CN106480080A - 用于改变基因产物的表达的crispr‑cas系统和方法 - Google Patents
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Abstract
本发明提供了用于改变靶基因序列以及相关基因产物的表达的系统、方法和组合物。提供了多种载体和载体系统、以及用于设计和使用此类载体的方法,其中一些载体和载体系统编码CRISPR复合物的一种或多种组分。还提供了指导CRISPR复合物在真核细胞中形成的方法以及用于利用CRISPR‑Cas系统的方法。
Description
本申请是2013年12月12日提交的题为“用于改变基因产物的表达的CRISPR-CAS系统和方法”的国家申请号为201380072715.1(PCT/US2013/074743)的发明专利申请的分案申请。
相关应用和引用参考
本申请要求分别于2013年7月2日和10月5日提交的均具有博德参考号BI-2011/008A、标题为“用于改变基因产物的表达的CRISPR-CAS系统和方法(CRISPR-CAS SYSTEMSAND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS)”的美国临时专利申请61/842,322和美国专利申请14/054,414的优先权。还要求分别于2012年12月12日、2013年1月2日、2013年3月15日以及2013年6月17日提交的分别具有博德参考号BI-2011/008/WSGR案卷号44063-701.101、BI-2011/008/WSGR案卷号44063-701.102、博德参考号BI-2011/008/VP案卷号44790.02.2003以及BI-2011/008/VP案卷号44790.03.2003、标题均为“用于序列操纵的系统、方法和组合物(SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION)”的美国临时专利申请61/736,527、61/748,427、61/791,409以及61/835,931的优先权。
参考了美国临时专利申请61/758,468;61/769,046;61/802,174;61/806,375;61/814,263;61/819,803以及61/828,130,这些临时专利申请各自的标题为“用于序列操纵的系统、方法以及组合物的工程化以及优化”,分别提交于2013年1月30日;2013年2月25;2013年3月15;2013年3月28;2013年4月20;2013年5月6日以及2013年5月28日。还参考了美国临时专利申请61/835,936、61/836,127、61/836,101、61/836,080以及61/835,973,每个提交于2013年6月17日。
前述申请、以及在其中或在它们的审查程序期间引用的所有文献或(“申请引用文献”)以及在这些申请引用文献中引用或参考的所有文献、以及在本文中引用或参考的所有文献(“本文引用的文献”)、在本文中引用的文献中引用或参考的所有文献,连同针对在本文中提及或通过引用结合在本文中的任何文献中的任何产品的任何制造商的说明书、说明、产品规格、和产品表,特此通过引用并入本文,并且可以在本发明的实践中采用。更具体地说,所有参考的文献均通过引用并入本文,其程度如同每个单独的文献被确切地并单独地指明通过引用而并入本文。
技术领域
本发明总体上涉及用于控制涉及序列靶向的基因表达的系统、方法、和组合物,该序列靶向例如可使用涉及规律间隔成簇短回文重复序列(CRISPR)及其组分的载体系统的、基因组微扰或基因编辑。
关于联邦资助研究的声明
本发明是在美国国立卫生研究院颁发的NIH先锋奖(Pioneer Award)DP1MH100706的政府支持下完成的。美国政府享有本发明的某些权利。
背景技术
在基因组测序技术和分析方法中的最新进展显著加速了对与范围广泛的生物功能和疾病相关联的遗传因子进行编目和映射的能力。精确的基因组靶向技术对于通过允许个体遗传元件的选择性干扰而使得因果性遗传变异的统性逆向工程成为可能、以及推进合成生物学、生物技术应用、和医学应用是需要的。虽然基因组编辑技术,如设计师锌指、转录激活子样效应因子(TALE)、或归巢大范围核酸酶(homing meganuclease)对于产生靶向的基因组干扰是可得的,但是仍然需要负担得起的、易于建立的、可扩展的、并且便于靶向真核基因组内的多个位置的新的基因组工程技术。
发明内容
对于具有一系列广泛应用的序列靶向的替代性且稳健的系统和技术存在着迫切需要。本发明着手解决这种需要并且提供了相关优点。CRISPR/Cas或CRISPR-Cas系统(两个术语在本申请通篇可互换地使用)不要求产生靶向特异性序列的定制蛋白,而是通过一个短RNA分子识别一个特异性DNA靶标,可以对单个Cas酶进行程序设计,换句话说可以使用所述短RNA分子将Cas酶募集到一个特异性DNA靶标。将该CRISPR-Cas系统添加到基因组测序技术和分析方法的组库中可以显著使该方法论简化并且提高对与范围广泛的生物功能和疾病相关联的遗传因子进行编目和映射的能力。为了有效而无有害作用地利用CRISPR-Cas系统用于基因组编辑,了解这些基因组工程工具的工程化和优化方面是至关重要的,这些方面是请求保护的本发明的方面。
