在其它方面，本发明提供编码本文中描述的融合蛋白的核酸以及包含所述核酸的表达载体，和表达所述融合蛋白的宿主细胞。
在另外的方面，本发明提供用于增加靶基因在细胞中的表达的方法。所述方法包括例如通过细胞与包括编码所述融合蛋白的序列的表达载体接触在所述细胞中表达如本文所述Cas9-HFD融合蛋白，以及还例如通过将细胞与一个或多个包含编码一个或多个引导RNA的核酸序列的表达载体接触来在细胞中表达一个或多个具有针对靶基因的互补性的引导RNA。
除非另有定义，否则本文所用的所有技术和/或科学术语具有与本发明所属领域的普通技术人员通常所理解的含义相同的含义。本文中描述了用于本发明的方法和材料；还可使用本领域中已知的其它合适的方法和材料。所述材料、方法和实施例仅为示例性的并非旨在限制。本文中提及的所有出版物、专利申请、专利、序列、数据库条目和其它参考文献通过引用整体并入。如发生矛盾，则以本专利说明书包括定义为准。
根据以下详细说明和附图以及权利要求，本发明特征和有利将是显而易见的。
附图概述
本专利或申请文件含有至少一个以彩色绘制的附图。具有彩色附图的本专利或专利申请公布的拷贝可在请求和支付必要的费用后由专利局提供。
图1A是显示单一引导RNA(sgRNA)将Cas9核酸酶招募至特定DNA序列，从而引入靶向改变的示意图。显示的引导RNA的序列为GGAGCGAGCGGAGCGGUACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(SEQ ID NO:9)。
图1B是显示用于将Cas9核酸酶招募至特定DNA序列，从而引入靶向改变的sgRNA的较长形式的示意图。显示的引导RNA的序列为GGAGCGAGCGGAGCGGUACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO:10)。
图1C是显示与转录激活结构域融合并被sgRNA招募至特定DNA序列的Cas9蛋白，所述Cas9蛋白含有D10A和H840A突变以使得蛋白质的核酸酶部分无催化活性。显示的引导RNA的序列为GGAGCGAGCGGAGCGGUACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU(SEQ ID NO:10)。
图1D是描绘dCas9-VP64融合蛋白被嵌合sgRNA招募至特定基因组靶序列的示意图。
图1E是举例说明16个sgRNA被靶向内源人VEGFA基因启动子的位置和取向的简图。小的水平箭头代表与基因组DNA序列互补的gRNA的前20nt，箭头从5’指向3’。灰色条指示先前在人293细胞中确定的相对于转录起始位点(直角箭头)编号的DNA酶I高敏位点(Liu等，JBiol Chem.2001年4月6日；276(14):11323-34)。
图2A是显示各种sgRNA对293细胞中的VEGFA蛋白表达的激活的条线图，每种与(灰色条块)或不与(黑色条块)dCas9-VP64一起表达。计算相对于如方法中描述的脱靶sgRNA对照的VEGFA的激活倍数。以一式三份进行每一个实验，误差条代表平均值的标准误差。星号表示如通过配对单侧t检验(p<0.05)测定的被显著升高高于脱靶对照的的样品。
图2B是显示多重sgRNA表达通过dCas9-VP64蛋白诱导VEGFA蛋白表达的协同激活的条线图。显示其中将指定的sgRNA组合与dCas9-VP64共表达的293细胞中的VEGFA蛋白的激活倍数。注意，在所有这些实验中，每一种用于转染的单独的sgRNA表达质粒的量相同。如2A中所述计算激活倍数值并以灰色条块显示。将每一个组合的由单独的sgRNA诱导的平均激活倍数值的计算的总和显示为黑色条块。星号表示被发现显著大于如通过方差分析(ANOVA)(p<0.05)测定的预期总和的所有组合。
图3A是举例说明被靶向内源人NTF3基因启动子的6个sgRNA的位置和取向的简图。水平箭头代表与基因组DNA序列互补的sgRNA的前20nt，箭头从5’指向3’。灰线表示从UCSC基因组浏览器上的ENCODE DNA酶I超敏反应跟踪鉴定的潜在开放染色质的区域，条的更粗部分表示首先转录的外显子。显示的编号是相对于转录起始位点的(+1，直角箭头)。
图3B是显示293细胞中sgRNA引导的dCas9-VP64对NTF3基因表达的激活的条线图。显示了用指定量的dCas9-VP64和NTF3靶向sgRNA表达质粒共转染的293细胞的通过定量RT-PCR检测的并针对GAPDH对照(ΔCt x 104)标准化的NTF3 mRNA的相对表达。以一式三份进行所有实验，误差条表示平均值的标准误差。星号表示如通过配对单侧T检验(P<0.05)测定比脱靶gRNA对照显著更大的样品。
图3C是显示多重gRNA表达通过dCas9-VP64蛋白诱导NTF3mRNA表达的协同激活。显示了用dCas9-VP64和指定的NTF3靶向gRNA表达质粒的组合共转染的293细胞的通过定量RT-PCR检测的并针对GAPDH对照(ΔCt x 104)标准化的NTF3 mRNA的相对表达。注意，在所有这些实验中，每一个用于转染的单独的gRNA表达质粒的量相同。以一式三份进行所有实验，误差条表示平均值的标准误差。显示了每一个组合的由单独的gRNA诱导的平均激活倍数值的计算的总和。
图4是sgRNA表达载体的示例性序列。
图5是CMV-T7-Cas9 D10A/H840A-3XFLAG-VP64表达载体的示例性序列。
图6是CMV-T7-Cas9记录的D10A/H840A-3XFLAG-VP64表达载体的示例性序列。
图7是Cas9-HFD即Cas9-激活物的示例性序列。任选的3xFLAG序列加以下划线；核定位信号PKKKRKVS(SEQ ID NO:11)以小写显示；两个接头以粗体显示；以及VP64转录激活物序列DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML(SEQ ID NO:12)加框表示。
图8A-8B是(8A)dCas9-NLS-3XFLAG-HP1α和(8B)dCas9-NLS-3XFLAG-HP1β的示例性序列。框＝核定位信号；下划线＝三重flag标签；双下划线＝HP1α铰链和chromoshadow结构域。
图9为dCas9-TET1的示例性序列。
图10是显示利用各种dCas9-VP64融合构建体获得的结果的条线图。所测试的那些构建体中，最优化的dCas9-VP64体系结构包括N末端NLS(NFN)和位于dCas9与VP64之间的另外的NLS(N)或FLAG标签/NLS(NF)。VEGFA基因在人HEK293细胞中的表达通过由RNA引导的dCas9-VP64融合物介导的转录激活来激活。将编码dCas9-VP64的变体的表达质粒与表达3个靶向VEGFA起始密码子的上游区域中的位点的gRNA(在本实验中，从单一gRNA表达gRNA并且通过Csy4内切核糖核酸酶加工出所述gRNA)的质粒共转染。VEGFA蛋白表达通过ELISA来测量，并且显示了两个重复的平均值，误差东躲西藏表示平均值的标准误差。
图11A-B是显示具有使Cas9催化功能灭活的替代性取代突变的dCas9-VP64激活物的活性的条线图。(11A)将表达具有对残基D10和H840的各种Cas9灭活取代的dCas9-VP64蛋白的质粒各自与单一gRNA或3个不同的靶向VEGFA上游区域的靶向gRNA(分别为蓝色和红色条块)共转染进HEK293细胞。(11B)还将表达这些dCas9-VP64变体的质粒转染进稳定地表达单个VEGFFA靶向gRNA的HEK293细胞系。通过ELISA测定VEGFA蛋白水平，显示了两个重复的平均值和平均值的标准误差(误差条)。
详述
本文中描述了与Cas9蛋白的无催化活性的形式融合(出于使得能够在细胞和活生物体中将这些功能性结构域RNA引导地靶向特定基因组位置的目的)的异源功能性结构域(例如，转录激活结构域)的融合蛋白。
CRISPR/Cas系统在细菌中已进化为保护细菌免受侵入质粒和病毒伤害的防御机制。来源于外来核酸的短的原型间隔序列被整合在CRISPR基因座中，随后被翻译和加工成短的CRISPR RNA(crRNA)。这些与第二tracrRNA复合的crRNA随后使用它们的与侵入核酸互补的序列来引导Cas9介导的断裂，和随后外来核酸的破坏。在2012年，Doudna和同事证明由crRNA与tracrRNA的融合物组成的单一引导RNA(sgRNA)可在体外介导Cas9核酸酶至特定DNA序列的招募(图1C；Jinek等，Science 2012)。
最近，sgRNA的更长形式已被用于在人细胞和斑马鱼中引入靶向改变(图1B；Mali等Science 2013,Hwang和Fu等，Nat Biotechnol.2013年3月；31(3):227-9)。Qi等证明Cas9的无催化活性突变形式(被称为dCas9)的gRNA介导的招募可导致大肠杆菌(E.coli)中的特定内源基因以及人细胞中的EGFP报告基因的抑制(Qi等，Cell 152,1173–1183(2013))。虽然本研究显示采用RNA引导的Cas9技术调控基因表达的潜能，但其不测试或显示异源功能性结构域(例如-转录激活结构域)是否可被融合于dCas9而不破坏其被可编程sgRNA或双重gRNA(dgRNA–即-定制的crRNA和tracrRNA)招募至特定基因组位点的能力。
如本文中所述，除了引导Cas9介导的核酸酶活性外，还可能使用CRISPR衍生的RNA来将与Cas9融合的异源功能性结构域(Cas9-HFD)靶向基因组中的特定位点(图1C)。例如，如本文中所述，可能使用单一引导RNA(sgRNA)来将Cas9-HFD例如Cas9转录激活物(在下文中被称为Cas9激活物)靶向特定基因的启动子，从而增加靶基因的表达。因此，可将Cas9-HFD定位至基因组中的位点，靶特异性由引导RNA的序列互补性确定。靶序列还包括PAM序列(与由RNA指定的序列相邻的由Cas9蛋白指定的2-5个核苷酸序列)。
Cas9-HFD通过将异源功能性结构域(例如，例如来自VP64或NF-κB p65的转录激活结构域)与无催化活性的Cas9蛋白的N末端或C末端融合来生成。
Cas9
许多细菌表达Cas9蛋白变体。来自化脓性链球菌(Streptococcus pyogenes)的Cas9是目前最常使用的；一些另外的Cas9蛋白与化脓性链球菌Cas9具有高水平的序列同一性，并且使用相同的引导RNA。其它的更加多样，使用不同的gRNA，并且同样地识别不同的PAM序列(与由RNA指定的序列相邻的由蛋白质指定的2-5个核苷酸的序列)。Chylinski等将来自一大组细菌的Cas9蛋白分类(RNA Biology 10:5,1–12；2013)，并且许多Cas9蛋白列于补充图1和其补充表1中，所述图表通过引用并入本文。另外的Cas9蛋白描述于Esvelt等，Nat Methods.2013年11月；10(11):1116-21和Fonfara等，“Phylogeny of Cas9determines functional exchangeability of dual-RNA and Cas9among orthologoustype II CRISPR-Cas systems.”Nucleic Acids Res.2013年11月22日中。[先于印刷的电子出版]doi:10.1093/nar/gkt1074。
许多物种的Cas9分子可用于本文中描述的方法和组合物。虽然化脓性链球菌和嗜热链球菌Cas9分子是本文中的许多公开内容的主题，但同样地可使用本文中所列的其它物种的Cas9分子、来源于或基于所述物种的Cas9蛋白的Cas9分子。换句话说，虽然本文中的许多描述使用化脓性链球菌和嗜热链球菌Cas9分子，但来自其它物种的Cas9分子可替代它们。此类物种包括下表中所示的那些物种，所述表是基于Chylinski等，2013的补充图1产生的。
Figure BDA0003133036850000101
Figure BDA0003133036850000111
Figure BDA0003133036850000121
本文中描述的构建体和方法包括任何那些Cas9蛋白以及它们对应的引导RNA或相容的其它引导RNA的使用。已显示来自嗜热链球菌LMD-9CRISPR1系统的Cas9在Cong等(Science 339,819(2013))中的人细胞中起作用。另外，Jinek等人在体外显示来自嗜热链球菌和英诺克李斯特菌(L.innocua)(但非来自奈瑟氏脑膜炎球菌或空肠弯曲菌(C.jejuni)的Cas9直系同源物(其可使用不同的引导RNA)可被双重化脓性链球菌gRNA导向裂解靶质粒DNA，虽然效率略有降低。
在一些实施方案中，本系统利用来自化脓性链球菌的Cas9蛋白(如在细菌中编码的或针对在哺乳动物细胞中的表达进行密码子最优化的)，其在D10、E762、H983或D986和H840或N863处含有突变，例如D10A/D10N和H840A/H840N/H840Y，以使得蛋白的核酸酶部分催化失活；这些位置上的取代可以是丙氨酸(如它们在Nishimasu等，Cell 156,935–949(2014)中一样)或它们可以是其它残基，例如谷氨酰胺、天冬酰胺、酪氨酸、丝氨酸或天冬氨酸，例如，E762Q、H983N、H983Y、D986N、N863D、N863S或N863H(图1C)。可用于本文中描述的方法和组合物的自由化来活的化脓性链球菌Cas9的序列如下；D10A和H840A的示例性突变以粗体表示并加以下划线。
Figure BDA0003133036850000141
Figure BDA0003133036850000151
在一些实施方案中，本文中使用的Cas9核酸酶与化脓性链球菌Cas9的序列具有至少约50％的同一性，即与SEQ ID NO:13具有至少50％的同一性。在一些实施方案中，所述核苷酸序列与SEQ ID NO:13具有约50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、99％或100％的同一性。
在一些实施方案中，本文中使用的无催化活性的Cas9与无催化活性的化脓性链球菌Cas9的序列具有至少约50％的同一性，即，与SEQ ID NO:13具有至少50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、99％或100％的同一性，其中D10和H840处的突变，例如D10A/D10N和H840A/H840N/H840Y得到保持。
在一些实施方案中，与SEQ ID NO:13的任何差异在非保守区中，如通过Chylinski等，RNA Biology 10:5,1–12；2013(例如，在补充图1及其补充表1中)；Esvelt等，NatMethods.2013年11月；10(11):1116-21和Fonfara等，Nucl.Acids Res.(2014)42(4):2577-2590中所示的序列的序列比对鉴定的。[2013年11月22日先于印刷的电子版]doi:10.1093/nar/gkt1074，并且其中D10和H840处的突变，例如，D10A/D10N和H840A/H840N/H840Y得到保持。
为了测定两个序列的百分比同一性，为了最佳比较目的，将所述序列比对(可根据需要在第一和第二氨基酸或核酸序列的一个或两个序列中引入缺口以进行最佳比对，并且为了比较目的可忽略非同源序列)。为了比较目的而比对的参照序列的长度为至少50％(在一些实施方案中，比对约50％、55％、60％、65％、70％、75％、85％、90％、95％或100％的参照序列的长度)。随后比较对应位置上的核苷酸或残基。当第一序列中的位置被与第二序列中的对应位置相同的核苷酸或残基占据时，则所述分子在该位置上是相同的。两个序列之间的百分比同一性是由序列序列共享的相同位置的数目的函数，该函数考虑了为一两个序列的最佳比对而需要引入的缺口数目和每一个缺口的长度。
序列的比较和两个序列之间的百分比同一性的测定可使用数学算法来实现。为了本申请的目的，两个氨基酸序列之间的百分比同一性使用Needleman和Wunsch((1970)J.Mol.Biol.48:444-453)算法，使用Blossum 62评分矩阵，利用为12的缺口罚分、为4的缺口延伸罚分和为5的移码缺口罚分来测定，该算法已被整合进GCG软件包中的GAP程序。
异源功能性结构域
可在Cas9的N或C末端上融合转录激活结构域。另外，尽管本说明书举例说明转录激活结构域，但还可使用如在本领域中是已知的其它异源功能性结构域(例如，转录阻遏物(例如，KRAB、ERD、SID和其它，例如ets2阻遏因子(ERF)阻遏结构域(ERD)的氨基酸473–530、KOX1的KRAB结构域的氨基酸1–97或Mad mSIN3相互作用结构域(SID)的氨基酸1–36；参见Beerli等，PNAS USA 95:14628-14633(1998))或沉默子诸如异染色质蛋白1(HP1，也称为swi6)，例如HP1α或HP1β；可招募与固定的RNA结合序列诸如被MS2衣壳蛋白、内切核糖核酸酶Csy4或λN蛋白结合的那些序列融合的长的非编码RNA(lncRNA)的蛋白质或肽；修饰DNA的甲基化状态的酶(例如，DNA甲基转移酶(DNMT)或TET蛋白)；或修饰组蛋白亚单位的酶(例如，组蛋白乙酰转移(HAT)、组蛋白脱乙酰酶(HDAC)、组蛋白甲基转移酶(例如，用于赖氨酸或精氨酸残基的甲基化)或组蛋白脱甲基酶(例如，用于赖氨酸或精氨酸残基的脱甲基化))。此类结构域的许多序列在本领域中是已知的，例如，催化DNA中的甲基化半胱氨酸的羟化的结构域。示例性蛋白包括10-11-易位(TET)1-3家族，将DNA中的5-甲基胞嘧啶(5-mC)转化成5-羟甲基胞嘧啶(5-hmC)的酶。
人TET1-3的序列在本领域中是已知的并且示于下表中：
Figure BDA0003133036850000171
*变体(1)代表较长的转录物并且编码较长的同种型(a)。变体(2)与变体1在5'UTR和3'UTR中以及在编码序列中不同。所得的同种型(b)是较短的并且相较于同种型a具有不同的C末端。
在一些实施方案中，可包括催化结构域的全长序列的全部或部分，例如包含富含半胱氨酸的延伸和由7个高度保守的外显子编码的2OGFeDO结构域，例如包含氨基酸1580-2052的Tet1催化结构域、包含氨基酸1290-1905的Tet2和包含氨基酸966-1678的Tet3的催化模块。关于举例说明所有3个Tet蛋白中的至关重要的催化残基的比对，参见，例如，Iyer等，Cell Cycle.2009年6月1日；8(11):1698-710.Epub 2009年6月27日的图1，并且关于全长序列(参见，例如，seq 2c)，参见其补充材料(可在ftp站点ftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html获得)；在一些实施方案中，所述序列包括Tet1的氨基酸1418-2136或Tet2/3中的对应区域。
其它催化分子可来自由Iyer等，2009鉴定的蛋白质。
在一些实施方案中，异源性功能结构域是生物系链，并且包含MS2衣壳蛋白、内切核糖核酸酶Csy4或λN蛋白的全部或部分(例如来自其的DNA结合结构域)。这些蛋白可用于将含有特定茎环结构的RNA分子招募至由dCas9 gRNA靶向序列指定的场所。例如，与MS2衣壳蛋白、内切核糖核酸酶Csy4或λN融合的dCas9可用于招募长的非编码RNA(lncRNA)诸如XIST或HOTAIR；参见，例如，Keryer-Bibens等，Biol.Cell 100:125–138(2008)，其连接于Csy4、MS2或λN结合序列。或者，可将Csy4、MS2或λN蛋白结合序列连接于另一种蛋白质，如Keryer-Bibens等(同上)中描述的，并且可使用本文中描述的方法和组合物将所述蛋白质靶向dCas9结合位点。在一些实施方案中，Csy4是无催化活性的。
在一些实施方案中，融合蛋白包括dCas9与异源功能性结构域之间的接头。可用于这些融合蛋白中(或串联结构中的融合蛋白之间)的接头可包括不干扰融合蛋白的功能的任何序列。在优选实施方案中，所述接头是短的，例如2-20个氨基酸，并且通常是柔性的(即，包含具有高度自由的氨基酸诸如甘氨酸、丙氨酸和丝氨酸)。在一些实施方案中，所述接头包含一个或多个由GGGS(SEQ ID NO:14)或GGGGS(SEQ ID NO:15)组成的单位，例如，2、3、4或更多个GGGS(SEQ ID NO:14)或GGGGS(SEQ ID NO:15)单位的重复。还可使用其它接头序列。
使用方法
所述Cas9-HFD系统是有用的并且是修饰内源基因的表达的通用工具。用于获得该系统的当前方法需要产生针对每一个待靶向的位点的新型工程化DNA结合蛋白(诸如工程化锌指或转录激活因子样效应子DNA结合结构域)。因为这些方法需要表达专门工程化来结合每一个靶位点的大蛋白质，因此它们在它们用于多路技术的能力上受到限制。然而，Cas9-HFD需要仅单一Cas9-HFD蛋白的表达，所述蛋白可通过表达多个短的gRNA来被靶向基因组中的多个位点。该系统因而可容易地用于同时诱导许多基因表达或将多个Cas9-HFD招募至单个基因、启动子或增强子。该能力将具有广泛的用途，例如用于基础生物学研究，其中其可用于研究基因功能和维持多个基因在单个途径中的表达，以及用于合成生物学，其中其将使得研究者能够在负责多个输出信号的细胞中产生回路。该技术可被执行和适合用于多路复用的相对容易性使得其成为具有许多广泛应用的广泛有用的技术。
本文所述方法包括将细胞与编码本文所述Cas9-HFD的核酸和编码一个或多个被导向选定的基因的引导RNA的核酸接触，从而调节该基因的表达。
引导RNA(gRNA)
引导RNA一般而言出现在两个不同的系统中：系统1，其使用一起指导Cas9进行裂解的单独的crRNA和tracrRNA，和系统2，其使用在单个系统中组合两个单独的引导RNA的嵌合crRNA-tracrRNA杂交体(被称为单一引导RNA或sgRNA，也参见Jinek等，Science 2012；337:816–821)。tracrRNA可被可变地截短，并且已显示许多长度在所述单独的系统(系统1)和嵌合gRNA系统(系统2)中都具有功能。例如，在一些实施方案中，可从其3’末端将tracrRNA截短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。在一些实施方案中，可将tracrRNA分子从其5’末端截短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。或者，可以从5’和3’末端截短tracrRNA分子，例如在5’末端截短至少1、2、3、4、5、6、7、8、9、10、15或20nt并且在3’末端鞭短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。参见，例如，Jinek等，Science2012；337:816–821；Mali等，Science.2013年2月15日；339(6121):823-6；Cong等，Science.2013年2月15日；339(6121):819-23；以及Hwang和Fu等，NatBiotechnol.2013年3月；31(3):227-9；Jinek等，Elife 2,e00471(2013))。对于系统2，一般地更长长度的嵌合gRNA已显示更大的中靶活性，但不同长度的gRNA的相对特异性目有仍未确定，从而在某些情况下可能期望使用较短的gRNA。在一些实施方案中，gRNA与在转录起始位点上游约100-800bp内，例如在转录起始位点的上游约500bp内，包括转录起始位点，或在转录起始位点下游约100-800bp内，例如约500bp内的区域互补。在一些实施方案中，使用编码不止一个gRNA的载体(例如，质粒)，例如编码导向靶基因的相同区域内的不同位点的2、3、4、5或更多个gRNA的质粒。
可使用在其5’末端上具有与基因组DNA靶位点的互补链互补的17-20nt的引导RNA(例如单一gRNA或tracrRNA/crRNA)将Cas9核酸酶导向具有例如序列NGG的另外的邻近的前间区序列邻近基序(PAM)的特定17-20nt的基因组靶。因此，本方法可包括单一引导RNA的使用，所述单一引导RNA包含与通常反式编码的tracrRNA融合的crRNA，例如Mali等，Science2013年2月15日；339(6121):823-6中描述的单一Cas9引导RNA，其在5’末端上具有例如25-17个，任选地20个或更少核苷酸(nt)的与靶序列互补的序列，例如，紧接前间区序列邻近基序(PAM)例如NGG、NAG或NNGG的5’的靶序列的互补链的20、19、18或17nt，优选地17或18nt。在一些实施方案中，所述单个Cas9引导RNA由如下序列组成：
Figure BDA0003133036850000201
Figure BDA0003133036850000202
或
Figure BDA0003133036850000203
其中X17-20是与靶序列的17-20个连续核苷酸互补的核苷酸序列。先前已在文献(Jinek等，Science.337(6096):816-21(2012)和Jinek等，Elife.2:e00471(2013))描述了编码单一引导RNA的DNA。
引导RNA可包含可以是不干扰核糖核酸对Cas9结合的任何序列的XN，其中N(在RNA中)可以是0-200例如0-100、0-50或0-20。
在一些实施方案中，所述引导RNA在3’末端上包含一个或多个腺嘌呤(A)或尿嘧啶(U)核苷酸。在一些实施方案中，作为用作终止RNA PolIII转录的终止信号的一个或多个T的任选的存在的结果，所述RNA在分子的3’末端包括一个或多个U，例如，1至8个或更多个U(例如，U、UU、UUU、UUUU、UUUUU、UUUUUU、UUUUUUU、UUUUUUUU)。
虽然本文中描述的一些实例利用单一gRNA，但还将所述方法与双重gRNA(例如在天然存在的系统中发现的crRNA和tracrRNA)一起使用。在该情况下，可将单一tracrRNA与多个不同的使用本系统表达的crRNA结合使用，例如下列序列：
(X17-20)GUUUUAGAGCUA(SEQ ID NO:102)；
(X17-20)GUUUUAGAGCUAUGCUGUUUUG(SEQ ID NO:103)；或(X17-20)GUUUUAGAGCUAUGCU(SEQ ID NO:104)；和tracrRNA序列。在该情况下，将crRNA在本文中描述的方法和分子中用作引导RNA，并且从相同或不同的DNA分子表达tracrRNA。在一些实施方案中，所述方法包括将细胞与tracrRNA接触，所述tracrRNA包含如下序列或由所述序列组成：GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQID NO:8)或其活性部分(活性部分是保持与Cas9或dCas9形成复合物的能力的部分)。在一些实施方案中，可从其3’末端将tracrRNA分子截短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。在另一个实施方案中，可从其5’末端将tracrRNA截短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。或者，可从5’和3’末端将tracrRNA分子截短，例如，在5’末端截短至少1、2、3、4、5、6、7、8、9、10、15或20nt并且在3’末端截短至少1、2、3、4、5、6、7、8、9、10、15、20、25、30、35或40nt。除了SEQ ID NO:8以外，示例性tracrRNA还包括下列序列：UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:105)或其活性部分；或AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ IDNO:106)或其活性部分。
在一些实施方案中，当将(X17-20)GUUUUAGAGCUAUGCUGUUUUG(SEQ ID NO:102)用作crRNA时，使用下列tracrRNA：GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:8)或其活性部分。
在一些实施方案中，当将(X17-20)GUUUUAGAGCUA(SEQ ID NO:102)用作crRNA时，使用下列tracrRNA：UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:105)或其活性部分。
在一些实施方案中，当将(X17-20)GUUUUAGAGCUAUGCU(SEQ ID NO:104)用作crRNA时，使用下列tracrRNA：AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(SEQ ID NO:106)或其活性部分。
在一些实施方案中，所述gRNA被靶向与基因组的其余部分中的任何序列相异至少3或更多个错配的位点以使脱靶效应减小至最小。
经修饰的RNA寡核苷酸诸如锁核酸(LNA)已被证明通过以更有利的(稳定的)构象锁定经修饰的寡核苷酸来提高RNA-DNA杂交的特异性。例如，2’-O-甲基RNA是其中在2’氧与4’碳之间存在另外的共价键联的经修饰的碱基，当被掺入寡核苷酸中时其可提高总体热稳定性和选择性(式I)。
Figure BDA0003133036850000231
因此，在一些实施方案中，本文中公开的tru-gRNA可包含一个或多个经修饰的RNA寡核苷酸。例如，本文中描述的截短的引导RNA分子可具有与靶序列互补的引导RNA的一个或一些或全部区域被修饰，例如锁定的(2’-O-4’-C亚甲基桥)、5'-甲基胞苷、2'-O-甲基-假尿苷，或其中磷酸核糖主链已被聚酰胺链(肽核酸)例如合成核糖核酸替代。
在其它实施方案中，tru-gRNA序列的一个、一些或全部核苷酸可被修饰，例如锁定的(2’-O-4’-C亚甲基桥)、5'-甲基胞苷、2'-O-甲基-假尿苷，或其中磷酸核糖主链已被聚酰胺链(肽核酸)例如合成核糖核酸替代。
在一些实施方案中，所述单一引导RNA和/或crRNA和/或tracrRNA可在3’末端上包含一个或多个腺嘌呤(A)或尿嘧啶(U)核苷酸。
现有基于Cas9的RGN使用gRNA-DNA异源双链体形成来引导靶向目标基因组位点。然而，RNA-DNA异源双链体可形成比它们的DNA-DNA对应物更加混杂的范围的结构。实际上，DNA-DNA双链体对错配更加敏感，从而表明DNA引导的核酸酶可能不不能容易地结合于脱靶序列，从而使得它们相较地比RNA引导的核酸酶更具特异性。因此，可用于本文中描述的方法的引导RNA可以是杂交体，即，即其中一个或多个脱氧核糖核苷酸例如短的DNA寡核苷酸替代gRNA的全部或部分，例如gRNA的互补区的全部或部分。该基于DNA的分子可替代单一gRNA系统中的gRNA的全部或部分或可选地可替代双重crRNA/tracrRNA系统中的crRNA和/或tracrRNA的全部或部分。将DNA整合进互补区的此类系统应当因DNA-DNA双链体对错配的总体不耐受性而相较于RNA-DNA双链体更容易靶向期望的基因组DNA序列。用于产生此类双链体的方法在本领域中是已知的，参见，例如，Barker等，BMC Genomics.2005年4月22日；6:57；和Sugimoto等，Biochemistry.2000年9月19日；39(37):11270-81。
另外，在使用单独的crRNA和tracrRNA的系统中，其一者或两者可以是合成的并且包含一个或多个经修饰的(例如，锁定的)核苷酸或脱氧核糖核苷酸。
在细胞背景中，Cas9与这些合成gRNA的复合物可用于提高CRISPR/Cas9核酸酶系统的全基因组特异性。
所述方法可包括在细胞中表达本文中描述的Cas9 gRNA加融合蛋白，或将所述细胞与其接触。
表达系统
为了使用描述的融合蛋白和引导RNA，可能期望从编码它们的核酸表达它们。这可以以多种方式来进行。例如，可将编码引导RNA或融合蛋白的核酸克隆入用于转化进用于复制和/或表达的原核或真核细胞的中间载体。中间载体通常是原核载体，例如，质粒或穿梭载体或昆虫载体，其用于贮存或操纵编码融合蛋白的核酸或用于产生融合蛋白。还可将编码引导RNA或融合蛋白的核酸克隆入表达载体，例如用于向植物细胞、动物细胞，优选地哺乳动物细胞或人细胞、真菌细胞、细菌细胞或原生动物细胞施用。
为了获得表达，通常将编码引导RNA或融合蛋白的序列亚克隆入含有指导转录的启动子的表达载体。合适的细菌和真核启动子在本领域中是公在的，并且描述于例如Sambrook等，Molecular Cloning,ALaboratory Manual(2001年第3版)；Kriegler,GeneTransfer and Expression:A Laboratory Manual(1990)和Current Protocols inMolecular Biology(Ausubel等，编辑，2010)中。用于表达工程化蛋白质的细菌表达系统可在例如大肠杆菌(E.coli)、芽孢杆菌属(Bacillus sp.)和沙门菌属(Salmonella)(Palva等，1983,Gene 22:229-235)中获得。此类表达系统的试剂盒是商购可得的。用于哺乳动物细胞、酵母和昆虫细胞的真核表达系统在本领域中是公知的并且也是商购可得的。
用于指导核酸表达的启动子取决于具体应用。例如，强组成型启动子通常用于融合蛋白的表达和纯化。相反地，当将体内施用融合蛋白以进行基因调控时，可使用组成型或诱导型启动子，这取决于融合蛋白的具体用途。另外，用于施用融合蛋白的优选启动子可以是弱启动子，诸如HSV TK或具有类似活性的启动子。启动子还可包括响应反式激活的元件，例如，缺氧应答元件、Gal4应答元件、lac阻遏应答元件和小分子控制系统诸如四环素调节的系统和RU-486系统(参见，例如，Gossen&Bujard,1992,Proc.Natl.Acad.Sci.USA,89:5547；Oligino等，1998,Gene Ther.,5:491-496；Wang等，1997,Gene Ther.,4:432-441；Neering等，1996,Blood,88:1147-55和Rendahl等，1998,Nat.Biotechnol.,16:757-761)。
除了启动子以外，表达载体通常还含有包含核酸在宿主细胞(原核或真核的)中表达所需的所有另外的元件的转录单位或表达盒。常见表达盒从而含有可操作地连接于例如编码融合蛋白的核酸序列的启动子和例如进行转录物的高效多腺苷酸化、转录终止、核糖体结合位点或翻译终止的所需的任何信号。表达盒的另外元件可包括例如增强子和异源剪接内含子信号。
根据融合蛋白的期望用途(例如，在植物、动物、细菌、真菌、原生动物等中表达)选择用于将遗传信息转运至细胞的特定表达载体。标准细菌表达载体包括质粒诸如基于pBR322的质粒、pSKF、pET23D和商购可得的靶-融合表达系统诸如GST和LacZ。优选的标签-融合蛋白是麦芽糖结合蛋白(MBP)。此类标签-融合蛋白可用于纯化工程化TALE重复蛋白。还可将表位标签例如c-myc或FLAG添加至重组蛋白以提供用于监测表达和用于监测细胞和亚细胞定位的方便的分离方法。
含有来自真核病毒的调控元件的表达载体通常用于真核表达载体，例如，SV40载体、乳头状瘤病毒载体和来源于爱泼斯坦-巴尔病毒的载体。其它示例性真核载体包括pMSG、pAV009/A+、pMTO10/A+、pMAMneo-5、杆状病毒pDSVE和允许在如下启动子指导下表达蛋白质的任何其它载体：SV40早期启动子、SV40晚期启动子、金属硫蛋白启动子、鼠乳腺肿瘤病毒动子、劳斯肉瘤病毒启动子、多角体蛋白启动子或经显示对于在真核细胞中的表达是有效的其它启动子。
用于表达引导RNA的载体可包括驱动引导RNA表达的RNA Pol III启动子，例如H1、U6或7SK启动子。这些人启动子允许在质粒转染后在哺乳动物细胞中表达gRNA。或者，T7启动子可用于例如体外转录，并且所述RNA可被体外转录和纯化。可使用适合用于短的RNA例如siRNA、shRNA或其它小的RNA表达的载体。
一些表达系统具有用于选择稳定地转染的细胞系的标志物诸如胸苷激酶、潮霉素B磷酸物转移酶和二氢叶酸还原酶。高产表达系统也是合适的，诸如在昆虫细胞中使用杆状病毒载体，利用在多角体蛋白启动子或其它强杆状病毒启动子的指导下的融合蛋白编码序列。
通常被包括在表达载体中的元件还包括在大肠杆菌中起作用的复制子、编码抗生素抗性以允许选择具有重组质粒的细菌的基因和允许重组序列插入的质粒的非必需区中的独特限制性位点。
标准转染法可用于产生表达大量蛋白质的细菌、哺乳动物、酵母或昆虫细胞系，随后使用标准技术(参见，例如，Colley等，1989,J.Biol.Chem.,264:17619-22；Guide toProtein Purification，于Methods in Enzymology，第182卷(Deutscher，编辑，1990)中)纯化所述蛋白质。真核和原核细胞的转化按照标准技术(参见，例如，Morrison,1977,J.Bacteriol.132:349-351；Clark-Curtiss&Curtiss,Methods in Enzymology 101:347-362(Wu等，编辑，1983)来进行。