在一个方面,本发明提供了一种改变或修饰一种或多种基因产物的表达的方法,该方法可以包括向包含并表达编码该一种或多种基因产物的DNA分子的细胞中引入一种工程化的非天然存在的CRISPR-Cas系统,该系统可以包括一种Cas蛋白和一种或多种指导RNA,该一种或多种指导RNA靶向这些DNA分子,由此该一种或多种指导RNA靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的该基因组座位,由此该一种或多种基因产物的表达被改变或修饰;并且,其中该Cas蛋白和该指导RNA并不共同天然存在。本发明包括,两种或更多种基因产物的表达可以被改变或修饰。本发明进一步包括,该指导RNA包括一种融合到tracr序列上的指导序列。在一个优选实施例中,该细胞是一种真核细胞,在一个更优选的实施例中,该细胞是一种哺乳动物细胞并且在一个仍更优选的实施例中,该哺乳动物细胞是一种人类细胞。本发明还包括,该Cas蛋白可以包括一个或多个核定位信号(NLS)。在一些实施例中,该Cas蛋白是一种II型CRISPR系统酶。在一些实施例中,该Cas蛋白是一种Cas9蛋白。在一些实施例中,该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物的突变的Cas9。该蛋白可以是一种Cas9同系物或直向同源物。在一些实施例中,该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。在一些实施例中,该Cas蛋白指导切割在该靶序列位置处的一条或两条链。在本发明的一个另外的方面,该基因产物的表达被降低并且该基因产物是一种蛋白质。本发明包括,引入细胞中是经由一种递送系统该递送系统可以包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
在另一个方面,本发明提供了一种改变或修饰一种或多种基因产物的表达的方法,该方法包括向包含并表达编码该一种或多种基因产物的DNA分子的细胞中引入一种工程化的非天然存在的载体系统,该载体系统可以包括一种或多种载体,该一种或多种载体包括:a)一种第一调节元件,该第一调节元件可操作地连接到一种或多种与编码该一种或多种基因产物的DNA分子的基因组座位中的靶序列杂交的CRISPR-Cas系统指导RNA上,b)一种第二调节元件,该第二调节元件可操作地连接到一种Cas蛋白上,其中组分(a)和(b)位于该系统的相同或不同载体上,由此这些指导RNA靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的该基因组座位,由此该一种或多种基因产物的表达被改变或修饰;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。本发明包括,两种或更多种基因产物的表达可以被改变或修饰。本发明进一步包括,该指导RNA包括一种融合到tracr序列上的指导序列。在一个优选实施例中,该细胞是一种真核细胞,在一个更优选的实施例中,该细胞是一种哺乳动物细胞并且在一个仍更优选的实施例中,该哺乳动物细胞是一种人类细胞。本发明还包括,该系统的载体可以进一步包括一个或多个NLS。在一些实施例中,该Cas蛋白是一种II型CRISPR系统酶。在一些实施例中,该Cas蛋白是一种Cas9蛋白。在一些实施例中,该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物的突变的Cas9。该蛋白可以是一种Cas9同系物或直向同源物。在一些实施例中,该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。在一些实施例中,该Cas蛋白指导切割在该靶序列位置处的一条或两条链。在本发明的一个另外的方面,该基因产物的表达被降低并且该基因产物是一种蛋白质。本发明包括,引入细胞中是经由一种递送系统该递送系统可以包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
本发明还提供了一种工程化的非天然存在的载体系统,该载体系统可以包括一种或多种载体,该一种或多种载体包括:a)一种第一调节元件,该第一调节元件可操作地连接到一种或多种与编码一种或多种基因产物的DNA分子的基因组座位中的靶序列杂交的CRISPR-Cas系统指导RNA上,b)一种第二调节元件,该第二调节元件可操作地连接到一种Cas蛋白上,其中组分(a)和(b)位于该系统的相同或不同载体上,由此这些指导RNA在细胞中靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的该基因组座位,由此该一种或多种基因产物的表达被改变或修饰;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。本发明包括,两种或更多种基因产物的表达可以被改变或修饰。本发明进一步包括,该指导RNA包括一种融合到tracr序列上的指导序列。在一个优选实施例中,该细胞是一种真核细胞,在一个更优选的实施例中,该细胞是一种哺乳动物细胞并且在一个仍更优选的实施例中,该哺乳动物细胞是一种人类细胞。本发明还包括,该系统的载体可以进一步包括一个或多个NLS。在一些实施例中,该Cas蛋白是一种II型CRISPR系统酶。在一些实施例中,该Cas蛋白是一种Cas9蛋白。在一些实施例中,该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物的突变的Cas9。该蛋白可以是一种Cas9同系物或直向同源物。在一些实施例中,该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。在一些实施例中,该Cas蛋白指导切割在该靶序列位置处的一条或两条链。在本发明的一个另外的方面,该基因产物的表达被降低并且该基因产物是一种蛋白质。本发明包括,引入细胞中是经由一种递送系统该递送系统可以包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