可使用用于将外来核苷酸序列引入宿主细胞的任何已知方法。这些方法包括使用磷酸钙转染、聚凝胺、原生质体融合、电穿孔、核转染、脂质体、显微注射、裸DNA、质粒载体、病毒载体(游离型和融合型)和用于将克隆的基因组DNA、cDNA、合成DNA或其它外来遗传物质引入宿主细胞的任何其它公知的方法(参见，例如，Sambrook等，同上)。唯一必需的是，使用的特定遗传工程方法能够成功地将至少一个基因引入能够表达选择的蛋白质的宿主细胞。
在一些实施方案中，所述融合蛋白包括提供给待被转运到细胞核的蛋白质的核定位结构域。几个核定位序列(NLS)是已知的，并且可使用任何适合的NLS。例如，许多NLS具有多个碱性氨基酸，称为二分基本重复(综述于Garcia-Bustos等，1991,Biochim.Biophys.Acta,1071:83-101)中。含NLS的二分基本重复可被置于嵌合蛋白的任何部分，并且导致嵌合蛋白被定位在细胞核内。在优选实施方案中，将核定位结构域整合进最终的融合蛋白内，因为本文中描述的融合蛋白的最终功能将通常需要被定位在细胞核中的蛋白质。然而，在其中DBD结构域本身或终嵌合蛋白内的另一个功能性结构域具有固有的核转运功能的情况下，可以不必添加单独的核定位结构域。
本发明包括所述载体和包含所述载体的细胞。
实施例
在下列实施例中进一步描述本发明，所述实施例不限制权利要求中描述的本发明的范围。
实施例1.工程化CRISPR/Cas激活系统:
假设RNA引导的转录激活物可通过将强合成VP64激活结构域(Beerli等，ProcNatl Acad Sci USA 95,14628–14633(1998))与无催化活性的dCas9蛋白的羧基末端融合来产生(图1D)。
为了在人细胞中表达引导RNA(gRNA)，工程化载体，所述载体可表达由U6启动子驱动的全长嵌合gRNA(最初由Jinek等(Science2012)描述的crRNA与tracrRNA的融合物)。如下进行gRNA表达质粒的构建。将成对的编码可变20nt gRNA靶向序列的DNA寡核苷酸退火在一起以产生具有4bp的悬突的短的双链DNA片段(表1)。
Figure BDA0003133036850000281
Figure BDA0003133036850000291
将这些片段连接入BsmBI消化的质粒pMLM3636以产生通过人U6启动子表达的编码嵌合的～102nt单链引导RNA的DNA(Mali等，Science.2013年2月15日；339(6121):823-6；Hwang等，Nat Biotechnol.2013年3月；31(3):227-9)。pMLM3636质粒及其完全DNA序列可获自Addgene。参见图4。
为了工程化Cas9激活物，将D10A、H840A催化突变(先前在Jinek等，2012；和Qi等，2013中描述的)引入野生型或密码子最优化的Cas9序列(图5)。这些突变使得Cas9无催化活性，从而使得其不再诱导双链断裂。在一个构建体中，将三重flag标签、核定位信号和VP64激活结构域与失活的Cas9的C末端融合(图6)。该融合蛋白的表达由CMV启动子驱动。
如下进行dCas-VP64表达质粒的构建。使用引物(该引物将T7启动子位点添加至起始密码子的5’并且将核定位信号添加在Cas9编码序列的羧基末端)通过PCR从质粒pMJ841(Addgene质粒#39318)扩增编码具有灭活D10A/H840A突变的Cas9核酸酶(dCas9)的DNA，并如先前所述(Hwang等，Nat Biotechnol 31,227–229(2013))将其克隆进含有CMV启动子质粒以产生质粒pMLM3629。将编码三重FLAG表位的寡核苷酸退火，并克隆进质粒pMLM3629中的XhoI和PstI位点以产生表达具有C末端flag FLAG标签的dCas9的质粒pMLM3647。将编码后接合成VP64激活结构域的Gly4Ser接头的DNA序列引入质粒pMLM3647中的FLAG-标记的dCas9的下游以产生质粒pSL690。通过QuikChange定点诱变(Agilent)将D10A/H840A突变引入质粒pJDS247，所述质粒编码已针对在人细胞中的表达进行了密码子最优化的FLAG标记的Cas9序列，以产生质粒pMLM3668。随后将编码Gly4Ser接头和VP64激活结构域的DNA序列克隆进pMLM3668以产生称为pMLM3705的密码子最优化的dCas9-VP64表达载体。
如下进行细胞培养、转染和ELISA测定。将Flp-In T-Rex 293细胞维持在补充有10％FBS、1％penstrep和1％Glutamax(Invitrogen)的高级DMEM中。按照制造商的说明书利用脂质体LTX(Invitrogen)转染细胞。简言之，将160,000个293细胞接种在24孔板中，第二天用250ng gRNA质粒、250ng Cas9-VP64质粒、30ng pmaxGFP质粒(Lonza)、0.5ul Plus试剂和1.65ul脂质体LTX转染所述细胞。在转染后40小时收获来自感染的293细胞的组织培养基，使用R&D System’sHuman VEGF-A ELISA试剂盒“人VEGF免疫测定”来测定分泌的VEGF-A蛋白。
16个sgRNA被构建来靶向293细胞中的位于人VEGFA基因的转录起始位点上游、下游或其上的3个DNA酶I高敏位点(HSS)内的序列(图1E)。
在测试16个VEGFA靶向gRNA招募新型dCas9-VP64融合蛋白的能力之前，首先测定这些gRNA的每一个的将Cas9核酸酶导向其在人293细胞中的期望的靶位点的能力。为了该目的，以1:3的比率转染gRNA和Cas9表达载体，因为先前的最优化实验表明使用该质粒比率在U2OS细胞中诱导了高水平的Cas9诱导的DNA裂解。
除用125ng编码VEGFA靶向gRNA的质粒和375ng编码活性Cas9核酸酶的质粒(pMLM3639)转染细胞外，如上文中针对dCas9-VP16 VEGFA实验所述进行293细胞的转染。转染后40小时，按照制造商的说明书，使用QIAamp DNA Blood Mini试剂盒(Qiagen)分离基因组DNA。使用Phusion Hot Start II高保真DNA聚合酶(NEB)，利用3％DMSO和下列降落PCR循环：98℃的10个循环，10秒；72–62℃，-1℃/循环，15秒；72℃，30秒，随后98℃的25个循环，10秒；62℃，15秒；72℃，30秒进行VEGFA启动子中的3个不同靶向区的PCR扩增。使用引物oFYF434(5’-TCCAGATGGCACATTGTCAG-3’(SEQ ID NO:82))和oFYF435(5’-AGGGAGCAGGAAAGTGAGGT-3’(SEQ ID NO:83))扩增-500区域。使用引物oFYF438(5’-GCACGTAACCTCACTTTCCT-3’(SEQ ID NO:84))和oFYF439(5’-CTTGCTACCTCTTTCCTCTTTCT-3’(SEQ ID NO:85))扩增转录起始位点周围的区域。使用引物oFYF444(5’-AGAGAAGTCGAGGAAGAGAGAG-3’(SEQ ID NO:86))和oFYF445(5’-CAGCAGAAAGTTCATGGTTTCG-3’(SEQ ID NO:87))扩增+500区域。使用Ampure XP珠(Agencourt)纯化PCR产物，随后进行T7内切核酸酶I测定，并如先前所述(Reyon等，Nat Biotech 30,460-465(2012))在QIAXCEL毛细管电泳系统上进行分析。
所有16个gRNA都能介导Cas9核酸酶诱导的插入缺失突变在它们各自的靶位点上的高效引入，如使用先前描述的T7E1基因分型测定评估的(表2)。因此，所有16个gRNA可与Cas9核酸酶复合并将其活性导向人细胞中的特定靶基因组位点。
表2.由VEGFA靶向的gRNA和Cas9核酸酶诱导的插入缺失突变的频率
Figure BDA0003133036850000321
为了测试dCas9-VP64蛋白是否也可被相同的gRNA靶向人细胞中的特定基因组位点，如下进行VEGFA蛋白的酶联免疫印迹测定。转染后40小时收获用编码VEGFA靶向sgRNA和dCas9-VP64的质粒转染的Flp-In T-Rex HEK293细胞的培养基，如先前所述(Maeder等，NatMethods 10,243–245(2013))测量VEGFA蛋白表达。通过将来自其中表达sgRNA和dCas9-VP64的细胞的培养基中的VEGFA蛋白的浓度除以来自其中表达脱靶sgRNA(被靶向EGFP报告基因中的序列)和dCas9-VP64的细胞的培养基中的VEGFA蛋白的浓度来计算VEGFA表达的激活倍数。
当与dCas9-VP64在人293细胞中共表达时，测试的16个gRNA中有15个诱导显著的VEGFA蛋白表达的增加(图2A)。观察到的VEGFA诱导的量级在2至18.7-激活倍数的范围内，平均值为5-倍激活。对照实验显示单独的16个gRNA、单独的dCas9-VP64以及与被设计来结合EGFP受体基因序列的“脱靶”gRNA一起的dCas9-VP64的每一种的表达全都不能诱导升高的VEGFA表达(图2A)，这表明特定gRNA和dCas9-VP64蛋白的共表达是启动子激活所需的。因此，dCas9-VP64被稳定地表达，并且可被gRNA导向激活人细胞中的特定基因组基因座的转录。在用gRNA3转染的细胞中观察到VEGFA的最大增加，所述gRNA3诱导蛋白质表达18.7倍。有趣地，3个最好的gRNA以及能够诱导3倍或更多倍的表达的9个gRNA中的6个靶向-500区域(转录起始位点上游离～500bp)。
因为在一个方面，本文中描述的系统使用可变的gRNA来招募共同的dCas9-VP64激活融合物，因此可设想多重引导RNA在单个细胞中的表达可能使得能够多重或组合激活内源基因靶。为了测试该可能性，用dCas9-VP64表达质粒与各自单独地诱导从VEGFA启动子的表达的4个gRNA(V1、V2、V3和V4)的表达质粒一起转染293细胞。所有4个gRNA与dCas9-VP64的共表达诱导VEGFA蛋白表达的协同激活(即，大于每一个单独的激活物的预期的累加效应的倍数-激活)(图2B)。另外，这4个激活物中的3个的各种组合也协同地激活VEGFA启动子(图2B)。因为转录的协同激活据信由多个激活结构域至单个启动子的招募引起，因此在这些实验中，多个gRNA/dCas9-VP64复合物可能同时结合VEGFA启动子。
这些实验表明，Cas9-HFD例如Cas9-激活蛋白(具有VP64转录激活结构域)和具有20nt的与人VEGF-A启动子中的位点互补的序列的sgRNA在人HEK293细胞中的共表达可导致VEGF-A表达的上调。通过ELISA测定测量VEGF-A蛋白的增加，并且发现单独gRNA可与Cas9-激活融合蛋白一起用于使VEGF-A蛋白水平升高达到～18倍(图2A)。另外，有可能通过转录协同(通过将多个靶向相同启动子中的不同位点的gRNA与Cas9-激活融合蛋白一起引入)来实现甚至更大的激活提高(图2B)。
实施例2.工程化靶向内源人NTF3基因的CRISPR/Cas激活系统
为了扩展本发现的一般性，我们测试RNA引导的激活物平台是否呆用于诱导人NTF3基因的表达。为了实现该目的，将6个sgRNA设计成人NTF3启动子中的预测的DNA酶I高敏位点(HSS)，并且将表达这些gRNA的每一个的质粒与已针对人细胞表达进行了密码子最优化的编码dCas9-VP64蛋白的质粒共转染(图3A)。
所有6个测试的gRNA诱导显著的NTF3转录物水平的升高，如通过定量RT-PCR检测的(图3B)。虽然不能精确地计算这6个RNA引导的激活物的倍数-激活值(因为转录物的基线水平基本上是不可检测的)，但激活的NTF3 mRNA表达的平均值在4倍的范围内变化。减少转染的gRNA和dCas9-VP64表达质粒的量导致更少的NTF3基因的激活(图3B)，这表明了明确的剂量依赖性效应。
另外，单独地以及以单一组合和双重组合用dCas9-VP64和NTF3靶向的gRNA表达质粒共转染293细胞。通过定量RT-PCR检测NTF3mRNA的相对表达，并将所述相对表达针对GAPDH对照(ΔCt x 104)进行标准化。在所有这些实验中，用于转染的每一个单独的gRNA表达质粒的量是相同的。图3B显示该多重gRNA表达通过dCas9-VP64蛋白诱导NTF3 mRNA表达的协同激活。
实施例3.工程化CRISPR/Cas-MS2、-Csy4和–λN融合系统–产生生物系链
产生融合蛋白，其中将MS2衣壳蛋白、Csy4核酸酶(优选地无催化活性的Csy4，例如Haurwitz等329(5997):1355-8(2010)中描述的H29A突变体)或λN与灭活的dCas9的N或C末端融合。MS2和λN是结合特定RNA序列的噬菌体蛋白，从而可用作将dCas9蛋白系连于利用特定MS2或λN RNA结合序列标记的异源RNA序列的衔接子。将dCas9-MS2融合物或dCas9-λN融合物与在它们的5’或3’末端与MS2或λN茎环识别序列融合的嵌合长非编码RNA(lncRNA)共表达。嵌合Xist或嵌合RepA lncRNA将被dCas9融合物专门招募，并且该策略诱导靶向沉默的能力将通过测量靶基因表达来测量。所述系统将通过测试对衣壳蛋白和嵌合RNA的各种改变来最优化。已前已显示对MS2衣壳蛋白的N55K和ΔFG突变阻止蛋白质聚集并增加对茎-环RNA的亲和力。另外，我们将测试据报导增加对于MS2衣壳蛋白的亲和力的高亲和力C-环RNA突变体。下文给出MS2和λN蛋白的示例性序列；MS2以二聚体形式起作用，从而MS2蛋白可包括融合单链二聚体序列。
1.单个MS2衣壳蛋白(wt、N55K或ΔFG)与dCas9的N末端或C末端的融合物的示例性序列
MS2衣壳蛋白氯基酸序列：
Figure BDA0003133036850000351
MS2 N55K：
Figure BDA0003133036850000352
MS2ΔFG：
Figure BDA0003133036850000353
2.融合的二聚MS2衣壳蛋白(wt、N55K或ΔFG)与dCas9的N末端或C末端的融合物的示例性序列
二聚MS2衣壳蛋白：
Figure BDA0003133036850000354
Figure BDA0003133036850000361
二聚MS2ΔFG：
Figure BDA0003133036850000362
3.λN与dCas9的N末端或C末端的融合物的示例性序列
λN氨基酸序列：
Figure BDA0003133036850000363
或
Figure BDA0003133036850000364
4.Csy4与dCas9的N末端或C末端的融合物的示例性序列
在Haurwitz等329(5997):1355-8(2010)中给出了Cys4(例如灭活的形式)的示例性序列。
将构建体在也表达已在其5’或3’末端与λN或MS2的同源茎-环识别序列融合的调控RNA例如长非编码RNA(lncRNA)诸如HOTAIR、HOTTIP、XIST或XIST RepA的细胞中表达。MS2的野生型和高亲和力序列分别为AAACAUGAGGAUUACCCAUGUCG(SEQ ID NO:96)和AAACAUGAGGAUCACCCAUGUCG(SEQ ID NO:97)(参见Keryer-Bibens等，同上，图2)；λN所结合的nutL和nutR BoxB分别为GCCCUGAAGAAGGGC(SEQ ID NO:98)和GCCCUGAAAAAGGGC(SEQ IDNO:99)。Csy4所结合的序列为GTTCACTGCCGTATAGGCAG(截短的20 nt)(SEQ ID NO:100)或GUUCACUGCCGUAUAGGCAGCUAAGAAA(SEQ ID NO:101)。
dCas9/MS2对表达MS2结合序列标记的lncRNA的细胞中的靶位点的结合将该lncRNA招募至dCas9结合位点；其中lncRNA是阻遏物，例如，XIST(dCas9结合位点附近的基因)被抑制。类似地，dCas9/λN对表达λN-结合序列标记的lncRNA的细胞中的靶位点的结合将该lncRNA招募至dCas9结合位点。
实施例4.工程化CRISPR/Cas-HP1融合系统-序列-特异性沉默
本文所述dCas9融合蛋白还可用于靶向沉默结构域，例如异染色质蛋白1(HP1，也称为swi6)，例如HP1α或HP1β。可将其中异染色质已被除去的HP1α或HP1β的截短的形式靶向特定基因座来诱导异染色质形成和基因沉默。与dCas9融合的截短的HP1的示例性序列示于图8A-8B中。可将HP1序列与如上所述的灭活的dCas9的N或C末端融合。
实施例5.工程化CRISPR/Cas-TET融合系统–序列-特异性脱甲基化
本文中描述的dCas9融合蛋白还可用于靶向修饰DNA的甲基化状态的酶(例如，DNA甲基转移酶(DNMT)或TET蛋白)。可将TET1的截短形式靶向特定基因座以催化DNA脱甲基化。与dCas9融合的截短的TET1的示例性序列示于图9中。可将TET1序列融合于如上所述的灭活的dCas9的N或C末端。
实施例6.工程化最优化的CRISPR/Cas-VP64融合物
通过改变这些融合物内的核定位信号(NLS)和3xFLAG-标签的数目和位置最优化具有VP64激活结构域的基于dCas9的转录激活物的活性(图10)。含有N末端NLS和位于dCas9与VP64序列之间的NLS的dCas9-VP64融合物一致地诱导较高水平的靶基因激活，可能因激活物的增加的核定位导致(图10)。此外，当将3xFLAG标签置于dCas9的C末端与VP64的N末端之间时，观察到甚至更高水平的激活。3xFLAG标签可用作人工接头，在dCas9与VP64之间提供必要的间隔，并且可能允许更好地折叠VP64结构域(当在dCas9附近受约束时这可能是无法进行的)或由招募RNA聚合酶II的转录中介体复合物更好地识别VP64。或者，带负电荷的3xFLAG标签还可用作偶然的转录激活结构域，从而增强VP64结构域的效应。
实施例7.最优化的无催化活性的Cas9蛋白(dCas9)
通过改变消除dCas9结构域中的Cas9的核酸酶活性的失活突变的性质来进行dCas9-VP64激活物的另外的最优化(图11A-B)。在迄今公开的研究中，将催化残基D10和H840突变成丙氨酸(D10A和H840A)来破坏介导DNA的水解的活性位点网络。有人假设，这些位置上的丙氨酸取代可能导致dCas9的去稳定，从而导致亚最佳活性。因此，测试D10或H840上结构更加保守的取代(例如，至天冬酰或酪氨酸残基：D10N、H840N和H840Y)以看它们是否可导致通过具有这些不同突变的dCas9-VP64融合物的更大基因激活。当将具有这些变异取代的dCas9-VP64变体与靶向内源人VEGFA基因的上游区域的3个gRNA共转染进HEK293细胞时，除一个变体外，对于所有这些变体观察到更多的VEGFA蛋白质表达(图11A)。然而，当将所述dCas9-VP64变体仅与这些gRNA中的一个共转染时(图11A)或当转染进表达单一VEGFA靶向的gRNA的HEK293来源的细胞系时(图11B)，该效应没有这样显著。
其它实施方案
应理解，虽然已结合其详细描述说明了本发明，但前述说明指在举例说明而不是限制本发明的范围，本发明的范围由所述权利要求的范围来界定。其它方面、有利方面和修改在下列权利要求的范围内。
序列表
<110> 通用医疗公司
<120> 遗传和表观遗传调节蛋白至特定基因组基因座的RNA引导的靶向
<130> 00786-0882WO1
<140> PCT/US2014/027335
<141> 2014-03-14
<150> 61/921,007
<151> 2013-12-26
<150> 61/838,178
<151> 2013-06-21
<150> 61/838,148
<151> 2013-06-21
<150> 61/799,647
<151> 2013-03-15
<160> 113
<170> PatentIn 3.5版
<210> 1
<211> 262
<212> RNA
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引导多核苷酸
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<222> (1)..(20)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
17-20个核苷酸,其中一些位置可以不存在
<220>
<221> 修饰的_碱基
<222> (63)..(262)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
0-200个核苷酸,其中一些位置可以不存在
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引导多核苷酸
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引导多核苷酸
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17-20个核苷酸,其中一些位置可以不存在
<220>
<221> 修饰的_碱基
<222> (88)..(287)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
0-200个核苷酸,其中一些位置可以不存在
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引导多核苷酸
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17-20个核苷酸,其中一些位置可以不存在
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<221> 修饰的_碱基
<222> (97)..(296)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
0-200个核苷酸,其中一些位置可以不存在
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引导寡核苷酸
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引导多核苷酸
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引导寡核苷酸
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cg 62
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引导多核苷酸
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核定位信号肽
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Pro Lys Lys Lys Arg Lys Val Ser
1 5
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<213> 人工序列
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VP64结构域多肽
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Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe
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Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu Asp
35 40 45
Met Leu
50
<210> 13
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Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
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Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
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Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 14
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
接头肽
<400> 14
Gly Gly Gly Ser
1
<210> 15
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
接头肽
<400> 15
Gly Gly Gly Gly Ser
1 5
<210> 16
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 16
gtgtgcagac ggcagtcact agg 23
<210> 17
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 17
gagcagcgtc ttcgagagtg agg 23
<210> 18
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 18
ggtgagtgag tgtgtgcgtg tgg 23
<210> 19
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 19
gttggagcgg ggagaaggcc agg 23
<210> 20
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 20
gggtgggggg agtttgctcc tgg 23
<210> 21
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 21
ggctttggaa agggggtggg ggg 23
<210> 22
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 22
ggggcggggt cccggcgggg cgg 23
<210> 23
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 23
gctcggaggt cgtggcgctg ggg 23
<210> 24
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 24
gactcaccgg ccagggcgct cgg 23
<210> 25
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 25
ggcgcagcgg ttaggtggac cgg 23
<210> 26
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 26
ggcgcatggc tccgccccgc cgg 23
<210> 27
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 27
gccacgacct ccgagctacc cgg 23
<210> 28
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 28
gcggcgtgag ccctccccct tgg 23
<210> 29
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 29
ggaggcgggg tggagggggt cgg 23
<210> 30
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 30
gggctcacgc cgcgctccgg cgg 23
<210> 31
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 31
gaccccctcc accccgcctc cgg 23
<210> 32
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 32
gagcgcggag ccatctggcc ggg 23
<210> 33
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 33
gcgcggcgcg gaaggggtta agg 23
<210> 34
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 34
gcggcgcggc gcgggccggc ggg 23
<210> 35
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 35
gccgcgccgc cctcccccgc cgg 23
<210> 36
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 36
gcggttataa ccagccaacc cgg 23
<210> 37
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 37
gtgcgcggag ctgttcggaa ggg 23
<210> 38
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 38
acaccgtgtg cagacggcag tcactg 26
<210> 39
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 39
acaccgagca gcgtcttcga gagtgg 26
<210> 40
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 40
acaccggtga gtgagtgtgt gcgtgg 26
<210> 41
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 41
acaccgttgg agcggggaga aggccg 26
<210> 42
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 42
acaccgggtg gggggagttt gctccg 26
<210> 43
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 43
acaccggctt tggaaagggg gtgggg 26
<210> 44
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 44
acaccggggc ggggtcccgg cggggg 26
<210> 45
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 45
acaccgctcg gaggtcgtgg cgctgg 26
<210> 46
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 46
acaccgactc accggccagg gcgctg 26
<210> 47
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 47
acaccggcgc agcggttagg tggacg 26
<210> 48
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 48
acaccggcgc atggctccgc cccgcg 26
<210> 49
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 49
acaccgccac gacctccgag ctaccg 26
<210> 50
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 50
acaccgcggc gtgagccctc cccctg 26
<210> 51
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 51
acaccggagg cggggtggag ggggtg 26
<210> 52
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 52
acaccgggct cacgccgcgc tccggg 26
<210> 53
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 53
acaccgaccc cctccacccc gcctcg 26
<210> 54
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 54
acaccgagcg cggagccatc tggccg 26
<210> 55
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 55
acaccgcgcg gcgcggaagg ggttag 26
<210> 56
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 56
acaccgcggc gcggcgcggg ccggcg 26
<210> 57
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 57
acaccgccgc gccgccctcc cccgcg 26
<210> 58
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 58
acaccgcggt tataaccagc caaccg 26
<210> 59
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 59
acaccgtgcg cggagctgtt cggaag 26
<210> 60
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 60
aaaacagtga ctgccgtctg cacacg 26
<210> 61
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 61
aaaaccactc tcgaagacgc tgctcg 26
<210> 62
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 62
aaaaccacgc acacactcac tcaccg 26
<210> 63
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 63
aaaacggcct tctccccgct ccaacg 26
<210> 64
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 64
aaaacggagc aaactccccc cacccg 26
<210> 65
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 65
aaaaccccac cccctttcca aagccg 26
<210> 66
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 66
aaaacccccg ccgggacccc gccccg 26
<210> 67
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 67
aaaaccagcg ccacgacctc cgagcg 26
<210> 68
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 68
aaaacagcgc cctggccggt gagtcg 26
<210> 69
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 69
aaaacgtcca cctaaccgct gcgccg 26
<210> 70
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 70
aaaacgcggg gcggagccat gcgccg 26
<210> 71
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 71
aaaacggtag ctcggaggtc gtggcg 26
<210> 72
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 72
aaaacagggg gagggctcac gccgcg 26
<210> 73
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 73
aaaacacccc ctccaccccg cctccg 26
<210> 74
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 74
aaaacccgga gcgcggcgtg agcccg 26
<210> 75
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 75
aaaacgaggc ggggtggagg gggtcg 26
<210> 76
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 76
aaaacggcca gatggctccg cgctcg 26
<210> 77
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 77
aaaactaacc ccttccgcgc cgcgcg 26
<210> 78
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 78
aaaacgccgg cccgcgccgc gccgcg 26
<210> 79
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 79
aaaacgcggg ggagggcggc gcggcg 26
<210> 80
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 80
aaaacggttg gctggttata accgcg 26
<210> 81
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
靶结合位点寡核苷酸
<400> 81
aaaacttccg aacagctccg cgcacg 26
<210> 82
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 82
tccagatggc acattgtcag 20
<210> 83
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 83
agggagcagg aaagtgaggt 20
<210> 84
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 84
gcacgtaacc tcactttcct 20
<210> 85
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 85
cttgctacct ctttcctctt tct 23
<210> 86
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 86
agagaagtcg aggaagagag ag 22
<210> 87
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
引物
<400> 87
cagcagaaag ttcatggttt cg 22
<210> 88
<211> 130
<212> PRT
<213> 肠杆菌噬菌体λ
<400> 88
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80
Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95
Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu
100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly
115 120 125
Ile Tyr
130
<210> 89
<211> 130
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
λ噬菌体MS2 N55K突变型多肽
<400> 89
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80
Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95
Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu
100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly
115 120 125
Ile Tyr
130
<210> 90
<211> 117
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
λ噬菌体MS2 ΔFG突变型多肽
<400> 90
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile
65 70 75 80
Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met
85 90 95
Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala
100 105 110
Asn Ser Gly Ile Tyr
115
<210> 91
<211> 262
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
二聚体MS2衣壳多肽
<400> 91
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80
Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95
Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu
100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly
115 120 125
Leu Tyr Gly Ala Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp
130 135 140
Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn
145 150 155 160
Gly Val Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys
165 170 175
Val Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr
180 185 190
Ile Lys Val Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val
195 200 205
Glu Leu Pro Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr
210 215 220
Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
225 230 235 240
Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala
245 250 255
Ala Asn Ser Leu Ile Asn
260
<210> 92
<211> 262
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
二聚体MS2 N55K突变型衣壳多肽
<400> 92
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80
Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95
Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu
100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly
115 120 125
Leu Tyr Gly Ala Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp
130 135 140
Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn
145 150 155 160
Gly Val Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys
165 170 175
Val Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr
180 185 190
Ile Lys Val Glu Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val
195 200 205
Glu Leu Pro Val Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr
210 215 220
Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala
225 230 235 240
Met Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala
245 250 255
Ala Asn Ser Leu Ile Asn
260
<210> 93
<211> 236
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
二聚体MS2 ΔFG突变型衣壳多肽
<400> 93
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile
65 70 75 80
Pro Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met
85 90 95
Gln Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala
100 105 110
Asn Ser Gly Leu Tyr Gly Ala Met Ala Ser Asn Phe Thr Gln Phe Val
115 120 125
Leu Val Asp Asn Gly Gly Thr Gly Asp Val Thr Val Ala Pro Ser Asn
130 135 140
Phe Ala Asn Gly Val Ala Glu Trp Ile Ser Ser Asn Ser Arg Ser Gln
145 150 155 160
Ala Tyr Lys Val Thr Cys Ser Val Arg Gln Ser Ser Ala Gln Lys Arg
165 170 175
Lys Tyr Thr Ile Lys Val Glu Val Pro Lys Gly Ala Trp Arg Ser Tyr
180 185 190
Leu Asn Met Glu Leu Thr Ile Pro Ile Phe Ala Thr Asn Ser Asp Cys
195 200 205
Glu Leu Ile Val Lys Ala Met Gln Gly Leu Leu Lys Asp Gly Asn Pro
210 215 220
Ile Pro Ser Ala Ile Ala Ala Asn Ser Leu Ile Asn
225 230 235
<210> 94
<211> 22
<212> PRT
<213> 肠杆菌噬菌体λ
<400> 94
Met Asp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala
1 5 10 15
Gln Trp Lys Ala Ala Asn
20
<210> 95
<211> 107
<212> PRT
<213> 肠杆菌噬菌体λ
<400> 95
Met Asp Ala Gln Thr Arg Arg Arg Glu Arg Arg Ala Glu Lys Gln Ala
1 5 10 15
Gln Trp Lys Ala Ala Asn Pro Leu Leu Val Gly Val Ser Ala Lys Pro
20 25 30
Val Asn Arg Pro Ile Leu Ser Leu Asn Arg Lys Pro Lys Ser Arg Val
35 40 45
Glu Ser Ala Leu Asn Pro Ile Asp Leu Thr Val Leu Ala Glu Tyr His
50 55 60
Lys Gln Ile Glu Ser Asn Leu Gln Arg Ile Glu Arg Lys Asn Gln Arg
65 70 75 80
Thr Trp Tyr Ser Lys Pro Gly Glu Arg Gly Ile Thr Cys Ser Gly Arg
85 90 95
Gln Lys Ile Lys Gly Lys Ser Ile Pro Leu Ile
100 105
<210> 96
<211> 23
<212> RNA
<213> 肠杆菌噬菌体λ
<400> 96
aaacaugagg auuacccaug ucg 23
<210> 97
<211> 23
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
高亲和力MS2结合寡核苷酸
<400> 97
aaacaugagg aucacccaug ucg 23
<210> 98
<211> 15
<212> RNA
<213> 肠杆菌噬菌体λ
<400> 98
gcccugaaga agggc 15
<210> 99
<211> 15
<212> RNA
<213> 肠杆菌噬菌体λ
<400> 99
gcccugaaaa agggc 15
<210> 100
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
截短的Csy4结合位点寡核苷酸
<220>
<223> 组合DNA/RNA分子的描述: 合成的
截短的Csy4结合位点寡核苷酸
<400> 100
gttcactgcc gtataggcag 20
<210> 101
<211> 28
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
Csy4结合位点寡核苷酸
<400> 101
guucacugcc guauaggcag cuaagaaa 28
<210> 102
<211> 32
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
crRNA寡核苷酸
<220>
<221> 修饰的_碱基
<222> (1)..(20)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
17-20个核苷酸,其中一些位置可以不存在
<400> 102
nnnnnnnnnn nnnnnnnnnn guuuuagagc ua 32
<210> 103
<211> 42
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
crRNA寡核苷酸
<220>
<221> 修饰的_碱基
<222> (1)..(20)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
17-20个核苷酸,其中一些位置可以不存在
<400> 103
nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcuguuu ug 42
<210> 104
<211> 36
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
crRNA寡核苷酸
<220>
<221> 修饰的_碱基
<222> (1)..(20)
<223> a, c, u, g, 未知的区域或其它区域和本区域可包括
17-20个核苷酸,其中一些位置可以不存在
<400> 104
nnnnnnnnnn nnnnnnnnnn guuuuagagc uaugcu 36
<210> 105
<211> 60
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
tracrRNA寡核苷酸
<400> 105
uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc gagucggugc 60
<210> 106
<211> 64
<212> RNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
tracrRNA寡核苷酸
<400> 106
agcauagcaa guuaaaauaa ggcuaguccg uuaucaacuu gaaaaagugg caccgagucg 60
gugc 64
<210> 107
<211> 2279
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
RNA表达载体多核苷酸
<220>
<221> 修饰的_碱基
<222> (331)..(350)
<223> a, c, t, g,未知的或其它
<400> 107
gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc 60
ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata 120
cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta 180
gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta 240
tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct 300
ttatatatct tgtggaaagg acgaaacacc nnnnnnnnnn nnnnnnnnnn gttttagagc 360
tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt 420
cggtgctttt tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa 480
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 540
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 600
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 660
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 720
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 780
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 840
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 900
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 960
acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 1020
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 1080
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 1140
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 1200
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 1260
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 1320
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 1380
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 1440
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 1500
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 1560
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 1620
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 1680
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 1740
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 1800
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 1860
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 1920
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 1980
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 2040
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 2100
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 2160
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 2220
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacct 2279
<210> 108
<211> 7786
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
CMV-T7-Cas9 D10A/H840A-3xFlag-VP64多核苷酸
<400> 108
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataagaaa 420
tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa 480
tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa 540
aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc 600
aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag 660
attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct 720
tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat 780
gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat 840
tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt 900
cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta 960
tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt 1020
ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat 1080
ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg 1140
tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta 1200
cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat 1260
caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat 1320
atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc 1380
tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca 1440
gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat 1500
gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat 1560
ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc 1620
tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga 1680
agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg 1740
acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg 1800
atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa 1860
ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat 1920
gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg 1980
acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag 2040
aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta 2100
aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa 2160
gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa 2220
gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc 2280
ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat 2340
gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga 2400
aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa 2460
tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt 2520
aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt 2580
gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt 2640
gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca 2700
cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc 2760
gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact 2820
caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg 2880
gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa 2940
agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt 3000
ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga 3060
caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa 3120
cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact 3180
cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat 3240
gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct 3300
gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc 3360
catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt 3420
gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag 3480
tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac 3540
ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa 3600
actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc 3660
aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga 3720
ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa 3780
gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta 3840
gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta 3900
gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct 3960
aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt 4020
gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat 4080
gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag 4140
ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat 4200
tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat 4260
gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa 4320
caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt 4380
aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat 4440
gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag 4500
ctaggaggtg acggttctcc caagaagaag aggaaagtct cgagcgacta caaagaccat 4560
gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga 4620
ggcggtggaa gcgggcgcgc cgacgcgctg gacgatttcg atctcgacat gctgggttct 4680
gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga tgactttgat 4740
ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt ataaccggtc 4800
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 4860
gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 4920
ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 4980
ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 5040
ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 5100
cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 5160
gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 5220
cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 5280
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 5340