在仍另一个方面,本发明提供了一种工程化的可编程的非天然存在的CRISPR-Cas系统,该系统可以包括一种Cas蛋白以及一种或多种指导RNA,该一种或多种指导RNA在细胞中靶向编码一种或多种基因产物的DNA分子的基因组座位,并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的基因组座位,由此该一种或多种基因产物的表达被改变或修饰;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。本发明包括,两种或更多种基因产物的表达可以被改变或修饰。本发明进一步包括,该指导RNA包括一种融合到tracr序列上的指导序列。在一个优选实施例中,该细胞是一种真核细胞,在一个更优选的实施例中,该细胞是一种哺乳动物细胞并且在一个仍更优选的实施例中,该哺乳动物细胞是一种人类细胞。本发明还包括,该CRISPR-Cas系统可以进一步包括一个或多个NLS。在一些实施例中,该Cas蛋白是一种II型CRISPR系统酶。在一些实施例中,该Cas蛋白是一种Cas9蛋白。在一些实施例中,该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物的突变的Cas9。该蛋白可以是一种Cas9同系物或直向同源物。在一些实施例中,该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。在一些实施例中,该Cas蛋白指导切割在该靶序列位置处的一条或两条链。在本发明的一个另外的方面,该基因产物的表达被降低并且该基因产物是一种蛋白质。本发明包括,引入细胞中是经由一种递送系统该递送系统可以包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
在一个方面,本发明提供了包括一种或多种载体的载体系统。在一些实施例中,该系统包括:(a)一种第一调节元件,该第一调节元件可操作地连接到一种tracr配对序列以及用于在该tracr配对序列的上游插入一个或多个指导序列的一个或多个插入位点,其中当表达时,该指导序列引导CRISPR复合物在真核细胞中与一个靶序列的序列特异性结合,其中该CRISPR复合物包括与以下各项复合的一种CRISPR酶:(1)杂交到该靶序列的指导序列,以及(2)杂交到该tracr序列的tracr配对序列;和(b)一种第二调节元件,该第二调节元件可操作地连接到编码所述CRISPR酶的酶编码序列,该CRISPR酶包括一个核定位序列;其中组分(a)和(b)位于该系统的相同或不同载体上。在一些实施例中,组分(a)进一步包括在该第一调节元件的控制之下在tracr配对序列的下游的tracr序列。在一些实施例中,组分(a)进一步包括可操作地连接到该第一调节元件的两个或更多个指导序列,其中当表达时,该两个或更多个指导序列中的每个引导CRISPR复合物在真核细胞中与一个不同靶序列的序列特异性结合。在一些实施例中,该系统包括在一种第三调节元件(诸如一个聚合酶III启动子)控制之下的tracr序列。在一些实施例中,当最佳比对时,沿着该tracr配对序列的长度,该tracr序列展现至少50%、60%、70%、80%、90%、95%、或99%序列互补性。确定最佳比对在本领域的普通技术人员的能力范围内。例如,存在公开和可商购的比对算法和程序,诸如但不限于ClustalW、matlab中的史密斯-沃特曼算法(Smith-Waterman)、Bowtie、Geneious、Biopython以及SeqMan。在一些实施例中,该CRISPR复合物包括一个或多个核定位序列,该一个或多个核定位序列具有足够强度来在真核细胞的细胞核中驱动所述CRISPR复合物以可检测到的量积聚。不希望受理论限制,认为核定位序列对于真核生物中的CRISPR复合物活性不是必要的,但包括此类序列增强该系统的活性,尤其对于靶向细胞核中的核酸分子而言。在一些实施例中,该CRISPR酶是II型CRISPR系统酶。在一些实施例中,该CRISPR酶是Cas9酶。在一些实施例中,该Cas9酶是肺炎链球菌、化脓链球菌、或嗜热链球菌Cas9,并且可包括源自于这些生物体的突变的Cas9。该酶可以是一种Cas9同系物或直向同源物。在一些实施例中,该CRISPR酶是密码子优化的以便在真核细胞中表达。在一些实施例中,该CRISPR酶引导在该靶序列位置处的一条或两条链的切割。在一些实施例中,该第一调节元件是一种聚合酶III启动子。在一些实施例中,该第二调节元件是一种聚合酶II启动子。在一些实施例中,该指导序列在长度上是至少15个、16个、17个、18个、19个、20个、25个核苷酸,或10-30个之间、或15-25个之间、或15-20个之间的核苷酸。通常,并且贯穿本说明书,术语“载体”是指一种核酸分子,它能够运送与其连接的另一种核酸分子。载体包括但不限于,单链、双链、或部分双链的核酸分子;包括一个或多个自由端、无自由端(例如环状的)的核酸分子;包括DNA、RNA、或两者的核酸分子;以及本领域已知的其他多种多样的多核苷酸。一种类型的载体是“质粒”,其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。另一种类型的载体是病毒载体,其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如,逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如,具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。其他载体(例如,非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中,并且由此与该宿主基因组一起复制。而且,某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。在重组DNA技术中使用的普通表达栽体通常是质粒形式。
重组表达载体可包含处于适合于在宿主细胞中的核酸表达的形式的本发明的核酸,这意味着这些重组表达载体包含基于待用于表达的宿主细胞而选择的一种或多种调节元件,所述调节元件可操作地连接至待表达的核酸序列。在重组表达载体内,“可操作地连接”旨在表示感兴趣的核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至该一种或多种调节元件(例如,处于一种体外转录/翻译系统中或当该载体被引入到宿主细胞中时,处于该宿主细胞中)。
术语“调节元件”旨在包括启动子、增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号,如多聚腺苷酸化信号和多聚U序列)。这样的调节序列例如描述于戈德尔(Goeddel),《基因表达技术:酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185,学术出版社(Academic Press),圣地亚哥(San Diego),加利福尼亚州(1990)中。调节元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如,组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达,所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。调节元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达,该方式可以是或者可以不是组织或细胞类型特异性的。在一些实施例中,一个载体包含一个或多个pol III启动子(例如1、2、3、4、5、或更多个pol III启动子)、一个或多个pol II启动子(例如1、2、3、4、5、或更多个pol II启动子)、一个或多个pol I启动子(例如1、2、3、4、5、或更多个pol I启动子)、或其组合。pol III启动子的实例包括但不限于U6和H1启动子。pol II启动子的实例包括但不限于逆转录劳斯肉瘤病毒(RSV)LTR启动子(任选地具有RSV增强子)、巨细胞病毒(CMV)启动子(任选地具有CMV增强子)[参见,例如,波沙特(Boshart)等人,《细胞》(Cell)41:521-530(1985)]、SV40启动子、二氢叶酸还原酶启动子、β-肌动蛋白启动子、磷酸甘油激酶(PGK)启动子、和EF1α启动子。还被术语“调节元件”涵盖的是增强子元件,如WPRE;CMV增强子;在HTLV-I的LTR中的R-U5’片段《分子细胞生物学》(Mol.Cell.Biol.,第8(1)卷,第466-472页,1988);SV40增强子;以及在兔β-珠蛋白的外显子2与3之间的内含子序列(《美国国家科学院院刊》(Proc.Natl.Acad.Sci.USA.),第78(3)卷,第1527-31页,1981)。本领域技术人员将理解,表达载体的设计可取决于诸如待转化的宿主细胞的选择、所希望的表达水平等因素。一种载体可以被引入到宿主细胞中而由此产生转录物、蛋白质、或肽,包括由如本文所述的核酸编码的融合蛋白或肽(例如,规律间隔成簇短回文重复序列(CRISPR)转录物、蛋白质、酶、其突变体形式、其融合蛋白,等等)。
有利的载体包括慢病毒以及腺相关病毒,并且也可选择此类型的载体以靶向具体类型的细胞。
在一个方面,本发明提供了一种真核宿主细胞,该真核宿主细胞包括:(a)一种第一调节元件,该第一调节元件可操作地连接到一种tracr配对序列以及用于在该tracr配对序列的上游插入一个或多个指导序列的一个或多个插入位点,其中当表达时,该指导序列引导CRISPR复合物在真核细胞中与一个靶序列的序列特异性结合,其中该CRISPR复合物包括与以下各项复合的一种CRISPR酶:(1)杂交到该靶序列的指导序列,以及(2)杂交到该tracr序列的tracr配对序列;和/或(b)一种第二调节元件,该第二调节元件可操作地连接到编码所述CRISPR酶的酶编码序列,该CRISPR酶包括一个核定位序列。在一些实施例中,该宿主细胞包括组分(a)以及(b)。在一些实施例中,组分(a)、组分(b)、或组分(a)和(b)稳定地整合到该宿主真核细胞的一个基因组中。在一些实施例中,组分(a)进一步包括在该第一调节元件的控制之下在tracr配对序列的下游的tracr序列。在一些实施例中,组分(a)进一步包括可操作地连接到该第一调节元件的两个或更多个指导序列,其中当表达时,该两个或更多个指导序列中的每个引导CRISPR复合物在真核细胞中与一个不同靶序列的序列特异性结合。