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5400
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5460
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5520
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 5580
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 5640
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5700
ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 5760
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5820
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5880
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5940
cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 6000
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 6060
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6120
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6180
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6240
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6300
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6360
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6420
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6480
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6540
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 6600
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 6660
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 6720
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 6780
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6840
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 6900
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 6960
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7020
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7080
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7140
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 7200
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 7260
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc 7320
ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 7380
atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 7440
caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 7500
cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 7560
aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 7620
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 7680
taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 7740
actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcc 7786
<210> 109
<211> 7785
<212> DNA
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
MV-T7-Cas9记录的D10A/H840A-3xFLAG-VP64多核苷酸
<400> 109
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag 420
tattctattg gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa 480
tacaaagtac cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa 540
aagaatctta tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg 600
aaacgaaccg ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa 660
atttttagca atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc 720
ttccttgtcg aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat 780
gaggtggcat atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac 840
tcaactgata aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc 900
cgtgggcact ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg 960
ttcatccagt tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt 1020
ggcgtggatg cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac 1080
ctgatcgcac aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc 1140
tcactaggcc tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg 1200
cagcttagta aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat 1260
cagtatgcgg acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac 1320
atactgagag ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg 1380
tacgatgaac atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct 1440
gagaaatata aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac 1500
ggcggagcga gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat 1560
gggacggaag agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact 1620
ttcgacaacg gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga 1680
aggcaggagg atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta 1740
acctttcgca taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg 1800
atgacaagaa agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa 1860
ggtgcgtcag ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac 1920
gaaaaagtat tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc 1980
acgaaagtta agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag 2040
aagaaagcaa tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg 2100
aaagaggact actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa 2160
gatcgattta atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag 2220
gacttcctgg ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc 2280
ctctttgaag atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac 2340
gataaggtta tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg 2400
aaacttatca acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag 2460
agcgacggct tcgccaatag gaactttatg cagctgatcc atgatgactc tttaaccttc 2520
aaagaggata tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt 2580
gcgaatcttg ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg 2640
gatgagctag ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca 2700
cgcgaaaatc aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata 2760
gaagagggta ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc 2820
caattgcaga acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt 2880
gatcaggaac tggacataaa ccgtttatct gattacgacg tcgatgccat tgtaccccaa 2940
tcctttttga aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga 3000
gggaaaagtg acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg 3060
cagctcctaa atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag 3120
aggggtggct tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc 3180
cgccaaatca caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac 3240
gagaacgata agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg 3300
gacttcagaa aggattttca attctataaa gttagggaga taaataacta ccaccatgcg 3360
cacgacgctt atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta 3420
gaaagtgagt ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa 3480
agcgaacagg agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat 3540
ttctttaaga cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa 3600
accaatgggg agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga 3660
aaagttttgt ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg 3720
ttttcaaagg aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag 3780
gactgggacc cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta 3840
gtagtggcaa aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg 3900
gggataacga ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg 3960
aaaggttaca aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt 4020
gagttagaaa atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac 4080
gaactcgcac taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag 4140
ttgaaaggtt cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat 4200
tatctcgacg aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat 4260
gccaatctgg acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag 4320
caggcggaaa atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc 4380
aagtattttg acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac 4440
gcgacactga ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag 4500
cttgggggtg acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat 4560
gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga 4620
ggcggtggaa gcgggcgcgc cgacgcgctg gacgatttcg atctcgacat gctgggttct 4680
gatgccctcg atgactttga cctggatatg ttgggaagcg acgcattgga tgactttgat 4740
ctggacatgc tcggctccga tgctctggac gatttcgatc tcgatatgtt ataaccggtc 4800
atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt 4860
gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 4920
ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 4980
ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 5040
ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 5100
cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 5160
gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 5220
cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 5280
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 5340
gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 5400
ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 5460
caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 5520
aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 5580
atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 5640
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 5700
ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca 5760
gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 5820
accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5880
cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5940
cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 6000
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 6060
aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 6120
aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6180
actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 6240
taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 6300
gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 6360
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 6420
ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 6480
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 6540
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 6600
acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 6660
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 6720
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 6780
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6840
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 6900
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 6960
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 7020
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 7080
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7140
cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 7200
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 7260
ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatcgatctc 7320
ccgatcccct agggtcgact ctcagtacaa tctgctctga tgccgcatag ttaagccagt 7380
atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta 7440
caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg 7500
cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt 7560
aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 7620
aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 7680
taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 7740
actatttacg gtaaactgcc cacttggcag tacatcaagt gtatc 7785
<210> 110
<211> 1461
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
Cas9-激活多肽
<400> 110
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp
1370 1375 1380
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
1385 1390 1395
Asp Asp Lys Ala Ala Gly Gly Gly Gly Ser Gly Arg Ala Asp Ala
1400 1405 1410
Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp
1415 1420 1425
Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe
1430 1435 1440
Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp Leu
1445 1450 1455
Asp Met Leu
1460
<210> 111
<211> 1527
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
dCas9-NLS-3xFLAG-HP1α多肽
<400> 111
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp
1370 1375 1380
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
1385 1390 1395
Asp Asp Lys Ala Ala Gly Gly Gly Gly Ser Met Lys Glu Gly Glu
1400 1405 1410
Asn Asn Lys Pro Arg Glu Lys Ser Glu Ser Asn Lys Arg Lys Ser
1415 1420 1425
Asn Phe Ser Asn Ser Ala Asp Asp Ile Lys Ser Lys Lys Lys Arg
1430 1435 1440
Glu Gln Ser Asn Asp Ile Ala Arg Gly Phe Glu Arg Gly Leu Glu
1445 1450 1455
Pro Glu Lys Ile Ile Gly Ala Thr Asp Ser Cys Gly Asp Leu Met
1460 1465 1470
Phe Leu Met Lys Trp Lys Asp Thr Asp Glu Ala Asp Leu Val Leu
1475 1480 1485
Ala Lys Glu Ala Asn Val Lys Cys Pro Gln Ile Val Ile Ala Phe
1490 1495 1500
Tyr Glu Glu Arg Leu Thr Trp His Ala Tyr Pro Glu Asp Ala Glu
1505 1510 1515
Asn Lys Glu Lys Glu Thr Ala Lys Ser
1520 1525
<210> 112
<211> 1521
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
dCas9-NLS-3xFLAG-HP1β多肽
<400> 112
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp
1370 1375 1380
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
1385 1390 1395
Asp Asp Lys Ala Ala Gly Gly Gly Gly Ser Thr Ala His Glu Thr
1400 1405 1410
Asp Lys Ser Glu Gly Gly Lys Arg Lys Ala Asp Ser Asp Ser Glu
1415 1420 1425
Asp Lys Gly Glu Glu Ser Lys Pro Lys Lys Lys Lys Glu Glu Ser
1430 1435 1440
Glu Lys Pro Arg Gly Phe Ala Arg Gly Leu Glu Pro Glu Arg Ile
1445 1450 1455
Ile Gly Ala Thr Asp Ser Ser Gly Glu Leu Met Phe Leu Met Lys
1460 1465 1470
Trp Lys Asn Ser Asp Glu Ala Asp Leu Val Pro Ala Lys Glu Ala
1475 1480 1485
Asn Val Lys Cys Pro Gln Val Val Ile Ser Phe Tyr Glu Glu Arg
1490 1495 1500
Leu Thr Trp His Ser Tyr Pro Ser Glu Asp Asp Asp Lys Lys Asp
1505 1510 1515
Asp Lys Asn
1520
<210> 113
<211> 2126
<212> PRT
<213> 人工序列
<220>
<223> 人工序列的说明: 合成的
dCas9-3xFLAG-TET1CD多肽
<400> 113
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser Asp Tyr Lys Asp
1370 1375 1380
His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
1385 1390 1395
Asp Asp Lys Ala Ala Gly Gly Gly Gly Ser Leu Pro Thr Cys Ser
1400 1405 1410
Cys Leu Asp Arg Val Ile Gln Lys Asp Lys Gly Pro Tyr Tyr Thr
1415 1420 1425
His Leu Gly Ala Gly Pro Ser Val Ala Ala Val Arg Glu Ile Met
1430 1435 1440
Glu Asn Arg Tyr Gly Gln Lys Gly Asn Ala Ile Arg Ile Glu Ile
1445 1450 1455
Val Val Tyr Thr Gly Lys Glu Gly Lys Ser Ser His Gly Cys Pro
1460 1465 1470
Ile Ala Lys Trp Val Leu Arg Arg Ser Ser Asp Glu Glu Lys Val
1475 1480 1485
Leu Cys Leu Val Arg Gln Arg Thr Gly His His Cys Pro Thr Ala
1490 1495 1500
Val Met Val Val Leu Ile Met Val Trp Asp Gly Ile Pro Leu Pro
1505 1510 1515
Met Ala Asp Arg Leu Tyr Thr Glu Leu Thr Glu Asn Leu Lys Ser
1520 1525 1530
Tyr Asn Gly His Pro Thr Asp Arg Arg Cys Thr Leu Asn Glu Asn
1535 1540 1545
Arg Thr Cys Thr Cys Gln Gly Ile Asp Pro Glu Thr Cys Gly Ala
1550 1555 1560
Ser Phe Ser Phe Gly Cys Ser Trp Ser Met Tyr Phe Asn Gly Cys
1565 1570 1575
Lys Phe Gly Arg Ser Pro Ser Pro Arg Arg Phe Arg Ile Asp Pro
1580 1585 1590
Ser Ser Pro Leu His Glu Lys Asn Leu Glu Asp Asn Leu Gln Ser
1595 1600 1605
Leu Ala Thr Arg Leu Ala Pro Ile Tyr Lys Gln Tyr Ala Pro Val
1610 1615 1620
Ala Tyr Gln Asn Gln Val Glu Tyr Glu Asn Val Ala Arg Glu Cys
1625 1630 1635
Arg Leu Gly Ser Lys Glu Gly Arg Pro Phe Ser Gly Val Thr Ala
1640 1645 1650
Cys Leu Asp Phe Cys Ala His Pro His Arg Asp Ile His Asn Met
1655 1660 1665
Asn Asn Gly Ser Thr Val Val Cys Thr Leu Thr Arg Glu Asp Asn
1670 1675 1680
Arg Ser Leu Gly Val Ile Pro Gln Asp Glu Gln Leu His Val Leu
1685 1690 1695
Pro Leu Tyr Lys Leu Ser Asp Thr Asp Glu Phe Gly Ser Lys Glu
1700 1705 1710
Gly Met Glu Ala Lys Ile Lys Ser Gly Ala Ile Glu Val Leu Ala
1715 1720 1725
Pro Arg Arg Lys Lys Arg Thr Cys Phe Thr Gln Pro Val Pro Arg
1730 1735 1740
Ser Gly Lys Lys Arg Ala Ala Met Met Thr Glu Val Leu Ala His
1745 1750 1755
Lys Ile Arg Ala Val Glu Lys Lys Pro Ile Pro Arg Ile Lys Arg
1760 1765 1770
Lys Asn Asn Ser Thr Thr Thr Asn Asn Ser Lys Pro Ser Ser Leu
1775 1780 1785
Pro Thr Leu Gly Ser Asn Thr Glu Thr Val Gln Pro Glu Val Lys
1790 1795 1800
Ser Glu Thr Glu Pro His Phe Ile Leu Lys Ser Ser Asp Asn Thr
1805 1810 1815
Lys Thr Tyr Ser Leu Met Pro Ser Ala Pro His Pro Val Lys Glu
1820 1825 1830
Ala Ser Pro Gly Phe Ser Trp Ser Pro Lys Thr Ala Ser Ala Thr
1835 1840 1845
Pro Ala Pro Leu Lys Asn Asp Ala Thr Ala Ser Cys Gly Phe Ser
1850 1855 1860
Glu Arg Ser Ser Thr Pro His Cys Thr Met Pro Ser Gly Arg Leu
1865 1870 1875
Ser Gly Ala Asn Ala Ala Ala Ala Asp Gly Pro Gly Ile Ser Gln
1880 1885 1890