在一些实施例中,该真核宿主细胞进一步包括一种第三调节元件,诸如一种聚合酶III启动子,该第三调节元件可操作地连接到所述tracr序列。在一些实施例中,当最佳比对时,沿着该tracr配对序列的长度,该tracr序列展现至少50%、60%、70%、80%、90%、95%、或99%序列互补性。该酶可以是一种Cas9同系物或直向同源物。在一些实施例中,该CRISPR酶是密码子优化的以便在真核细胞中表达。在一些实施例中,该CRISPR酶引导在该靶序列位置处的一条或两条链的切割。在一些实施例中,该CRISPR酶缺少DNA链切割活性。在一些实施例中,该第一调节元件是一种聚合酶III启动子。在一些实施例中,该第二调节元件是一种聚合酶II启动子。在一些实施例中,该指导序列在长度上是至少15个、16个、17个、18个、19个、20个、25个核苷酸,或10-30个之间、或15-25个之间、或15-20个之间的核苷酸。在一个方面,本发明提供一种非人的真核生物;优选地一种多细胞真核生物,包括根据所述实施例中任一个的一种真核宿主细胞。在其他方面,本发明提供一种真核生物;优选地一种多细胞真核生物,包括根据所述实施例中任一个的一种真核宿主细胞。在这些方面的一些实施例中,该生物体可以是一种动物;例如一种哺乳动物。另外,该生物体可以是一种节肢动物,诸如一种昆虫。该生物体也可以是一种植物。进一步,该生物体可以是一种真菌。
在一个方面,本发明提供了一种试剂盒,该试剂盒包括在此所述的组分中的一种或多种。在一些实施例中,该试剂盒包括一种载体系统以及用于使用该试剂盒的说明书。在一些实施例中,该载体系统包括:(a)一种第一调节元件,该第一调节元件可操作地连接到一种tracr配对序列以及用于在该tracr配对序列的上游插入一个或多个指导序列的一个或多个插入位点,其中当表达时,该指导序列引导CRISPR复合物在真核细胞中与一个靶序列的序列特异性结合,其中该CRISPR复合物包括与以下各项复合的一种CRISPR酶:(1)杂交到该靶序列的指导序列,以及(2)杂交到该tracr序列的tracr配对序列;和/或(b)一种第二调节元件,该第二调节元件可操作地连接到编码所述CRISPR酶的酶编码序列,该CRISPR酶包括一个核定位序列。在一些实施例中,该试剂盒包括位于该系统的相同或不同载体上的组分(a)和(b)。在一些实施例中,组分(a)进一步包括在该第一调节元件的控制之下在tracr配对序列的下游的tracr序列。在一些实施例中,组分(a)进一步包括可操作地连接到该第一调节元件的两个或更多个指导序列,其中当表达时,该两个或更多个指导序列中的每个引导CRISPR复合物在真核细胞中与一个不同靶序列的序列特异性结合。在一些实施例中,该系统进一步包括一种第三调节元件,诸如一种聚合酶III启动子,该第三调节元件可操作地连接到所述tracr序列。在一些实施例中,当最佳比对时,沿着该tracr配对序列的长度,该tracr序列展现至少50%、60%、70%、80%、90%、95%、或99%序列互补性。在一些实施例中,该CRISPR酶包括一个或多个核定位序列,该一个或多个核定位序列具有足够强度来在真核细胞的细胞核中驱动所述CRISPR酶以可检测到的量积聚。在一些实施例中,该CRISPR酶是II型CRISPR系统酶。在一些实施例中,该CRISPR酶是Cas9酶。在一些实施例中,该Cas9酶是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物体的突变的Cas9。该酶可以是一种Cas9同系物或直向同源物。在一些实施例中,该CRISPR酶是密码子优化的以便在真核细胞中表达。在一些实施例中,该CRISPR酶引导在该靶序列位置处的一条或两条链的切割。在一些实施例中,该CRISPR酶缺少DNA链切割活性。在一些实施例中,该第一调节元件是一种聚合酶III启动子。在一些实施例中,该第二调节元件是一种聚合酶II启动子。在一些实施例中,该指导序列在长度上是至少15个、16个、17个、18个、19个、20个、25个核苷酸,或10-30个之间、或15-25个之间、或15-20个之间的核苷酸。
在一个方面,本发明提供了修饰在一种真核细胞中的靶多核苷酸的方法。在一些实施例中,该方法包括允许一种CRISPR复合物结合到该靶多核苷酸上以实施所述靶多核苷酸的切割,由此修饰该靶多核苷酸,其中该CRISPR复合物包括与杂交到在所述靶多核苷酸内的一个靶序列上的指导序列复合的CRISPR酶,其中所述指导序列连接到一种tracr配对序列上,该tracr配对序列进而杂交到一种tracr序列上。在一些实施例中,所述切割包括通过所述CRISPR酶切割在该靶序列位置的一条或两条链。在一些实施例中,所述切割导致靶基因的转录降低。在一些实施例中,该方法进一步包括通过与外源模板多核苷酸同源重组修复所述切割的靶多核苷酸,其中所述修复导致一种突变,包括所述靶多核苷酸的一个或多个核苷酸的插入、缺失、或取代。在一些实施例中,所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。在一些实施例中,该方法进一步包括将一种或多种载体递送到所述真核细胞,其中该一种或多种载体驱动下列一者或多者的表达:该CRISPR酶、连接到该tracr配对序列上的指导序列、以及该tracr序列。在一些实施例中,所述载体被递送到受试者内的真核细胞中。在一些实施例中,所述修饰发生在细胞培养物中的所述真核细胞中。在一些实施例中,该方法进一步包括在所述修饰之前从受试者中分离所述真核细胞。在一些实施例中,该方法进一步包括使所述真核细胞和/或从中衍生的细胞返回到所述受试者中。