Leu Gly Glu Val Ala Pro Leu Pro Thr Leu Ser Ala Pro Val Met
1895 1900 1905
Glu Pro Leu Ile Asn Ser Glu Pro Ser Thr Gly Val Thr Glu Pro
1910 1915 1920
Leu Thr Pro His Gln Pro Asn His Gln Pro Ser Phe Leu Thr Ser
1925 1930 1935
Pro Gln Asp Leu Ala Ser Ser Pro Met Glu Glu Asp Glu Gln His
1940 1945 1950
Ser Glu Ala Asp Glu Pro Pro Ser Asp Glu Pro Leu Ser Asp Asp
1955 1960 1965
Pro Leu Ser Pro Ala Glu Glu Lys Leu Pro His Ile Asp Glu Tyr
1970 1975 1980
Trp Ser Asp Ser Glu His Ile Phe Leu Asp Ala Asn Ile Gly Gly
1985 1990 1995
Val Ala Ile Ala Pro Ala His Gly Ser Val Leu Ile Glu Cys Ala
2000 2005 2010
Arg Arg Glu Leu His Ala Thr Thr Pro Val Glu His Pro Asn Arg
2015 2020 2025
Asn His Pro Thr Arg Leu Ser Leu Val Phe Tyr Gln His Lys Asn
2030 2035 2040
Leu Asn Lys Pro Gln His Gly Phe Glu Leu Asn Lys Ile Lys Phe
2045 2050 2055
Glu Ala Lys Glu Ala Lys Asn Lys Lys Met Lys Ala Ser Glu Gln
2060 2065 2070
Lys Asp Gln Ala Ala Asn Glu Gly Pro Glu Gln Ser Ser Glu Val
2075 2080 2085
Asn Glu Leu Asn Gln Ile Pro Ser His Lys Ala Leu Thr Leu Thr
2090 2095 2100
His Asp Asn Val Val Thr Val Ser Pro Tyr Ala Leu Thr His Val
2105 2110 2115
Ala Gly Pro Tyr Asn His Trp Val
2120 2125

Claims (25)

1.一种融合蛋白，其包含连接于异源功能性结构域的无催化活性的CRISPR相关9(dCas9)蛋白。
2.根据权利要求1所述的融合蛋白，其中所述异源功能性结构域是转录激活结构域。
3.根据权利要求2所述的融合蛋白，其中所述转录激活结构域来自VP64或NF-κB p65。
4.根据权利要求1所述的融合蛋白，其中所述异源功能性结构域是转录沉默子或转录抑制结构域。
5.根据权利要求4所述的融合蛋白，其中所述转录抑制结构域是Krueppel相关盒(KRAB)结构域、ERF抑制结构域(ERD)或mSin3A相互作用结构域(SID)。
6.根据权利要求4所述的融合蛋白，其中所述转录沉默子是异染色质蛋白1(HP1)，例如HP1α或HP1β。
7.根据权利要求1所述的融合蛋白，其中所述异源功能性结构域是修饰DNA的甲基化状态的酶。
8.根据权利要求7所述的融合蛋白，其中修饰DNA的甲基化状态的酶是DNA甲基转移酶(DNMT)或TET蛋白。
9.根据权利要求8所述的融合蛋白，其中所述TET蛋白是TET1。
10.根据权利要求1所述的融合蛋白，其中所述异源功能性结构域是修饰组蛋白亚单位的酶。
11.根据权利要求1所述的融合蛋白，其中修饰组蛋白亚单位的酶是组蛋白乙酰转移酶(HAT)、组蛋白脱乙酰基酶(HDAC)、组蛋白甲基转移酶(HMT)或组蛋白脱甲基酶。
12.根据权利要求1所述的融合蛋白，其中所述异源功能性结构域是生物系链。
13.根据权利要求12所述的融合蛋白，其中所述生物系链是MS2、Csy4或λN蛋白。
14.根据权利要求1所述的融合蛋白，其中所述无催化活性的Cas9蛋白来自化脓性链球菌。
15.根据权利要求1所述的融合蛋白，其中所述无催化活性的Cas9蛋白在D10、E762、H983或D986；以及在H840或N863处包含突变。
16.根据权利要求15所述的融合蛋白，其中所述突变是：
(i)D10A或D10N，和
(ii)H840A、H840N或H840Y。
17.根据权利要求1所述的融合蛋白，其中通过任选的间插接头将所述异源功能性结构域连接于无催化活性的Cas9蛋白的N末端或C末端，其中所述接头不干扰所述融合蛋白的活性。
18.根据权利要求1所述的融合蛋白，其还任选地通过一个或多个间插接头在N末端、C末端上和/或在所述无催化活性的CRISPR相关9(Cas9)蛋白与所述异源功能性结构域之间中包含核定位序列和一个或多个表位标签的一者或两者。
19.根据权利要求18所述的融合蛋白，其中所述表位标签是c-myc、6His或FLAG。
20.一种核酸，其编码权利要求1-19的任一项的融合蛋白。
21.一种表达载体，其包含权利要求20的核酸。
22.一种增加靶基因在细胞中的表达的方法，所述方法包括在所述细胞中表达权利要求2-3的融合蛋白，和一个或多个被导向所述靶基因的引导RNA。
23.一种减少细胞中的靶基因的方法，所述方法包括在所述细胞中表达权利要求4-6的融合蛋白，和一个或多个被导向所述靶基因的引导RNA。
24.一种减少细胞中的靶基因或其启动子或增强子的DNA甲基化的方法，所述方法包括在所述细胞中表达权利要求7-9的融合蛋白，和一个或多个被导向所述相关靶基因序列的引导RNA。
25.一种修饰细胞中与靶基因或其启动子或增强子相关的组蛋白的方法，所述方法包括在所述细胞中表达权利要求10-11的融合蛋白，和一个或多个被导向所述相关靶基因序列的引导RNA。
CN202110709688.0A 2013-03-15 2014-03-14 遗传和表观遗传调节蛋白至特定基因组基因座的rna引导的靶向 Pending CN113563476A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024192914A1 (zh) * 2023-03-21 2024-09-26 深圳赫兹生命科学技术有限公司 去势ms2病毒样颗粒亚单位疫苗及其制备方法

Families Citing this family (365)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8053191B2 (en) 2006-08-31 2011-11-08 Westend Asset Clearinghouse Company, Llc Iterative nucleic acid assembly using activation of vector-encoded traits
US20140031250A1 (en) 2010-10-07 2014-01-30 David Tsai Ting Biomarkers of Cancer
WO2012064975A1 (en) 2010-11-12 2012-05-18 Gen9, Inc. Protein arrays and methods of using and making the same
AU2011338841B2 (en) 2010-11-12 2017-02-16 Gen9, Inc. Methods and devices for nucleic acids synthesis
JP6214530B2 (ja) 2011-07-15 2017-10-18 ザ ジェネラル ホスピタル コーポレイション 転写活性化因子様エフェクターの組立て方法
WO2013066438A2 (en) 2011-07-22 2013-05-10 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
EP2748318B1 (en) 2011-08-26 2015-11-04 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US11021737B2 (en) 2011-12-22 2021-06-01 President And Fellows Of Harvard College Compositions and methods for analyte detection
GB201122458D0 (en) * 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
US10039777B2 (en) 2012-03-20 2018-08-07 Neuro-Lm Sas Methods and pharmaceutical compositions of the treatment of autistic syndrome disorders
US9150853B2 (en) 2012-03-21 2015-10-06 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
LT2841601T (lt) 2012-04-24 2019-07-10 Gen9, Inc. Nukleorūgščių rūšiavimo būdai ir multipleksinis preparatyvinis in vitro klonavimas
WO2013163628A2 (en) 2012-04-27 2013-10-31 Duke University Genetic correction of mutated genes
DE202013012241U1 (de) 2012-05-25 2016-01-18 Emmanuelle Charpentier Zusammensetzungen für die durch RNA gesteuerte Modifikation einer Ziel-DNA und für die durch RNA gesteuerte Modulation der Transkription
US9890364B2 (en) 2012-05-29 2018-02-13 The General Hospital Corporation TAL-Tet1 fusion proteins and methods of use thereof
WO2014004393A1 (en) 2012-06-25 2014-01-03 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
EP3789405A1 (en) 2012-10-12 2021-03-10 The General Hospital Corporation Transcription activator-like effector (tale) - lysine-specific demethylase 1 (lsd1) fusion proteins
PL2928496T3 (pl) 2012-12-06 2020-04-30 Sigma-Aldrich Co. Llc Modyfikacja i regulacja genomu w oparciu o CRISPR
BR112015013784A2 (pt) 2012-12-12 2017-07-11 Massachusetts Inst Technology aplicação, manipulação e otimização de sistemas, métodos e composições para manipulação de sequência e aplicações terapêuticas
EP2931892B1 (en) 2012-12-12 2018-09-12 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
EP3919505B1 (en) 2013-01-16 2023-08-30 Emory University Uses of cas9-nucleic acid complexes
WO2014124284A1 (en) 2013-02-07 2014-08-14 The General Hospital Corporation Tale transcriptional activators
EP2971184B1 (en) 2013-03-12 2019-04-17 President and Fellows of Harvard College Method of generating a three-dimensional nucleic acid containing matrix
NZ712727A (en) 2013-03-14 2017-05-26 Caribou Biosciences Inc Compositions and methods of nucleic acid-targeting nucleic acids
KR102210322B1 (ko) 2013-03-15 2021-02-01 더 제너럴 하스피탈 코포레이션 Rna-안내 게놈 편집의 특이성을 증가시키기 위한 rna-안내 foki 뉴클레아제(rfn)의 용도
US10760064B2 (en) 2013-03-15 2020-09-01 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
CN105518146B (zh) * 2013-04-04 2022-07-15 哈佛学院校长同事会 利用CRISPR/Cas系统的基因组编辑的治疗性用途
US20140356956A1 (en) * 2013-06-04 2014-12-04 President And Fellows Of Harvard College RNA-Guided Transcriptional Regulation
EP3003392B1 (en) 2013-06-04 2019-10-23 President and Fellows of Harvard College Rna-guideded transcriptional regulation
AU2014274840B2 (en) * 2013-06-05 2020-03-12 Duke University RNA-guided gene editing and gene regulation
WO2014204727A1 (en) 2013-06-17 2014-12-24 The Broad Institute Inc. Functional genomics using crispr-cas systems, compositions methods, screens and applications thereof
KR20160034901A (ko) 2013-06-17 2016-03-30 더 브로드 인스티튜트, 인코퍼레이티드 서열 조작에 최적화된 ｃｒｉｓｐｒ-ｃａｓ 이중 닉카아제 시스템, 방법 및 조성물
EP3011029B1 (en) 2013-06-17 2019-12-11 The Broad Institute, Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
CN105793425B (zh) 2013-06-17 2021-10-26 布罗德研究所有限公司 使用病毒组分靶向障碍和疾病的crispr-cas系统和组合物的递送、用途和治疗应用
CN107995927B (zh) 2013-06-17 2021-07-30 布罗德研究所有限公司 用于肝靶向和治疗的crispr-cas系统、载体和组合物的递送与用途
US10011850B2 (en) 2013-06-21 2018-07-03 The General Hospital Corporation Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
JP2016528890A (ja) 2013-07-09 2016-09-23 プレジデント アンド フェローズ オブ ハーバード カレッジ ＣＲＩＳＰＲ／Ｃａｓ系を用いるゲノム編集の治療用の使用
US20150044772A1 (en) * 2013-08-09 2015-02-12 Sage Labs, Inc. Crispr/cas system-based novel fusion protein and its applications in genome editing
US20150044192A1 (en) 2013-08-09 2015-02-12 President And Fellows Of Harvard College Methods for identifying a target site of a cas9 nuclease
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
EP3036334A1 (en) * 2013-08-22 2016-06-29 E. I. du Pont de Nemours and Company A soybean u6 polymerase iii promoter and methods of use
US9322037B2 (en) * 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
DE202014010413U1 (de) 2013-09-18 2015-12-08 Kymab Limited Zellen und Organismen
WO2015066119A1 (en) 2013-10-30 2015-05-07 North Carolina State University Compositions and methods related to a type-ii crispr-cas system in lactobacillus buchneri
JP2016536021A (ja) 2013-11-07 2016-11-24 エディタス・メディシン，インコーポレイテッド ＣＲＩＳＰＲ関連方法および支配ｇＲＮＡのある組成物
US9068179B1 (en) 2013-12-12 2015-06-30 President And Fellows Of Harvard College Methods for correcting presenilin point mutations
WO2015089364A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crystal structure of a crispr-cas system, and uses thereof
EP3835419A1 (en) 2013-12-12 2021-06-16 The Regents of The University of California Methods and compositions for modifying a single stranded target nucleic acid
KR20160089527A (ko) 2013-12-12 2016-07-27 더 브로드 인스티튜트, 인코퍼레이티드 게놈 편집을 위한 ｃｒｉｓｐｒ-ｃａｓ 시스템 및 조성물의 전달, 용도 및 치료적 응용
WO2015089486A2 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems
CA2932472A1 (en) 2013-12-12 2015-06-18 Massachusetts Institute Of Technology Compositions and methods of use of crispr-cas systems in nucleotide repeat disorders
EP3985124A1 (en) * 2013-12-26 2022-04-20 The General Hospital Corporation Multiplex guide rnas
US10787654B2 (en) 2014-01-24 2020-09-29 North Carolina State University Methods and compositions for sequence guiding Cas9 targeting
EP4063503A1 (en) 2014-02-11 2022-09-28 The Regents of the University of Colorado, a body corporate Crispr enabled multiplexed genome engineering
US11028388B2 (en) 2014-03-05 2021-06-08 Editas Medicine, Inc. CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa
US11339437B2 (en) 2014-03-10 2022-05-24 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
US11141493B2 (en) 2014-03-10 2021-10-12 Editas Medicine, Inc. Compositions and methods for treating CEP290-associated disease
ES2745769T3 (es) 2014-03-10 2020-03-03 Editas Medicine Inc Procedimientos y composiciones relacionados con CRISPR/CAS para tratar la amaurosis congénita de Leber 10 (LCA10)
US11242525B2 (en) 2014-03-26 2022-02-08 Editas Medicine, Inc. CRISPR/CAS-related methods and compositions for treating sickle cell disease
CN106460003A (zh) 2014-04-08 2017-02-22 北卡罗来纳州立大学 用于使用crispr相关基因rna引导阻遏转录的方法和组合物
BR112016028023A2 (pt) 2014-05-30 2017-08-22 Univ Leland Stanford Junior Composições e métodos de administração de tratamentos para infecções virais latentes
EP3919621A1 (en) 2014-06-23 2021-12-08 The General Hospital Corporation Genomewide unbiased identification of dsbs evaluated by sequencing (guide-seq)
US20150376587A1 (en) * 2014-06-25 2015-12-31 Caribou Biosciences, Inc. RNA Modification to Engineer Cas9 Activity
US20170198268A1 (en) * 2014-07-09 2017-07-13 Gen9, Inc. Compositions and Methods for Site-Directed DNA Nicking and Cleaving
CA2954626A1 (en) 2014-07-11 2016-01-14 E. I. Du Pont De Nemours And Company Compositions and methods for producing plants resistant to glyphosate herbicide
WO2016022363A2 (en) 2014-07-30 2016-02-11 President And Fellows Of Harvard College Cas9 proteins including ligand-dependent inteins
US9932566B2 (en) 2014-08-07 2018-04-03 Agilent Technologies, Inc. CIS-blocked guide RNA
EP3633032A3 (en) 2014-08-28 2020-07-29 North Carolina State University Novel cas9 proteins and guiding features for dna targeting and genome editing
EP3191595B1 (en) 2014-09-12 2019-12-25 E. I. du Pont de Nemours and Company Generation of site-specific-integration sites for complex trait loci in corn and soybean, and methods of use
WO2016049258A2 (en) * 2014-09-25 2016-03-31 The Broad Institute Inc. Functional screening with optimized functional crispr-cas systems
WO2016054106A1 (en) * 2014-09-29 2016-04-07 The Regents Of The University Of California SCAFFOLD RNAs
AU2015330699B2 (en) * 2014-10-10 2021-12-02 Editas Medicine, Inc. Compositions and methods for promoting homology directed repair
US9816080B2 (en) 2014-10-31 2017-11-14 President And Fellows Of Harvard College Delivery of CAS9 via ARRDC1-mediated microvesicles (ARMMs)
AU2015342749B2 (en) 2014-11-07 2022-01-27 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
US11352666B2 (en) 2014-11-14 2022-06-07 Institute For Basic Science Method for detecting off-target sites of programmable nucleases in a genome
CN107109486B (zh) * 2014-11-14 2021-08-13 基础科学研究院 用于检测基因组中遗传剪刀的脱靶位点的方法
CN104531633A (zh) * 2014-11-18 2015-04-22 李云英 Cas9-scForkI融合蛋白及其应用
US10858662B2 (en) 2014-11-19 2020-12-08 Institute For Basic Science Genome editing with split Cas9 expressed from two vectors
CA2969619A1 (en) * 2014-12-03 2016-06-09 Agilent Technologies, Inc. Guide rna with chemical modifications
US10975392B2 (en) 2014-12-05 2021-04-13 Abcam Plc Site-directed CRISPR/recombinase compositions and methods of integrating transgenes
CN116059378A (zh) 2014-12-10 2023-05-05 明尼苏达大学董事会 用于治疗疾病的遗传修饰的细胞、组织和器官
WO2016094872A1 (en) * 2014-12-12 2016-06-16 The Broad Institute Inc. Dead guides for crispr transcription factors
EP3985115A1 (en) * 2014-12-12 2022-04-20 The Broad Institute, Inc. Protected guide rnas (pgrnas)
JP2017538427A (ja) 2014-12-18 2017-12-28 インテグレイテッド ディーエヌエイ テクノロジーズ インコーポレイテッド Ｃｒｉｓｐｒ系組成物及び使用方法
US20180179523A1 (en) * 2014-12-18 2018-06-28 Integrated Dna Technologies, Inc. Crispr-based compositions and methods of use
US10190106B2 (en) 2014-12-22 2019-01-29 Univesity Of Massachusetts Cas9-DNA targeting unit chimeras
US11208638B2 (en) 2015-01-12 2021-12-28 The Regents Of The University Of California Heterodimeric Cas9 and methods of use thereof
EP3250689B1 (en) 2015-01-28 2020-11-04 The Regents of The University of California Methods and compositions for labeling a single-stranded target nucleic acid
RS64527B1 (sr) 2015-01-28 2023-09-29 Caribou Biosciences Inc Hibridni polinukleotidi crispr dnk/rnk i postupci upotrebe
EP3256487A4 (en) 2015-02-09 2018-07-18 Duke University Compositions and methods for epigenome editing
CN107532148B (zh) 2015-02-18 2022-06-24 衣阿华州立大学研究基金公司 修饰nf-yc4启动子的转录抑制子结合位点以增加蛋白质含量和抗应力
AU2016226077B2 (en) 2015-03-03 2021-12-23 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases with altered PAM specificity
CA2976387A1 (en) 2015-03-27 2016-10-06 E I Du Pont De Nemours And Company Soybean u6 small nuclear rna gene promoters and their use in constitutive expression of small rna genes in plants
CA2981715A1 (en) 2015-04-06 2016-10-13 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide rnas for crispr/cas-mediated gene regulation
EP3286571B1 (en) 2015-04-24 2021-08-18 Editas Medicine, Inc. Evaluation of cas9 molecule/guide rna molecule complexes
CA2988854A1 (en) 2015-05-08 2016-11-17 President And Fellows Of Harvard College Universal donor stem cells and related methods
US11390884B2 (en) 2015-05-11 2022-07-19 Editas Medicine, Inc. Optimized CRISPR/cas9 systems and methods for gene editing in stem cells
US10117911B2 (en) 2015-05-29 2018-11-06 Agenovir Corporation Compositions and methods to treat herpes simplex virus infections
WO2016196361A1 (en) 2015-05-29 2016-12-08 North Carolina State University Methods for screening bacteria, archaea, algae, and yeast using crispr nucleic acids
US20160346360A1 (en) 2015-05-29 2016-12-01 Agenovir Corporation Compositions and methods for cell targeted hpv treatment
EP3303634B1 (en) 2015-06-03 2023-08-30 The Regents of The University of California Cas9 variants and methods of use thereof
US20180296537A1 (en) 2015-06-05 2018-10-18 Novartis Ag Methods and compositions for diagnosing, treating, and monitoring treatment of shank3 deficiency associated disorders
KR20180031671A (ko) 2015-06-09 2018-03-28 에디타스 메디신, 인코포레이티드 이식의 개선을 위한 crispr/cas-관련 방법 및 조성물
JP6961494B2 (ja) * 2015-06-10 2021-11-05 フイルメニツヒ ソシエテ アノニムFirmenich Sa 匂い物質および芳香の受容体をスクリーニングするための細胞株
IL298524B2 (en) 2015-06-12 2024-03-01 Lonza Walkersville Inc Methods for nuclear reprogramming using synthetic transcription factors
ES2960226T3 (es) 2015-06-15 2024-03-01 Univ North Carolina State Métodos y composiciones para la administración eficiente de ácidos nucleicos y antimicrobianos basados en ARN
WO2016205759A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of cas9 orthologs and variants for sequence manipulation
MX2017016289A (es) 2015-06-18 2018-08-15 Broad Inst Inc Mutaciones de la enzima crispr que reducen los efectos fuera del blanco.