在一个方面,本发明提供了修饰一种多核苷酸在真核细胞中的表达的方法。在一些实施例中,该方法包括允许一种CRISPR复合物结合到该多核苷酸上,这样使得所述结合导致所述多核苷酸的表达增加或降低;其中该CRISPR复合物包括与杂交到在所述多核苷酸内的一个靶序列上的指导序列复合的CRISPR酶,其中所述指导序列连接到一种tracr配对序列上,该tracr配对序列进而杂交到一种tracr序列上。在一些实施例中,该方法进一步包括将一种或多种载体递送到所述真核细胞,其中该一种或多种载体驱动下列一者或多者的表达:该CRISPR酶、连接到该tracr配对序列上的指导序列、以及该tracr序列。
在一个方面,本发明提供了一种产生包含突变的疾病基因的模式真核细胞的方法。在一些实施例中,疾病基因是与患有或产生一种疾病的风险的增加相关联的任何基因。在一些实施例中,该方法包括(a)将一种或多种载体引入真核细胞中,其中该一种或多种载体驱动以下一者或多者的表达:CRISPR酶、连接到tracr配对序列上的指导序列、以及tracr序列;以及(b)允许一种CRISPR复合物结合到一个靶多核苷酸上以实施在所述疾病基因内的该靶多核苷酸的切割,其中该CRISPR复合物包含与(1)杂交到该靶多核苷酸内的该靶序列上的指导序列、和(2)杂交到该tracr序列上的该tracr配对序列复合的CRISPR酶,由此产生包含突变的疾病基因的模式真核细胞。在一些实施例中,所述切割包括通过所述CRISPR酶切割在该靶序列位置的一条或两条链。在一些实施例中,所述切割导致靶基因的转录降低。在一些实施例中,该方法进一步包括通过与外源模板多核苷酸同源重组修复所述切割的靶多核苷酸,其中所述修复导致一种突变,包括所述靶多核苷酸的一个或多个核苷酸的插入、缺失、或取代。在一些实施例中,所述突变导致在从包含该靶序列的基因表达的蛋白质中的一个或多个氨基酸改变。
在一个方面,本发明提供了一种用于研发生物活性剂的方法,该生物活性剂调制与疾病基因相关的细胞信号传导事件。在一些实施例中,疾病基因是与患有或产生一种疾病的风险的增加相关联的任何基因。在一些实施例中,该方法包括(a)使一种测试化合物与所述实施例中的任一项的一种模式细胞接触;并且(b)检测读数变化,该变化指示与所述疾病基因的所述突变关联的细胞信号传导事件的减少或增加,从而开发调制与所述疾病基因关联的所述细胞信号传导事件的所述生物活性剂。
在一个方面,本发明提供包括在tracr配对序列上游的指导序列的一种重组多核苷酸,其中当表达时该指导序列引导一种CRISPR复合物与存在于真核细胞中的一个对应的靶序列的序列特异性结合。在一些实施例中,该靶序列是存在于真核细胞中的病毒序列。在一些实施例中,该靶序列是原癌基因或癌基因。
在一个方面,本发明提供了一种通过在一个或多个细胞中的基因中引入一个或多个突变来选择一个或多个细胞的方法,该方法包括:将一种或多种载体引入一个或多个细胞中,其中该一种或多种载体驱动以下一者或多者的表达:CRISPR酶、连接到tracr配对序列上的指导序列、tracr序列、以及编辑模板;其中该编辑模板包含消除CRISPR酶切的一个或多个突变;允许该编辑模板与在有待选择的一个或多个细胞中的靶多核苷酸同源重组;允许CRISPR复合物结合到靶多核苷酸上以实施在所述基因内的靶多核苷酸的切割,其中该CRISPR复合物包含与(1)杂交到该靶多核苷酸内的靶序列上的指导序列、和(2)杂交到该tracr序列上的tracr配对序列复合的CRISPR酶,其中该CRISPR复合物到该靶多核苷酸上的结合诱导细胞死亡,由此允许选择其中已经引入一个或多个突变的一个或多个细胞。在一些实施例中,该CRISPR酶是II型CRISPR系统酶。在一些实施例中,该CRISPR酶是一种Cas9蛋白。在一些实施例中,该Cas9蛋白是肺炎链球菌、化脓链球菌或嗜热链球菌Cas9,并且可包括源自于这些生物的突变的Cas9。该酶可以是一种Cas9同系物或直向同源物。在一些实施例中,该酶是密码子优化的,用于在真核细胞中进行表达。在一些实施例中,该酶指导切割在该靶序列位置处的一条或两条链。在一个优选的实施例中,该CRISPR酶是Cas9。在本发明的另一个优选实施例中,该有待选择的细胞可以是真核细胞。本发明的方面允许选择特异细胞,而不需要选择标记或可能包括反选择系统的两步法。
本发明的方面包括内源基因组中的位点特异性基因敲除:本发明与使用基于锌指和TAL效应物的位点特异性核酸酶技术相比是有利的,因为它无需详尽的设计并且可用于同时敲除同一基因组内的多个基因。在一个另外的方面,本发明包括位点特异性基因组编辑。本发明与使用天然或人工位点特异性核酸酶或重组酶相比是有利的,因为它可以能够引入位点特异性双链断裂,以促进靶向的基因组座位处的同源重组。在另一个方面,本发明包括DNA序列特异性干扰。可以使用本发明失活基于有害DNA的生物(如微生物、病毒或甚至癌细胞)的基因组,通过直接造这些生物的基因组中的特定位点处引入断裂。本发明提供了多元基因组工程化的方法和组合物,因为本发明的CRISPR-Cas系统可以通过使用多个序列特异性CRISPR间隔子元件或指导序列而容易地靶向基因组中的多个位点。
因此,本发明的目的在于,在本发明中不涵盖任何先前已知的产品、制造该产品的过程、或使用该产品的方法,使得申请人保留和特此披露放弃任何先前已知的产品、过程、或方法的权利。进一步指出的是,在本发明的范围之内,本发明并不旨在涵盖任何产品、过程、或该产品的制造或使用该产品的方法,其不符合USPTO(35U.S.C.§112,第一段)或EPO(EPC的第83条)的书面说明和可实施性要求,使得申请人保留和特此公开放弃任何先前描述的产品、制造该产品的过程、或使用该产品的方法的权利。
应当指出的是,在本披露中并且尤其是在权利要求书和/或段落中,术语如“包含(comprises)”、“包含(comprised)”、“包含(comprising)”等等可具有在美国专利法中属于它的含义;例如,它们可以表示“包括(includes)”、“包括(included)”、“包括(including)”等等;并且术语如“基本上由……组成(consisting essentially of)”和“基本上由……组成(consists essentially of)”具有在美国专利法中归于它们的含义,例如,它们允许未被清楚叙述的要素,但是排除在现有技术中发现或者影响本发明的基本或新颖特征的要素。这些和其他实施例披露于以下详细说明中,或者据其显而易见并且由其涵盖。
附图简述
本发明的新颖特征在所附权利要求书中具体提出。通过参考对说明性实施例进行叙述的以下详细说明,将获得对本发明的特征和优点的更好理解,在这些实施例中利用了本发明的原理,并且在这些附图中:
图1显示了该CRISPR系统的示意模型。来自化脓链球菌(黄色)的Cas9核酸酶经由合成的指导RNA(sgRNA)而被靶向基因组DNA,该指导RNA包含一个20个nt的指导序列(蓝色)和一个支架(红色)。与DNA靶(蓝色)的指导序列碱基对,直接地在必要的5’-NGG原型间隔子邻近基序(PAM;红紫色)的上游,并且Cas9在该PAM(红色三角形)的上游约3bp介导了双链断裂(DSB)。
图2A-F显示了一个示例性CRISPR系统、一个可能的作用机制、一个在真核细胞中的表达的示例性适应性改变、以及评估核定位和CRISPR活性的试验的结果。图2C按照出现次序分别披露了SEQ ID NOS 23-24。图2E按照出现次序分别披露了SEQ ID NOS 25-27。图2F按照出现次序分别披露了SEQ ID NOS 28-32。
图3A-D显示了针对示例性靶标的SpCas9特异性的评估结果。图3A按照出现次序分别披露了SEQ ID NOS 33、26和34-44。图3C披露了SEQ ID NO:33。
图4A-G显示了一个示例性载体系统以及它在指导真核细胞中的同源重组中的使用结果。图4E披露了SEQ ID NO:45。图4F按照出现次序分别披露了SEQ ID NOS 46-47。图4G按照出现次序分别披露了SEQ ID NOS 48-52。
图5提供了原型间隔子序列的表(按照出现次序分别是SEQ ID NOS 16、15、14、53-58、18、17以及59-63),并且总结了基于示例性脓链球菌和嗜热链球菌CRISPR系统设计的原型间隔子靶标的修饰效率结果,这些CRISPR系统具有针对人类和小鼠基因组中的座位的相应PAM。用Cas9以及前-crRNA/tracrRNA或嵌合RNA转染细胞,在转染72小时之后进行分析。基于来自指示细胞系的Surveyor分析结果,计算了indel(插入缺失)百分比(对于所有原型间隔子靶标N=3,误差为S.E.M.,N.D.表示使用Surveyor测定未检测到,并且N.T.表示在本研究中未检测)。
图6A-C显示了对于Cas9介导的基因靶向而言的不同tracrRNA转录本的比较。图6A按照出现次序分别披露了SEQ ID NOS 64-65。
图7显示了对于双链断裂诱导的微插入和微缺失的检测的surveyor核酸酶试验的示意图。
图8A-B显示了用于CRISPR系统元件在真核细胞中的表达的示例性双顺反子表达载体。图8A按照出现次序分别披露了SEQ ID NOS 66-68。图8B按照出现次序分别披露了SEQID NOS 69-71。
图9A-C显示了在人类基因组中在邻近的化脓链球菌SF370座位1PAM(NGG)(图9A)和嗜热链球菌LMD9座位2PAM(NNAGAAW)(图9B)之间的距离、以及对于每个PAM就染色体而言的距离(Chr)(图9C)的直方图。
图10A-D显示了一个示例性CRISPR系统、一个针对在真核细胞中的表达的示例性适应性改变、以及评估CRISPR活性的试验的结果。图10B按照出现次序分别披露了SEQ IDNOS 72-73。图10C披露了SEQ ID NO:74。
图11A-C显示了用于靶向哺乳动物细胞中的基因组座位的CRISPR系统的示例性操纵。图11A披露了SEQ ID NO:75。图11B按照出现次序分别披露了SEQ ID NOS 76-78。
图12A-B显示了在哺乳动物中的crRNA加工的Northern印迹分析的结果。图12A披露了SEQ ID NO:79。
图13A-B显示了在人PVALB和小鼠Th座位中的原型间隔子的示例性选择。图13A披露了SEQ ID NO:80。图13B披露了SEQ ID NO:81。
图14显示了在人EMX1座位中的嗜热链球菌CRISPR系统的示例性原型间隔子和相应PAM序列靶标。图14披露了SEQ ID NO:74。
图15提供了用于Surveyor、RFLP、基因组测序、以及Northern印迹分析的引物和探针的序列表(按照出现次序分别是SEQ ID NOS 82-93)。
图16A-C显示了具有嵌合RNA的CRISPR系统的示例性操纵以及在真核细胞中的系统活性的SURVEYOR分析的结果。图16A披露了SEQ ID NO:94。
图17A-B显示了在真核细胞中的CRISPR系统活性的SURVEYOR分析的结果的图示。