EP3313989A4 (en) * 2015-06-29 2018-12-05 Ionis Pharmaceuticals, Inc. Modified crispr rna and modified single crispr rna and uses thereof
WO2017004279A2 (en) * 2015-06-29 2017-01-05 Massachusetts Institute Of Technology Compositions comprising nucleic acids and methods of using the same
JP7044373B2 (ja) 2015-07-15 2022-03-30 ラトガース，ザ ステート ユニバーシティ オブ ニュージャージー ヌクレアーゼ非依存的な標的化遺伝子編集プラットフォームおよびその用途
WO2017015637A1 (en) 2015-07-22 2017-01-26 Duke University High-throughput screening of regulatory element function with epigenome editing technologies
US20170119820A1 (en) 2015-07-31 2017-05-04 Regents Of The University Of Minnesota Modified cells and methods of therapy
WO2017024047A1 (en) * 2015-08-03 2017-02-09 Emendobio Inc. Compositions and methods for increasing nuclease induced recombination rate in cells
WO2017023974A1 (en) * 2015-08-03 2017-02-09 President And Fellows Of Harvard College Cas9 genome editing and transcriptional regulation
KR20240132120A (ko) * 2015-08-25 2024-09-02 듀크 유니버시티 Rna-가이드된 엔도뉴클레아제를 이용하는 게놈 조작에서 특이성을 개선하는 조성물 및 방법
US9512446B1 (en) 2015-08-28 2016-12-06 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
CN114875012A (zh) * 2015-08-28 2022-08-09 通用医疗公司 工程化的CRISPR-Cas9核酸酶
US9926546B2 (en) 2015-08-28 2018-03-27 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
KR102691636B1 (ko) * 2015-08-31 2024-08-02 애질런트 테크놀로지스, 인크. 상동 재조합에 의한 crispr/cas-기반 게놈 편집을 위한 화합물 및 방법
WO2017044776A1 (en) * 2015-09-10 2017-03-16 Texas Tech University System Single-guide rna (sgrna) with improved knockout efficiency
JP2018530536A (ja) 2015-09-11 2018-10-18 ザ ジェネラル ホスピタル コーポレイション ヌクレアーゼＤＳＢの完全照合およびシーケンシング（ＦＩＮＤ−ｓｅｑ）
WO2017053879A1 (en) 2015-09-24 2017-03-30 Editas Medicine, Inc. Use of exonucleases to improve crispr/cas-mediated genome editing
US11286480B2 (en) 2015-09-28 2022-03-29 North Carolina State University Methods and compositions for sequence specific antimicrobials
US9850484B2 (en) 2015-09-30 2017-12-26 The General Hospital Corporation Comprehensive in vitro reporting of cleavage events by sequencing (Circle-seq)
AU2016336565B2 (en) 2015-10-06 2019-07-18 Aict Method for producing genome-modified plants from plant protoplasts at high efficiency
EP4089175A1 (en) 2015-10-13 2022-11-16 Duke University Genome engineering with type i crispr systems in eukaryotic cells
IL294014B2 (en) 2015-10-23 2024-07-01 Harvard College Nucleobase editors and their uses
WO2017070598A1 (en) 2015-10-23 2017-04-27 Caribou Biosciences, Inc. Engineered crispr class 2 cross-type nucleic-acid targeting nucleic acids
AU2016349288A1 (en) 2015-11-03 2018-05-31 President And Fellows Of Harvard College Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix
EP3374501B1 (en) 2015-11-11 2023-07-12 Lonza Ltd Crispr-associated (cas) proteins with reduced immunogenicity
AU2016357319B2 (en) 2015-11-20 2022-03-10 Sage Science, Inc. Preparative electrophoretic method for targeted purification of genomic DNA fragments
WO2017090724A1 (ja) * 2015-11-25 2017-06-01 国立大学法人 群馬大学 Ｄｎａメチル化編集用キットおよびｄｎａメチル化編集方法
US11345931B2 (en) 2015-12-14 2022-05-31 President And Fellows Of Harvard College Cas discrimination using tuned guide RNA
WO2017112620A1 (en) 2015-12-22 2017-06-29 North Carolina State University Methods and compositions for delivery of crispr based antimicrobials
EP4053277A1 (en) * 2015-12-28 2022-09-07 Novartis AG Compositions and methods for the treatment of hemoglobinopathies
EP3402494B1 (en) 2016-01-11 2021-04-07 The Board of Trustees of the Leland Stanford Junior University Chimeric proteins and methods of immunotherapy
KR20180096800A (ko) 2016-01-11 2018-08-29 더 보드 어브 트러스티스 어브 더 리랜드 스탠포드 주니어 유니버시티 키메라 단백질 및 유전자 발현을 조절하는 방법
US11845933B2 (en) 2016-02-03 2023-12-19 Massachusetts Institute Of Technology Structure-guided chemical modification of guide RNA and its applications
JP2019513345A (ja) 2016-02-10 2019-05-30 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン 核酸の検知
WO2017143071A1 (en) 2016-02-18 2017-08-24 The Regents Of The University Of California Methods and compositions for gene editing in stem cells
WO2017151444A1 (en) * 2016-02-29 2017-09-08 Agilent Technologies, Inc. Methods and compositions for blocking off-target nucleic acids from cleavage by crispr proteins
WO2017160600A1 (en) 2016-03-15 2017-09-21 Apse, Inc Methods and compositions for increased double stranded rna production
JP2019515654A (ja) 2016-03-16 2019-06-13 ザ ジェイ． デヴィッド グラッドストーン インスティテューツ 肥満及び／又は糖尿病を処置するための方法及び組成物、並びに候補処置薬剤を識別するための方法及び組成物
EP3219799A1 (en) 2016-03-17 2017-09-20 IMBA-Institut für Molekulare Biotechnologie GmbH Conditional crispr sgrna expression
WO2017165862A1 (en) 2016-03-25 2017-09-28 Editas Medicine, Inc. Systems and methods for treating alpha 1-antitrypsin (a1at) deficiency
EP3433363A1 (en) 2016-03-25 2019-01-30 Editas Medicine, Inc. Genome editing systems comprising repair-modulating enzyme molecules and methods of their use
CN106701765A (zh) * 2016-04-11 2017-05-24 广东赤萌医疗科技有限公司 用于hiv感染治疗的多核苷酸及其制备药物应用
US11236313B2 (en) 2016-04-13 2022-02-01 Editas Medicine, Inc. Cas9 fusion molecules, gene editing systems, and methods of use thereof
US20190127713A1 (en) * 2016-04-13 2019-05-02 Duke University Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use
CN116200465A (zh) 2016-04-25 2023-06-02 哈佛学院董事及会员团体 用于原位分子检测的杂交链反应方法
CN107326046A (zh) * 2016-04-28 2017-11-07 上海邦耀生物科技有限公司 一种提高外源基因同源重组效率的方法
RU2745563C2 (ru) 2016-05-20 2021-03-29 Регенерон Фармасьютикалс, Инк. Способы преодоления иммунологической толерантности с использованием множества направляющих рнк
US20190100732A1 (en) 2016-06-02 2019-04-04 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Assay for the removal of methyl-cytosine residues from dna
GB2552861B (en) * 2016-06-02 2019-05-15 Sigma Aldrich Co Llc Using programmable DNA binding proteins to enhance targeted genome modification
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
EP3473728B1 (en) * 2016-06-15 2021-10-20 Toolgen Incorporated Method for screening targeted genetic scissors by using multiple target system of on-target and off-target activity and use thereof
LT3474669T (lt) 2016-06-24 2022-06-10 The Regents Of The University Of Colorado, A Body Corporate Barkodu pažymėtų kombinatorinių bibliotekų generavimo būdai
MX2019000088A (es) 2016-06-27 2019-08-29 Broad Inst Inc Composiciones y metodos para detectar y tratar la diabetes.
US11359234B2 (en) 2016-07-01 2022-06-14 Microsoft Technology Licensing, Llc Barcoding sequences for identification of gene expression
EP3478852B1 (en) * 2016-07-01 2020-08-12 Microsoft Technology Licensing, LLC Storage through iterative dna editing
US20180004537A1 (en) 2016-07-01 2018-01-04 Microsoft Technology Licensing, Llc Molecular State Machines
AU2017295720B2 (en) 2016-07-13 2021-07-22 Vertex Pharmaceuticals Incorporated Methods, compositions and kits for increasing genome editing efficiency
CN109477086A (zh) * 2016-07-13 2019-03-15 陈奇涵 一种基因组dna特异性编辑方法和应用
AU2017302551B2 (en) * 2016-07-26 2023-04-27 The General Hospital Corporation Variants of CRISPR from Prevotella and Francisella 1 (Cpf1)
CN109789185A (zh) * 2016-07-28 2019-05-21 基础科学研究院 用于治疗眼部疾病的含cas9蛋白和向导rna的药物组合物
US10548302B2 (en) 2016-07-29 2020-02-04 Regeneron Pharmaceuticals, Inc. Fibrillin-1 mutations for modeling neonatal progeroid syndrome with congenital lipodystrophy
AU2017305404B2 (en) 2016-08-02 2023-11-30 Editas Medicine, Inc. Compositions and methods for treating CEP290 associated disease
CA3032699A1 (en) 2016-08-03 2018-02-08 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
AU2017308889B2 (en) 2016-08-09 2023-11-09 President And Fellows Of Harvard College Programmable Cas9-recombinase fusion proteins and uses thereof
US11905549B2 (en) 2016-08-10 2024-02-20 Genahead Bio, Inc. Method for modifying target site in genome of eukaryotic cell, and method for detecting presence or absence of nucleic acid sequence to be detected at target site
WO2018034554A1 (ko) * 2016-08-19 2018-02-22 주식회사 툴젠 인위적으로 조작된 신생혈관형성 조절 시스템
AU2017313912B2 (en) * 2016-08-19 2024-01-04 Whitehead Institute For Biomedical Research Methods of editing DNA methylation
CN110418841A (zh) 2016-08-24 2019-11-05 桑格摩生物治疗股份有限公司 工程化的靶特异性核酸酶
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
JP7203014B2 (ja) 2016-08-24 2023-01-12 サンガモ セラピューティクス， インコーポレイテッド 工学操作されたヌクレアーゼを使用した遺伝子発現の調節
WO2018048194A1 (ko) * 2016-09-07 2018-03-15 울산대학교 산학협력단 dCas9 단백질 및 표적 핵산 서열에 결합하는 gRNA를 이용한 핵산 검출의 민감도 및 특이도 향상용 조성물 및 방법
WO2018049073A1 (en) * 2016-09-07 2018-03-15 Flagship Pioneering, Inc. Methods and compositions for modulating gene expression
CN107880132B (zh) * 2016-09-30 2022-06-17 北京大学 一种融合蛋白及使用其进行同源重组的方法
GB2569733B (en) 2016-09-30 2022-09-14 Univ California RNA-guided nucleic acid modifying enzymes and methods of use thereof
US10669539B2 (en) 2016-10-06 2020-06-02 Pioneer Biolabs, Llc Methods and compositions for generating CRISPR guide RNA libraries
US11242542B2 (en) 2016-10-07 2022-02-08 Integrated Dna Technologies, Inc. S. pyogenes Cas9 mutant genes and polypeptides encoded by same
WO2018068053A2 (en) 2016-10-07 2018-04-12 Integrated Dna Technologies, Inc. S. pyogenes cas9 mutant genes and polypeptides encoded by same
AU2017341926B2 (en) 2016-10-14 2022-06-30 The General Hospital Corporation Epigenetically regulated site-specific nucleases
WO2018071868A1 (en) 2016-10-14 2018-04-19 President And Fellows Of Harvard College Aav delivery of nucleobase editors
CN110520530A (zh) 2016-10-18 2019-11-29 明尼苏达大学董事会 肿瘤浸润性淋巴细胞和治疗方法
WO2018083606A1 (en) 2016-11-01 2018-05-11 Novartis Ag Methods and compositions for enhancing gene editing
US20180282722A1 (en) * 2016-11-21 2018-10-04 Massachusetts Institute Of Technology Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing
CA3044101A1 (en) 2016-11-22 2018-05-31 Integrated Dna Technologies, Inc. Crispr/cpf1 systems and methods
IL267024B2 (en) 2016-12-08 2023-12-01 Intellia Therapeutics Inc Adapted leader RNAs for genomic editing
WO2018111947A1 (en) 2016-12-12 2018-06-21 Integrated Dna Technologies, Inc. Genome editing enhancement
US20180179553A1 (en) 2016-12-14 2018-06-28 Ligandal, Inc. Compositions and methods for nucleic acid and/or protein payload delivery
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
WO2018125964A1 (en) * 2016-12-28 2018-07-05 Ionis Pharmaceuticals, Inc. Modified crispr rna and uses thereof
JP7219972B2 (ja) 2017-01-05 2023-02-09 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー Ｄｎａ二本鎖切断に非依存的な標的化遺伝子編集プラットフォームおよびその用途
EP4095263A1 (en) 2017-01-06 2022-11-30 Editas Medicine, Inc. Methods of assessing nuclease cleavage
EP4249501A3 (en) 2017-01-09 2024-01-03 Whitehead Institute for Biomedical Research Methods of altering gene expression by perturbing transcription factor multimers that structure regulatory loops
KR102084186B1 (ko) * 2017-01-17 2020-03-03 기초과학연구원 Dna 단일가닥 절단에 의한 염기 교정 비표적 위치 확인 방법
CN110199032A (zh) 2017-01-23 2019-09-03 雷杰纳荣制药公司 羟基类固醇17-β脱氢酶13(HSD17B13)变体及其用途
TW201839136A (zh) 2017-02-06 2018-11-01 瑞士商諾華公司 治療血色素異常症之組合物及方法
EP3580337A4 (en) * 2017-02-10 2020-12-02 Zymergen, Inc. MODULAR UNIVERSAL PLASMID DESIGN STRATEGY FOR COMPILING AND PROCESSING SEVERAL DNA CONSTRUCTS FOR SEVERAL HOST
US11518991B2 (en) 2017-02-24 2022-12-06 Georg-August-Universitaet Goettingen Stiftung Oeffentlichen Rechts, Universitaetsmedizin Method for re-expression of different hypermethylated genes involved in fibrosis, like hypermethylated RASAL1 and use thereof in treatment of fibrosis as well as kit of parts for re-expression of hypermethylated genes including RASAL1 in a subject
EP3592853A1 (en) 2017-03-09 2020-01-15 President and Fellows of Harvard College Suppression of pain by gene editing
JP2020510439A (ja) 2017-03-10 2020-04-09 プレジデント アンド フェローズ オブ ハーバード カレッジ シトシンからグアニンへの塩基編集因子
WO2018170184A1 (en) 2017-03-14 2018-09-20 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
IL269458B2 (en) 2017-03-23 2024-02-01 Harvard College Nucleic base editors that include nucleic acid programmable DNA binding proteins
CN108660161B (zh) * 2017-03-31 2023-05-09 中国科学院脑科学与智能技术卓越创新中心 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法
EP3607308A1 (en) 2017-04-07 2020-02-12 Sage Science, Inc. Systems and methods for detection of genetic structural variation using integrated electrophoretic dna purification
BR112019021719A2 (pt) 2017-04-21 2020-06-16 The General Hospital Corporation Variantes de cpf1 (cas12a) com especificidade para pam alterada
AU2018254616B2 (en) 2017-04-21 2022-07-28 The General Hospital Corporation Inducible, tunable, and multiplex human gene regulation using crispr-Cpf1
EP3615672A1 (en) 2017-04-28 2020-03-04 Editas Medicine, Inc. Methods and systems for analyzing guide rna molecules
EP3622070A2 (en) 2017-05-10 2020-03-18 Editas Medicine, Inc. Crispr/rna-guided nuclease systems and methods
WO2018209320A1 (en) 2017-05-12 2018-11-15 President And Fellows Of Harvard College Aptazyme-embedded guide rnas for use with crispr-cas9 in genome editing and transcriptional activation
CN110959040A (zh) 2017-05-25 2020-04-03 通用医疗公司 具有改进的精度和特异性的碱基编辑器
JP2020527329A (ja) 2017-06-05 2020-09-10 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． Ｂ４ｇａｌｔ１バリアント及びその使用
CN108977442B (zh) * 2017-06-05 2023-01-06 广州市锐博生物科技有限公司 用于dna编辑的系统及其应用
CN110997908A (zh) 2017-06-09 2020-04-10 爱迪塔斯医药公司 工程化的cas9核酸酶
CA3065326A1 (en) 2017-06-14 2018-12-20 Wisconsin Alumni Research Foundation Modified guide rnas, crispr-ribonucleotprotein complexes and methods of use
WO2018232356A1 (en) 2017-06-15 2018-12-20 The Regents Of The University Of California Targeted non-viral dna insertions
US10011849B1 (en) 2017-06-23 2018-07-03 Inscripta, Inc. Nucleic acid-guided nucleases
US9982279B1 (en) 2017-06-23 2018-05-29 Inscripta, Inc. Nucleic acid-guided nucleases
WO2019006418A2 (en) 2017-06-30 2019-01-03 Intima Bioscience, Inc. ADENO-ASSOCIATED VIRAL VECTORS FOR GENE THERAPY
US20200140896A1 (en) 2017-06-30 2020-05-07 Novartis Ag Methods for the treatment of disease with gene editing systems
KR20240046316A (ko) * 2017-07-11 2024-04-08 시그마-알드리치 컴퍼니., 엘엘씨 표적된 게놈 변형을 개선하기 위한 뉴클레오솜 상호작용 단백질 도메인 사용
US11866726B2 (en) 2017-07-14 2024-01-09 Editas Medicine, Inc. Systems and methods for targeted integration and genome editing and detection thereof using integrated priming sites
JP2020534795A (ja) 2017-07-28 2020-12-03 プレジデント アンド フェローズ オブ ハーバード カレッジ ファージによって支援される連続的進化（ｐａｃｅ）を用いて塩基編集因子を進化させるための方法および組成物
SG11201911619YA (en) 2017-07-31 2020-01-30 Regeneron Pharma Assessment of crispr/cas-induced recombination with an exogenous donor nucleic acid in vivo
CN111182790A (zh) * 2017-07-31 2020-05-19 瑞泽恩制药公司 Crispr报告体非人类动物及其用途
US11130999B2 (en) 2017-07-31 2021-09-28 Regeneron Pharmaceuticals, Inc. Cas-ready mouse embryonic stem cells and mice and uses thereof
JP7207665B2 (ja) 2017-08-04 2023-01-18 北京大学 メチル化により修飾されたｄｎａ塩基を特異的に認識するｔａｌｅ ｒｖｄおよびその使用
JP7109009B2 (ja) 2017-08-08 2022-07-29 北京大学 遺伝子ノックアウト方法
AU2018320865B2 (en) 2017-08-23 2023-09-14 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases with altered PAM specificity
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
AU2018338790B2 (en) 2017-09-29 2022-09-15 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized TTR locus and methods of use
WO2019075197A1 (en) 2017-10-11 2019-04-18 The General Hospital Corporation METHODS OF DETECTION OF INDIVIDUAL SITE-SPECIFIC PARASITE GENOMIC DEAMINATION BY BASE EDITING TECHNOLOGIES
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN107602707B (zh) * 2017-10-17 2021-04-23 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
AU2018355587B2 (en) 2017-10-27 2023-02-02 The Regents Of The University Of California Targeted replacement of endogenous T cell receptors
US20210180053A1 (en) 2017-11-01 2021-06-17 Novartis Ag Synthetic rnas and methods of use
CN111448313A (zh) 2017-11-16 2020-07-24 阿斯利康(瑞典)有限公司 用于改善基于Cas9的敲入策略的有效性的组合物和方法
EP3728589A4 (en) * 2017-12-22 2021-11-03 G+Flas Life Sciences CHEMICAL GENOMIC ENGINEERING MOLECULES AND PROCESSES
CN109504711A (zh) * 2018-02-14 2019-03-22 复旦大学 基于CRISPR/cas9和过氧化物酶APEX2系统识别分析特异性基因组位点相互作用DNA的方法
EP3755798A4 (en) 2018-02-19 2022-04-06 Yale University PHOSPHOPEPTIDE-ENCODED OLIGONUCLEOTIDE BANKS AND METHODS FOR DETECTING PHOSPHORYLATION-DEPENDENT MOLECULAR INTERACTIONS
KR20200124702A (ko) 2018-02-23 2020-11-03 파이어니어 하이 부렛드 인터내쇼날 인코포레이팃드 신규한 cas9 오르소로그
AU2019236209A1 (en) 2018-03-14 2020-10-01 Editas Medicine, Inc. Systems and methods for the treatment of hemoglobinopathies
CN111885915B (zh) 2018-03-19 2023-04-28 瑞泽恩制药公司 使用crispr/cas系统对动物进行转录调制
AU2019247490A1 (en) 2018-04-06 2020-10-22 Children's Medical Center Corporation Compositions and methods for somatic cell reprogramming and modulating imprinting
JP7460539B2 (ja) 2018-04-17 2024-04-02 ザ ジェネラル ホスピタル コーポレイション 核酸を結合、修飾、および切断する物質の基質選択性および部位のためのｉｎ ｖｉｔｒｏでの高感度アッセイ
WO2019204766A1 (en) 2018-04-19 2019-10-24 The Regents Of The University Of California Compositions and methods for gene editing
WO2019213430A1 (en) * 2018-05-03 2019-11-07 The Board Of Trustees Of The Leland Stanford Junior University Compositions and methods for nicking target dna sequences
CN108588123A (zh) * 2018-05-07 2018-09-28 南京医科大学 CRISPR/Cas9载体组合在制备基因敲除猪的血液制品中的应用
GB2589246A (en) 2018-05-16 2021-05-26 Synthego Corp Methods and systems for guide RNA design and use
US10227576B1 (en) 2018-06-13 2019-03-12 Caribou Biosciences, Inc. Engineered cascade components and cascade complexes
CN110592141B (zh) * 2018-06-13 2023-07-07 中国科学院上海有机化学研究所 用于调控基因编辑效率的化合物及其应用
JP2021530212A (ja) 2018-07-13 2021-11-11 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニアThe Regents Of The University Of California レトロトランスポゾンベースの送達媒体及びその使用方法
EP3821020A4 (en) 2018-08-15 2022-05-04 Zymergen Inc. APPLICATIONS OF CRISPRI IN HIGH-THROUGHPUT METABOLIC ENGINEERING
AU2019326408A1 (en) 2018-08-23 2021-03-11 Sangamo Therapeutics, Inc. Engineered target specific base editors
EP3847251A1 (en) 2018-09-07 2021-07-14 Astrazeneca AB Compositions and methods for improved nucleases
EP3861120A4 (en) 2018-10-01 2023-08-16 North Carolina State University RECOMBINANT TYPE I CRISPR-CAS SYSTEM
WO2020076976A1 (en) 2018-10-10 2020-04-16 Readcoor, Inc. Three-dimensional spatial molecular indexing
US11407995B1 (en) 2018-10-26 2022-08-09 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US11434477B1 (en) 2018-11-02 2022-09-06 Inari Agriculture Technology, Inc. RNA-guided nucleases and DNA binding proteins
US11739320B2 (en) 2018-11-05 2023-08-29 Wisconsin Alumni Research Foundation Gene correction of Pompe disease and other autosomal recessive disorders via RNA-guided nucleases
JP2022514493A (ja) 2018-12-14 2022-02-14 パイオニア ハイ－ブレッド インターナショナル， インコーポレイテッド ゲノム編集のための新規なｃｒｉｓｐｒ－ｃａｓシステム
WO2020131632A1 (en) 2018-12-20 2020-06-25 Regeneron Pharmaceuticals, Inc. Nuclease-mediated repeat expansion
EP3911746A1 (en) 2019-01-14 2021-11-24 Institut National de la Santé et de la Recherche Médicale (INSERM) Methods and kits for generating and selecting a variant of a binding protein with increased binding affinity and/or specificity
US11946040B2 (en) 2019-02-04 2024-04-02 The General Hospital Corporation Adenine DNA base editor variants with reduced off-target RNA editing
US20220145330A1 (en) 2019-02-10 2022-05-12 The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Glads Modified mitochondrion and methods of use thereof
MX2021010559A (es) 2019-03-07 2021-12-15 Univ California Polipéptidos efectores de crispr-cas y métodos de uso de estos.
SG11202108091YA (en) 2019-03-18 2021-08-30 Regeneron Pharma Crispr/cas screening platform to identify genetic modifiers of tau seeding or aggregation
US11781131B2 (en) 2019-03-18 2023-10-10 Regeneron Pharmaceuticals, Inc. CRISPR/Cas dropout screening platform to reveal genetic vulnerabilities associated with tau aggregation
DE112020001342T5 (de) 2019-03-19 2022-01-13 President and Fellows of Harvard College Verfahren und Zusammensetzungen zum Editing von Nukleotidsequenzen
SG11202108451VA (en) 2019-04-03 2021-09-29 Regeneron Pharma Methods and compositions for insertion of antibody coding sequences into a safe harbor locus
ES2923629T3 (es) 2019-04-04 2022-09-29 Regeneron Pharma Métodos para la introducción sin cicatrices de modificaciones dirigidas en vectores de direccionamiento
CN114206108B (zh) 2019-04-04 2023-09-29 瑞泽恩制药公司 包括人源化凝血因子12基因座的非人动物
JP2022528722A (ja) 2019-04-12 2022-06-15 アストラゼネカ・アクチエボラーグ 改善された遺伝子編集のための組成物及び方法
WO2020247452A1 (en) 2019-06-04 2020-12-10 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized ttr locus with a beta-slip mutation and methods of use
SG11202111256XA (en) 2019-06-07 2021-11-29 Regeneron Pharma Non-human animals comprising a humanized albumin locus
JP2022536606A (ja) 2019-06-14 2022-08-18 リジェネロン・ファーマシューティカルズ・インコーポレイテッド タウオパチーのモデル
EP3783104A1 (en) * 2019-08-20 2021-02-24 Kemijski Institut Coiled-coil mediated tethering of crispr-cas and exonucleases for enhanced genome editing
CN114616002A (zh) 2019-09-13 2022-06-10 瑞泽恩制药公司 使用由脂质纳米颗粒递送的crispr/cas系统在动物中进行的转录调控
AU2020352552A1 (en) 2019-09-23 2022-03-17 Omega Therapeutics, Inc. Compositions and methods for modulating hepatocyte nuclear factor 4-alpha (HNF4α) gene expression
EP3812472B1 (en) 2019-10-21 2022-11-23 Albert-Ludwigs-Universität Freiburg A truly unbiased in vitro assay to profile off-target activity of one or more target-specific programmable nucleases in cells (abnoba-seq)
US11331333B2 (en) 2019-11-08 2022-05-17 Georg-August-Universität Göttingen Stiftung Öffentichen Rechts, Universitätsmadizin Treatment of aberrant fibroblast proliferation
CN114746125A (zh) 2019-11-08 2022-07-12 瑞泽恩制药公司 用于x连锁青少年型视网膜劈裂症疗法的crispr和aav策略
WO2021108561A1 (en) * 2019-11-25 2021-06-03 La Jolla Institute For Immunology Methods and compositions for modulating heterochromatin dysfunction, genomic instability, and associated conditions
WO2021108363A1 (en) 2019-11-25 2021-06-03 Regeneron Pharmaceuticals, Inc. Crispr/cas-mediated upregulation of humanized ttr allele
AU2020401206A1 (en) 2019-12-11 2022-07-14 Intellia Therapeutics, Inc. Modified guide RNAs for gene editing
CN111088357B (zh) * 2019-12-31 2022-09-20 深圳大学 针对escc的肿瘤标志物及其应用
WO2021151073A2 (en) * 2020-01-24 2021-07-29 The General Hospital Corporation Unconstrained genome targeting with near-pamless engineered crispr-cas9 variants
EP4093863A4 (en) * 2020-01-24 2024-04-10 The General Hospital Corporation CRISPR-CAS ENZYMES WITH IMPROVED ON-TARGET ACTIVITY
AU2021232598A1 (en) 2020-03-04 2022-09-08 Regeneron Pharmaceuticals, Inc. Methods and compositions for sensitization of tumor cells to immune therapy
WO2021195079A1 (en) 2020-03-23 2021-09-30 Regeneron Pharmaceuticals, Inc. Non-human animals comprising a humanized ttr locus comprising a v30m mutation and methods of use
AU2021253959A1 (en) * 2020-04-09 2022-11-17 Verve Therapeutics, Inc. Base editing of PCSK9 and methods of using same for treatment of disease
EP4146813A4 (en) * 2020-05-04 2024-09-04 Editas Medicine Inc KNOCK-IN SELECTION OF AN ESSENTIAL GENE
US20230193212A1 (en) 2020-05-06 2023-06-22 Orchard Therapeutics (Europe) Limited Treatment for neurodegenerative diseases
DE112021002672T5 (de) 2020-05-08 2023-04-13 President And Fellows Of Harvard College Vefahren und zusammensetzungen zum gleichzeitigen editieren beider stränge einer doppelsträngigen nukleotid-zielsequenz
JP2023526007A (ja) 2020-05-13 2023-06-20 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル β－ヘモグロビン異常症の処置のための塩基編集アプローチ
WO2021246165A1 (ja) * 2020-06-03 2021-12-09 国立大学法人広島大学 Ｏａｓｉｓ遺伝子の脱メチル化のための核酸及びそれを用いた脱メチル化方法
EP4179079A1 (en) 2020-07-10 2023-05-17 Horizon Discovery Limited Method for producing genetically modified cells
JP2023537093A (ja) 2020-08-11 2023-08-30 イッサム リサーチ デベロップメント カンパニー オブ ザ ヘブライ ユニバーシティ オブ エルサレム リミテッド Ｗｗｏｘ関連疾患の治療方法
KR102674574B1 (ko) * 2020-09-02 2024-06-13 한국과학기술연구원 Cas9을 위한 신규 tracrRNA 시스템
WO2022079020A1 (en) 2020-10-13 2022-04-21 Centre National De La Recherche Scientifique (Cnrs) Targeted-antibacterial-plasmids combining conjugation and crispr /cas systems and uses thereof
CN112430622A (zh) * 2020-10-26 2021-03-02 扬州大学 一种FokI和dCpf1融合蛋白表达载体及其介导的定点基因编辑方法
RU2762831C1 (ru) * 2020-10-26 2021-12-23 Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский институт сельскохозяйственной биотехнологии" (ФГБНУ ВНИИСБ) Молекула рнк-проводника для геномного редактирования протомоторной области гена vrn-a1 однодольных зерновых с применением системы crispr/cas9
WO2022120022A1 (en) 2020-12-02 2022-06-09 Regeneron Pharmaceuticals, Inc. Crispr sam biosensor cell lines and methods of use thereof
KR20220082186A (ko) 2020-12-10 2022-06-17 한세준 유,무선 충전이 가능한 보조배터리형 uv-led살균기
WO2022148955A1 (en) 2021-01-05 2022-07-14 Horizon Discovery Limited Method for producing genetically modified cells
US20240052372A1 (en) 2021-02-25 2024-02-15 INSERM (Institut National de la Santé et de la Recherche Médicale) Allele-specific genome editing of the nr2e3 mutation g56r
KR20220124652A (ko) 2021-03-03 2022-09-14 중앙대학교 산학협력단 CRISPR/Cas9 시스템을 이용한 유전체 단일염기 편집 방법 및 이의 용도
CN113846019B (zh) * 2021-03-05 2023-08-01 海南师范大学 一种海洋微拟球藻靶向表观基因组遗传调控方法
EP4095243A1 (en) 2021-05-25 2022-11-30 European Molecular Biology Laboratory System for hybridization-based precision genome cleavage and editing, and uses thereof
CN117396602A (zh) 2021-05-27 2024-01-12 阿斯利康(瑞典)有限公司 具有增强的稳定性的cas9效应蛋白
TW202317764A (zh) 2021-06-10 2023-05-01 美商英特利亞醫療公司 用於基因編輯之包含內部連接子之經修飾引導rna
EP4377460A1 (en) 2021-07-30 2024-06-05 Tune Therapeutics, Inc. Compositions and methods for modulating expression of methyl-cpg binding protein 2 (mecp2)
CA3227103A1 (en) 2021-07-30 2023-02-02 Matthew P. GEMBERLING Compositions and methods for modulating expression of frataxin (fxn)
CA3229450A1 (en) 2021-08-20 2023-02-23 Wisconsin Alumni Research Foundation Nonviral generation of genome edited chimeric antigen receptor t cells
KR20240055811A (ko) 2021-09-10 2024-04-29 애질런트 테크놀로지스, 인크. 프라임 편집을 위한 화학적 변형을 갖는 가이드 rna
WO2023052366A1 (en) 2021-09-28 2023-04-06 INSERM (Institut National de la Santé et de la Recherche Médicale) Base editing approaches for the treatment of beta-hemoglobinopathies
WO2023052508A2 (en) 2021-09-30 2023-04-06 Astrazeneca Ab Use of inhibitors to increase efficiency of crispr/cas insertions
MX2024003887A (es) 2021-10-14 2024-07-09 Arsenal Biosciences Inc Células inmunitarias que tienen arnch coespresados y sistemas de compuerta lógica.
AU2022371414A1 (en) 2021-10-20 2024-05-02 University Of Copenhagen Isolated glial progenitor cells for use in the competition treatment of age-related white matter loss
CN118251491A (zh) 2021-10-28 2024-06-25 瑞泽恩制药公司 用于敲除C5的CRISPR/Cas相关方法及组合物
EP4426832A1 (en) 2021-11-03 2024-09-11 The J. David Gladstone Institutes, A Testamentary Trust Established under The Will of J. David Gladstone Precise genome editing using retrons
CA3237303A1 (en) 2021-11-03 2023-05-11 Intellia Therapeutics, Inc. Polynucleotides, compositions, and methods for genome editing
WO2023099591A1 (en) 2021-12-01 2023-06-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for increasing fetal hemoglobin content by editing the +55-kb region of the erythroid-specific bcl11a enhancer
CA3238939A1 (en) 2021-12-08 2023-06-15 Gaurang Patel Mutant myocilin disease model and uses thereof
US20230279442A1 (en) 2021-12-15 2023-09-07 Versitech Limited Engineered cas9-nucleases and method of use thereof
WO2023137471A1 (en) 2022-01-14 2023-07-20 Tune Therapeutics, Inc. Compositions, systems, and methods for programming t cell phenotypes through targeted gene activation
WO2023137472A2 (en) 2022-01-14 2023-07-20 Tune Therapeutics, Inc. Compositions, systems, and methods for programming t cell phenotypes through targeted gene repression
WO2023141487A1 (en) * 2022-01-20 2023-07-27 Inari Agriculture Technology, Inc. Improved soybean explant preparation and transformation
WO2023141602A2 (en) 2022-01-21 2023-07-27 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2023144104A1 (en) 2022-01-25 2023-08-03 INSERM (Institut National de la Santé et de la Recherche Médicale) Base editing approaches for the treatment of βeta-thalassemia
WO2023150623A2 (en) 2022-02-02 2023-08-10 Regeneron Pharmaceuticals, Inc. Anti-tfr:gaa and anti-cd63:gaa insertion for treatment of pompe disease
WO2023152351A1 (en) 2022-02-14 2023-08-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Treatment of liver cancers by disrupting the beta-catenin/tcf-4 binding site located upstream of meg3 in the dlk1/dio3 locus
WO2023212677A2 (en) 2022-04-29 2023-11-02 Regeneron Pharmaceuticals, Inc. Identification of tissue-specific extragenic safe harbors for gene therapy approaches
WO2023220603A1 (en) 2022-05-09 2023-11-16 Regeneron Pharmaceuticals, Inc. Vectors and methods for in vivo antibody production
WO2023217888A1 (en) 2022-05-10 2023-11-16 Institut National de la Santé et de la Recherche Médicale Base editing approaches for correcting the cd39 (cag>tag) mutation in patients suffering from βeta-thalassemia
WO2023235726A2 (en) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Crispr interference therapeutics for c9orf72 repeat expansion disease
WO2023235725A2 (en) 2022-05-31 2023-12-07 Regeneron Pharmaceuticals, Inc. Crispr-based therapeutics for c9orf72 repeat expansion disease
WO2023250511A2 (en) 2022-06-24 2023-12-28 Tune Therapeutics, Inc. Compositions, systems, and methods for reducing low-density lipoprotein through targeted gene repression
WO2024015881A2 (en) 2022-07-12 2024-01-18 Tune Therapeutics, Inc. Compositions, systems, and methods for targeted transcriptional activation
WO2024020346A2 (en) 2022-07-18 2024-01-25 Renagade Therapeutics Management Inc. Gene editing components, systems, and methods of use
WO2024018056A1 (en) 2022-07-22 2024-01-25 Institut National de la Santé et de la Recherche Médicale Base editing approaches for correcting the ivs2-1 (g>a) mutation in patients suffering from βeta-thalassemia
WO2024026474A1 (en) 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
US20240067968A1 (en) 2022-08-19 2024-02-29 Tune Therapeutics, Inc. Compositions, systems, and methods for regulation of hepatitis b virus through targeted gene repression
WO2024044723A1 (en) 2022-08-25 2024-02-29 Renagade Therapeutics Management Inc. Engineered retrons and methods of use
WO2024047247A1 (en) 2022-09-02 2024-03-07 Institut National de la Santé et de la Recherche Médicale Base editing approaches for the treatment of amyotrophic lateral sclerosis
WO2024064642A2 (en) 2022-09-19 2024-03-28 Tune Therapeutics, Inc. Compositions, systems, and methods for modulating t cell function
WO2024073606A1 (en) 2022-09-28 2024-04-04 Regeneron Pharmaceuticals, Inc. Antibody resistant modified receptors to enhance cell-based therapies
WO2024084025A1 (en) 2022-10-21 2024-04-25 Keygene N.V. Rna transfection in plant cells with modified rna
US20240182561A1 (en) 2022-11-04 2024-06-06 Regeneron Pharmaceuticals, Inc. Calcium voltage-gated channel auxiliary subunit gamma 1 (cacng1) binding proteins and cacng1-mediated delivery to skeletal muscle
WO2024107765A2 (en) 2022-11-14 2024-05-23 Regeneron Pharmaceuticals, Inc. Compositions and methods for fibroblast growth factor receptor 3-mediated delivery to astrocytes
CN115820603B (zh) * 2022-11-15 2024-07-05 吉林大学 一种基于dCasRx-NSUN6单基因特异性M5C修饰编辑方法
WO2024121354A1 (en) 2022-12-08 2024-06-13 Keygene N.V. Duplex sequencing with covalently closed dna ends
WO2024131940A1 (zh) * 2022-12-23 2024-06-27 益杰立科(上海)生物科技有限公司 融合物及其用途
WO2024163683A2 (en) 2023-02-01 2024-08-08 Tune Therapeutics, Inc. Systems, compositions, and methods for modulating expression of methyl-cpg binding protein 2 (mecp2) and x-inactive specific transcript (xist)
WO2024163678A2 (en) 2023-02-01 2024-08-08 Tune Therapeutics, Inc. Fusion proteins and systems for targeted activation of frataxin (fxn) and related methods
WO2024165484A1 (en) 2023-02-06 2024-08-15 Institut National de la Santé et de la Recherche Médicale Enrichment of genetically modified hematopoietic stem cells through multiplex base editing
CN116376975B (zh) * 2023-02-27 2024-05-14 中国科学院脑科学与智能技术卓越创新中心 激活异染色质基因的方法及应用
WO2024186890A1 (en) 2023-03-06 2024-09-12 Intellia Therapeutics, Inc. Compositions and methods for hepatitis b virus (hbv) genome editing
WO2024201368A1 (en) 2023-03-29 2024-10-03 Astrazeneca Ab Use of inhibitors to increase efficiency of crispr/cas insertions
WO2024209000A1 (en) 2023-04-04 2024-10-10 Keygene N.V. Linkers for duplex sequencing

Family Cites Families (166)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4603044A (en) 1983-01-06 1986-07-29 Technology Unlimited, Inc. Hepatocyte Directed Vesicle delivery system
US4957773A (en) 1989-02-13 1990-09-18 Syracuse University Deposition of boron-containing films from decaborane
US5436150A (en) 1992-04-03 1995-07-25 The Johns Hopkins University Functional domains in flavobacterium okeanokoities (foki) restriction endonuclease
USRE45721E1 (en) 1994-08-20 2015-10-06 Gendaq, Ltd. Relating to binding proteins for recognition of DNA
US20030017149A1 (en) 1996-10-10 2003-01-23 Hoeffler James P. Single chain monoclonal antibody fusion reagents that regulate transcription in vivo
US6534261B1 (en) 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20020164575A1 (en) 1999-09-14 2002-11-07 Sangamo Biosciences, Inc., A Delaware Corporation Gene identification
DE60023936T2 (de) 1999-12-06 2006-05-24 Sangamo Biosciences Inc., Richmond Methoden zur verwendung von randomisierten zinkfingerprotein-bibliotheken zur identifizierung von genfunktionen
WO2001083819A2 (en) 2000-04-28 2001-11-08 Sangamo Biosciences, Inc. Methods for designing exogenous regulatory molecules
EP1276859B1 (en) 2000-04-28 2007-02-07 Sangamo Biosciences Inc. Targeted modification of chromatin structure
US20030198627A1 (en) 2001-09-01 2003-10-23 Gert-Jan Arts siRNA knockout assay method and constructs
WO2003072788A1 (en) * 2002-02-21 2003-09-04 The Wistar Institute Of Anatomy And Biology Methods and compositions for reversibly controlling expression of target genes in cells
WO2003104414A2 (en) * 2002-06-11 2003-12-18 The Scripps Research Institute Artificial transcription factors
WO2004099366A2 (en) 2002-10-23 2004-11-18 The General Hospital Corporation Context sensitive parallel optimization of zinc finger dna binding domains
US7021555B2 (en) 2004-01-06 2006-04-04 Zoo Med Laboratories, Inc. Spraying/misting for plants and animals
US7919277B2 (en) 2004-04-28 2011-04-05 Danisco A/S Detection and typing of bacterial strains
EP1858920B1 (en) 2005-02-18 2016-02-03 GlaxoSmithKline Biologicals SA Proteins and nucleic acids from meningitis/sepsis-associated escherichia coli
WO2007014181A2 (en) * 2005-07-25 2007-02-01 Johns Hopkins University Site-specific modification of the human genome using custom-designed zinc finger nucleases
SG10201508995QA (en) 2005-07-26 2015-11-27 Sangamo Biosciences Inc Targeted integration and expression of exogenous nucleic acid sequences
WO2007025097A2 (en) 2005-08-26 2007-03-01 Danisco A/S Use
US7833784B2 (en) 2005-11-28 2010-11-16 The Scripps Research Institute Zinc finger binding domains for TNN
ATE530669T1 (de) 2006-05-19 2011-11-15 Danisco Markierte mikroorganismen und entsprechende markierungsverfahren
EP2213731B1 (en) 2006-05-25 2013-12-04 Sangamo BioSciences, Inc. Variant foki cleavage half-domains
AU2007258872A1 (en) 2006-06-16 2007-12-21 Danisco A/S Bacterium
US9201063B2 (en) 2006-11-16 2015-12-01 General Electric Company Sequential analysis of biological samples
WO2008093152A1 (en) * 2007-02-01 2008-08-07 Cellectis Obligate heterodimer meganucleases and uses thereof
PL2126130T3 (pl) 2007-03-02 2015-10-30 Dupont Nutrition Biosci Aps Hodowle o ulepszonej fagooporności
WO2008118394A1 (en) 2007-03-23 2008-10-02 New York University Methods of affecting nitrogen assimilation in plants
JP5258874B2 (ja) 2007-04-10 2013-08-07 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｒｎａ干渉タグ
WO2008151032A2 (en) 2007-05-31 2008-12-11 Washington University In St. Louis Arrays and methods comprising m. smithii gene products
AR068551A1 (es) 2007-09-25 2009-11-18 Pastoral Greenhouse Gas Res Lt Peptidos y polipeptidos permeabilizantes celulares para celulas microbianas
FR2925918A1 (fr) 2007-12-28 2009-07-03 Pasteur Institut Typage et sous-typage moleculaire de salmonella par identification des sequences nucleotidiques variables des loci crispr
FR2930264B1 (fr) 2008-04-18 2013-02-22 Gervais Danone Sa Nouvelle souche de lactobacillus paracasei subsp. paracasei dotee de proprietes antimicrobiennes et immunomodulatrices.
JP2010017178A (ja) 2008-06-11 2010-01-28 Sumitomo Chemical Co Ltd Ｄｎａを定量又は検出する方法
JP2010017179A (ja) 2008-06-11 2010-01-28 Sumitomo Chemical Co Ltd Ｄｎａを定量又は検出する方法
WO2010011961A2 (en) 2008-07-25 2010-01-28 University Of Georgia Research Foundation, Inc. Prokaryotic rnai-like system and methods of use
JP2010048566A (ja) 2008-08-19 2010-03-04 Sumitomo Chemical Co Ltd Ｄｎａを定量又は検出する方法
JP2010068800A (ja) 2008-08-19 2010-04-02 Sumitomo Chemical Co Ltd Ｄｎａを定量又は検出する方法
US20100076057A1 (en) 2008-09-23 2010-03-25 Northwestern University TARGET DNA INTERFERENCE WITH crRNA
WO2010037001A2 (en) * 2008-09-26 2010-04-01 Immune Disease Institute, Inc. Selective oxidation of 5-methylcytosine by tet-family proteins
WO2010046648A2 (en) 2008-10-21 2010-04-29 Animal Health Trust Diagnostic test for streptococcus equi
WO2010046493A2 (en) 2008-10-23 2010-04-29 Université de Lausanne Gene transfer vectors comprising at least one isolated dna molecule having insulator and or boundary properties and methods to identify the same
US9404098B2 (en) 2008-11-06 2016-08-02 University Of Georgia Research Foundation, Inc. Method for cleaving a target RNA using a Cas6 polypeptide
WO2010054154A2 (en) 2008-11-07 2010-05-14 Danisco A/S Bifidobacteria crispr sequences
CN102984955B (zh) 2008-11-11 2015-01-07 营养健康有限公司 长双歧杆菌
GB2466177A (en) 2008-12-03 2010-06-16 Arab Science & Technology Found Bacteriophage selection and breeding
WO2010066907A1 (en) 2008-12-12 2010-06-17 Danisco A/S Genetic cluster of strains of streptococcus thermophilus having unique rheological properties for dairy fermentation
KR20100093626A (ko) 2009-02-17 2010-08-26 서강대학교산학협력단 슈도모나스 애루지노사에 대한 파아지 치료
WO2010113037A1 (en) 2009-04-03 2010-10-07 Centre National De La Recherche Scientifique Gene transfer vectors comprising genetic insulator elements and methods to identify genetic insulator elements
CA2760155A1 (en) 2009-04-27 2010-11-11 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
WO2010144151A2 (en) 2009-06-12 2010-12-16 Pacific Biosciences Of California, Inc. Single-molecule real-time analysis of protein synthesis
US20120178647A1 (en) 2009-08-03 2012-07-12 The General Hospital Corporation Engineering of zinc finger arrays by context-dependent assembly
KR101754083B1 (ko) 2009-09-25 2017-07-05 바스프 플랜트 사이언스 컴퍼니 게엠베하 향상된 수확량 관련 형질을 갖는 식물 및 이의 제조 방법
US9677125B2 (en) 2009-10-21 2017-06-13 General Electric Company Detection of plurality of targets in biological samples
US20110269119A1 (en) 2009-10-30 2011-11-03 Synthetic Genomics, Inc. Encoding text into nucleic acid sequences
PT2534173T (pt) 2010-02-08 2019-10-31 Sangamo Therapeutics Inc Semidomínios de clivagem manipulados
WO2011101696A1 (en) * 2010-02-18 2011-08-25 Cellectis Improved meganuclease recombination system
US20120027786A1 (en) 2010-02-23 2012-02-02 Massachusetts Institute Of Technology Genetically programmable pathogen sense and destroy
US10087431B2 (en) 2010-03-10 2018-10-02 The Regents Of The University Of California Methods of generating nucleic acid fragments
AU2011224331C1 (en) 2010-03-12 2014-11-27 Brookhaven Science Associates/Brookhaven National Laboratory Enterobacter sp. 638 and methods of use thereof
SG185481A1 (en) 2010-05-10 2012-12-28 Univ California Endoribonuclease compositions and methods of use thereof
US20110201118A1 (en) 2010-06-14 2011-08-18 Iowa State University Research Foundation, Inc. Nuclease activity of tal effector and foki fusion protein
WO2012047726A1 (en) 2010-09-29 2012-04-12 The Broad Institute, Inc. Methods for chromatin immuno-precipitations
NZ607870A (en) 2010-10-20 2015-09-25 Dupont Nutrition Biosci Aps Lactococcus crispr-cas sequences
US20130337454A1 (en) 2010-10-27 2013-12-19 Philippe Duchateau Method for increasing the efficiency of double-strand break-induced mutagenesis
KR101556359B1 (ko) 2011-01-03 2015-10-01 주식회사 툴젠 디자인된 tal 이펙터 뉴클레아제를 통한 게놈 엔지니어링
WO2012097353A1 (en) 2011-01-14 2012-07-19 Life Technologies Corporation Methods, compositions, and kits for detecting rare cells
US20140113376A1 (en) 2011-06-01 2014-04-24 Rotem Sorek Compositions and methods for downregulating prokaryotic genes
EP2543255B2 (en) 2011-07-04 2022-12-28 DSM IP Assets B.V. Anti-listerial mixed culture and method for producing cheese
JP6214530B2 (ja) 2011-07-15 2017-10-18 ザ ジェネラル ホスピタル コーポレイション 転写活性化因子様エフェクターの組立て方法
GB201122458D0 (en) 2011-12-30 2012-02-08 Univ Wageningen Modified cascade ribonucleoproteins and uses thereof
IN2014DN07853A (zh) 2012-02-24 2015-04-24 Hutchinson Fred Cancer Res
KR102084539B1 (ko) 2012-02-29 2020-03-04 상가모 테라퓨틱스, 인코포레이티드 헌팅턴병을 치료하기 위한 방법 및 조성물
WO2013141680A1 (en) 2012-03-20 2013-09-26 Vilnius University RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX
US9637739B2 (en) 2012-03-20 2017-05-02 Vilnius University RNA-directed DNA cleavage by the Cas9-crRNA complex
JP6352250B2 (ja) 2012-05-02 2018-07-04 ダウ アグロサイエンシィズ エルエルシー リンゴ酸デヒドロゲナーゼの標的改変
WO2013169802A1 (en) 2012-05-07 2013-11-14 Sangamo Biosciences, Inc. Methods and compositions for nuclease-mediated targeted integration of transgenes
US11120889B2 (en) 2012-05-09 2021-09-14 Georgia Tech Research Corporation Method for synthesizing a nuclease with reduced off-site cleavage
DE202013012241U1 (de) * 2012-05-25 2016-01-18 Emmanuelle Charpentier Zusammensetzungen für die durch RNA gesteuerte Modifikation einer Ziel-DNA und für die durch RNA gesteuerte Modulation der Transkription
US9102936B2 (en) 2012-06-11 2015-08-11 Agilent Technologies, Inc. Method of adaptor-dimer subtraction using a CRISPR CAS6 protein
EP2674501A1 (en) 2012-06-14 2013-12-18 Agence nationale de sécurité sanitaire de l'alimentation,de l'environnement et du travail Method for detecting and identifying enterohemorrhagic Escherichia coli
US10025647B2 (en) * 2012-06-30 2018-07-17 Intel Corporation Memory poisoning with hints
EP2872154B1 (en) 2012-07-11 2017-05-31 Sangamo BioSciences, Inc. Methods and compositions for delivery of biologics
ES2757623T3 (es) 2012-07-25 2020-04-29 Broad Inst Inc Proteínas de unión a ADN inducibles y herramientas de perturbación genómica y aplicaciones de las mismas
EP3406715B1 (en) 2012-09-07 2023-12-13 Corteva Agriscience LLC Fad3 performance loci and corresponding target site specific binding proteins capable of inducing targeted breaks
WO2014059025A1 (en) 2012-10-09 2014-04-17 Liposcience, Inc. Nmr quantification of branched chain amino acids
EP3789405A1 (en) 2012-10-12 2021-03-10 The General Hospital Corporation Transcription activator-like effector (tale) - lysine-specific demethylase 1 (lsd1) fusion proteins
US20150315576A1 (en) 2012-11-01 2015-11-05 Massachusetts Institute Of Technology Genetic device for the controlled destruction of dna
PL2928496T3 (pl) * 2012-12-06 2020-04-30 Sigma-Aldrich Co. Llc Modyfikacja i regulacja genomu w oparciu o CRISPR
MX2015007549A (es) 2012-12-12 2017-01-20 Broad Inst Inc Modificaciones de sistemas, métodos y composiciones guía optimizadas para la manipulación de secuencias.
BR112015013784A2 (pt) * 2012-12-12 2017-07-11 Massachusetts Inst Technology aplicação, manipulação e otimização de sistemas, métodos e composições para manipulação de sequência e aplicações terapêuticas
PL2896697T3 (pl) 2012-12-12 2016-01-29 Broad Inst Inc Projektowanie systemów, sposoby i optymalizowane kompozycje kierujące do manipulacji sekwencją
PL2898075T3 (pl) 2012-12-12 2016-09-30 PROJEKTOWANIE i OPTYMALIZACJA ULEPSZONYCH SYSTEMÓW, SPOSOBY I KOMPOZYCJE ENZYMÓW DO MANIPULACJI SEKWENCJĄ
EP2931892B1 (en) 2012-12-12 2018-09-12 The Broad Institute, Inc. Methods, models, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
WO2014093694A1 (en) 2012-12-12 2014-06-19 The Broad Institute, Inc. Crispr-cas nickase systems, methods and compositions for sequence manipulation in eukaryotes
US8993233B2 (en) 2012-12-12 2015-03-31 The Broad Institute Inc. Engineering and optimization of systems, methods and compositions for sequence manipulation with functional domains
EP2931899A1 (en) 2012-12-12 2015-10-21 The Broad Institute, Inc. Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof
EP2825654B1 (en) 2012-12-12 2017-04-26 The Broad Institute, Inc. Crispr-cas component systems, methods and compositions for sequence manipulation
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
BR102013032129B1 (pt) 2012-12-13 2022-06-07 Dow Agrosciences Llc Método para identificar a presença de um polinucleotídeo de dna doador exógeno inserido dentro de um único locus genômico eucariótico alvo
RU2699523C2 (ru) * 2012-12-17 2019-09-05 Президент Энд Фэллоуз Оф Харвард Коллидж Рнк-направляемая инженерия генома человека
US20140173780A1 (en) * 2012-12-19 2014-06-19 Dow Agrosciences Llc Soybean transformation for efficient and high-throughput transgenic event production
EP2943060A4 (en) 2013-01-14 2016-11-09 Recombinetics Inc LIVESTOCK WITHOUT HORN
US20140212869A1 (en) 2013-01-25 2014-07-31 Agilent Technologies, Inc. Nucleic Acid Proximity Assay Involving the Formation of a Three-way junction
CN103233028B (zh) 2013-01-25 2015-05-13 南京徇齐生物技术有限公司 一种无物种限制无生物安全性问题的真核生物基因打靶方法及螺旋结构dna序列
WO2014124284A1 (en) 2013-02-07 2014-08-14 The General Hospital Corporation Tale transcriptional activators
WO2014124226A1 (en) 2013-02-07 2014-08-14 The Rockefeller University Sequence specific antimicrobials
WO2014127287A1 (en) 2013-02-14 2014-08-21 Massachusetts Institute Of Technology Method for in vivo tergated mutagenesis
US10227610B2 (en) 2013-02-25 2019-03-12 Sangamo Therapeutics, Inc. Methods and compositions for enhancing nuclease-mediated gene disruption
WO2014131833A1 (en) 2013-02-27 2014-09-04 Helmholtz Zentrum München Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh) Gene editing in the oocyte by cas9 nucleases
CA2901545C (en) 2013-03-08 2019-10-08 Oxford Nanopore Technologies Limited Use of spacer elements in a nucleic acid to control movement of a helicase
US10612043B2 (en) 2013-03-09 2020-04-07 Agilent Technologies, Inc. Methods of in vivo engineering of large sequences using multiple CRISPR/cas selections of recombineering events
NZ712727A (en) 2013-03-14 2017-05-26 Caribou Biosciences Inc Compositions and methods of nucleic acid-targeting nucleic acids
KR102210322B1 (ko) 2013-03-15 2021-02-01 더 제너럴 하스피탈 코포레이션 Rna-안내 게놈 편집의 특이성을 증가시키기 위한 rna-안내 foki 뉴클레아제(rfn)의 용도
US10760064B2 (en) 2013-03-15 2020-09-01 The General Hospital Corporation RNA-guided targeting of genetic and epigenomic regulatory proteins to specific genomic loci
US20140349400A1 (en) 2013-03-15 2014-11-27 Massachusetts Institute Of Technology Programmable Modification of DNA
US20140273235A1 (en) 2013-03-15 2014-09-18 Regents Of The University Of Minnesota ENGINEERING PLANT GENOMES USING CRISPR/Cas SYSTEMS
US20140273230A1 (en) 2013-03-15 2014-09-18 Sigma-Aldrich Co., Llc Crispr-based genome modification and regulation
US9234213B2 (en) 2013-03-15 2016-01-12 System Biosciences, Llc Compositions and methods directed to CRISPR/Cas genomic engineering systems
US11332719B2 (en) 2013-03-15 2022-05-17 The Broad Institute, Inc. Recombinant virus and preparations thereof
KR102223568B1 (ko) 2013-04-05 2021-03-04 다우 아그로사이언시즈 엘엘씨 식물의 게놈 내의 외인성 서열의 통합을 위한 방법 및 조성물
US20150056629A1 (en) 2013-04-14 2015-02-26 Katriona Guthrie-Honea Compositions, systems, and methods for detecting a DNA sequence
AU2014253942B9 (en) 2013-04-16 2020-08-13 Regeneron Pharmaceuticals, Inc. Targeted modification of rat genome
CN103224947B (zh) 2013-04-28 2015-06-10 陕西师范大学 一种基因打靶系统
WO2014182700A1 (en) 2013-05-10 2014-11-13 Sangamo Biosciences, Inc. Delivery methods and compositions for nuclease-mediated genome engineering
US11414695B2 (en) 2013-05-29 2022-08-16 Agilent Technologies, Inc. Nucleic acid enrichment using Cas9
US20150067922A1 (en) 2013-05-30 2015-03-05 The Penn State Research Foundation Gene targeting and genetic modification of plants via rna-guided genome editing
US20150315252A1 (en) 2013-06-11 2015-11-05 Clontech Laboratories, Inc. Protein enriched microvesicles and methods of making and using the same
EP3011029B1 (en) 2013-06-17 2019-12-11 The Broad Institute, Inc. Delivery, engineering and optimization of tandem guide systems, methods and compositions for sequence manipulation
US10011850B2 (en) 2013-06-21 2018-07-03 The General Hospital Corporation Using RNA-guided FokI Nucleases (RFNs) to increase specificity for RNA-Guided Genome Editing
CN103343120B (zh) 2013-07-04 2015-03-04 中国科学院遗传与发育生物学研究所 一种小麦基因组定点改造方法
JP6482546B2 (ja) 2013-07-19 2019-03-13 ラリクス・バイオサイエンス・リミテッド・ライアビリティ・カンパニーＬａｒｉｘ Ｂｉｏｓｃｉｅｎｃｅ， Ｌｌｃ 二重対立遺伝子ノックアウトを生成するための方法および組成物
US20150044772A1 (en) 2013-08-09 2015-02-12 Sage Labs, Inc. Crispr/cas system-based novel fusion protein and its applications in genome editing
SG11201601313TA (en) 2013-08-29 2016-03-30 Univ Temple Methods and compositions for rna-guided treatment of hiv infection
US10167466B2 (en) 2013-09-04 2019-01-01 Csir Site-specific nuclease single-cell assay targeting gene regulatory elements to silence gene expression
US9322037B2 (en) 2013-09-06 2016-04-26 President And Fellows Of Harvard College Cas9-FokI fusion proteins and uses thereof
US9074199B1 (en) 2013-11-19 2015-07-07 President And Fellows Of Harvard College Mutant Cas9 proteins
RU2725520C2 (ru) 2013-12-11 2020-07-02 Регенерон Фармасьютикалс, Инк. Способы и композиции для направленной модификации генома
JP2017501149A (ja) 2013-12-12 2017-01-12 ザ・ブロード・インスティテュート・インコーポレイテッド 粒子送達構成成分を用いた障害及び疾患の標的化のためのｃｒｉｓｐｒ−ｃａｓ系及び組成物の送達、使用及び治療適用
WO2015089364A1 (en) 2013-12-12 2015-06-18 The Broad Institute Inc. Crystal structure of a crispr-cas system, and uses thereof
US20150191744A1 (en) 2013-12-17 2015-07-09 University Of Massachusetts Cas9 effector-mediated regulation of transcription, differentiation and gene editing/labeling
EP3985124A1 (en) 2013-12-26 2022-04-20 The General Hospital Corporation Multiplex guide rnas
EP3105327A4 (en) 2014-02-12 2017-10-18 Thomas Jefferson University Compositions and methods of using microrna inhibitors
ES2745769T3 (es) 2014-03-10 2020-03-03 Editas Medicine Inc Procedimientos y composiciones relacionados con CRISPR/CAS para tratar la amaurosis congénita de Leber 10 (LCA10)
JP6737714B2 (ja) 2014-03-14 2020-08-12 ユニヴァーシティ オブ ワシントン ゲノムインスレーターエレメントおよびその使用
WO2015139139A1 (en) 2014-03-20 2015-09-24 UNIVERSITé LAVAL Crispr-based methods and products for increasing frataxin levels and uses thereof
JP2017509350A (ja) 2014-04-03 2017-04-06 マサチューセッツ インスティテュート オブ テクノロジー ガイドｒｎａの生成のための方法および組成物
AU2015342749B2 (en) 2014-11-07 2022-01-27 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
MA41349A (fr) 2015-01-14 2017-11-21 Univ Temple Éradication de l'herpès simplex de type i et d'autres virus de l'herpès associés guidée par arn
RS64527B1 (sr) 2015-01-28 2023-09-29 Caribou Biosciences Inc Hibridni polinukleotidi crispr dnk/rnk i postupci upotrebe
KR102605464B1 (ko) 2015-01-30 2023-11-22 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 일차 조혈 세포에서의 단백질 전달
EP3256487A4 (en) 2015-02-09 2018-07-18 Duke University Compositions and methods for epigenome editing
AU2016226077B2 (en) 2015-03-03 2021-12-23 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases with altered PAM specificity
EP3302575A4 (en) 2015-05-28 2019-01-16 Coda Biotherapeutics GENOME EDITION VECTORS
WO2016205759A1 (en) 2015-06-18 2016-12-22 The Broad Institute Inc. Engineering and optimization of systems, methods, enzymes and guide scaffolds of cas9 orthologs and variants for sequence manipulation
US9790490B2 (en) 2015-06-18 2017-10-17 The Broad Institute Inc. CRISPR enzymes and systems
EP3337908A4 (en) 2015-08-18 2019-01-23 The Broad Institute, Inc. METHOD AND COMPOSITIONS FOR CHANGING THE FUNCTION AND STRUCTURE OF CHROMATIN GRINDING AND / OR DOMAINS
US9926546B2 (en) 2015-08-28 2018-03-27 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
CN114875012A (zh) 2015-08-28 2022-08-09 通用医疗公司 工程化的CRISPR-Cas9核酸酶
US9512446B1 (en) 2015-08-28 2016-12-06 The General Hospital Corporation Engineered CRISPR-Cas9 nucleases
AU2017341926B2 (en) 2016-10-14 2022-06-30 The General Hospital Corporation Epigenetically regulated site-specific nucleases
WO2018148256A1 (en) 2017-02-07 2018-08-16 The Regents Of The University Of California Gene therapy for haploinsufficiency
AU2018254616B2 (en) 2017-04-21 2022-07-28 The General Hospital Corporation Inducible, tunable, and multiplex human gene regulation using crispr-Cpf1
JP2021523719A (ja) 2018-05-17 2021-09-09 ザ ジェネラル ホスピタル コーポレイション Ｃｃｃｔｃ結合因子バリアント
JP2023503618A (ja) 2019-11-27 2023-01-31 ザ ジェネラル ホスピタル コーポレイション 遺伝子発現を活性化するためのシステムおよび方法
EP4176434A4 (en) 2020-05-29 2024-09-11 Massachusetts Gen Hospital SYSTEMS AND METHODS FOR STABLE AND HERITABLE CHANGE THROUGH PRECISION MACHINING (FORM)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LEI S. QI等: "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression", CELL, vol. 152, pages 1173 - 105 *
MARTIN JINEK等: "A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity", SCIENCE, vol. 337, pages 816 - 821, XP093068947, DOI: 10.1126/science.1225829 *
MORGAN L MAEDER等: "Robust, synergistic regulation of human gene expression using TALE activators", NATURE METHODS, pages 10 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024192914A1 (zh) * 2023-03-21 2024-09-26 深圳赫兹生命科学技术有限公司 去势ms2病毒样颗粒亚单位疫苗及其制备方法

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