图18显示了使用UCSC基因组浏览器进行的在人类基因组中的一些化脓链球菌Cas9靶位点的示例性可视化。图18按照出现次序分别披露了SEQ ID NOS 95-173。
图19A-D显示了揭示五个Cas9家族的系统发生分析的环状描绘,这五个家族包括三组大的Cas9(约1400个氨基酸)和两组小的Cas9(约1100个氨基酸)。
图20A-F显示了揭示五个Cas9家族的系统发生分析的线性描绘,这五个家族包括三组大的Cas9(约1400个氨基酸)和两组小的Cas9(约1100个氨基酸)。
图21A-D显示了经由同源重组的基因组编辑。(a)SpCas9切口酶的示意图,具有在RuvC I催化结构域中的D10A突变。(b)示意图,表示使用有义或反义单链寡核苷酸作为修复模板在人EMX1座位处的同源重组(HR)。以上红色箭头表示sgRNA切割位点;用于基因分型的PCR引物(表J和K)表示为右图中的箭头。图21C按照出现次序分别披露了SEQ ID NOS 174-176、174、177以及176。(c)由HR修饰的区域的序列。d,对于野生型(wt)和切口酶(D10A)SpCas9介导的在EMX1靶标1座位的indel的SURVEYOR分析(n=3)。箭头指示预期片段大小的位置。
图22A-B显示了用于SpCas9的单个载体设计。图22A按照出现次序分别披露了SEQID NOS 178-180。图22B披露了SEQ ID NO:181。
本文的这些图仅仅是出于说明性的目的,并且不一定是按比例绘制的。
具体实施方式
术语“多核苷酸”、“核苷酸”、“核苷酸序列”、“核酸”和“寡核苷酸”可互换地使用。它们是指具有任何长度的核苷酸的聚合形式,是脱氧核糖核苷酸或核糖核苷酸、或其类似物。多核苷酸可具有任何三维结构,并且可以执行已知或未知的任何功能。以下是多核苷酸的非限制性实例:基因或基因片段的编码区或非编码区、根据连接分析定义的多个座位(一个座位)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、短发夹RNA(shRNA)、micro-RNA(miRNA)、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针、和引物。多核苷酸可以包含一个或多个经修饰的核苷酸,如甲基化的核苷酸和核苷酸类似物。如果存在,可以在聚合物组装之前或之后进行核苷酸结构的修饰。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后,如通过与标记的组分缀合来进一步修饰。
在本发明的多个方面,术语“嵌合RNA”、“嵌合指导RNA”、“指导RNA”、“单个指导RNA”以及“合成指导RNA”是可互换地使用的并且是指包括指导序列、tracr序列以及tracr配对序列的多核苷酸序列。术语“指导序列”是指在指定靶位点的指导RNA内约20bp的序列,并且可与术语“指导”或“间隔子”互换地使用。术语“tracr配对序列”也可与术语“(一个或多个)同向重复”互换地使用。一种示例性CRISPR-Cas系统示于图1中。
如本文所用的术语“野生型”是本领域技术人员所理解的术语,并且表示生物、菌株、基因的典型形式或者当它在自然界存在时区别于突变体或变体形式的特征。
如本文所用的术语“变体”应当被理解为表示具有衍生自在自然界中存在的模式的性质的展示。
术语“非天然存在的”或“工程化的”可互换地使用并且表面人工的参与。这些术语,当指核酸分子或多肽时,表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
“互补性”是指核酸与另一个核酸序列借助于传统的沃森-克里克或其他非传统类型形成一个或多个氢键的能力。互补百分比表示一个核酸分子中可与一个第二核酸序列形成氢键(例如,沃森-克里克碱基配对)的残基的百分比(例如,10个之中有5、6、7、8、9、10个即为50%、60%、70%、80%、90%、和100%互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60%、65%、70%、75%、80%、85%、90%、95%、97%、98%、99%、或100%的互补程度,或者是指在严格条件下杂交的两个核酸。
如本文使用的对于杂交的“严格条件”是指与靶序列具有互补性的一个核酸主要地与该靶序列杂交并且基本上不杂交到非靶序列上的条件。严格条件通常是序列依赖性的,并且取决于许多因素而变化。一般而言,该序列越长,则该序列特异性地杂交到其靶序列上的温度就越高。严格条件的非限制性实例描述于蒂森(Tijssen)(1993)的《生物化学和分子生物学中的实验室技术-核酸探针杂交》(Laboratory Techniques In BiochemistryAnd Molecular Biology-Hybridization With Nucleic Acid Probes),第I部分,第二章,“杂交原理概述和核酸探针分析策略”(“Overview of principles of hybridization andthe strategy of nucleic acid probe assay”),爱思唯尔(Elsevier),纽约。
“杂交”是指其中一个或多个多核苷酸反应形成一种复合物的反应,该复合物经由这些核苷酸残基之间的碱基的氢键键合而稳定化。氢键键合可以借助于沃森-克里克碱基配对、Hoogstein结合或以任何其他序列特异性方式而发生。该复合物可包含形成一个双链体的两条链、形成多链复合物的三条或多条链、单个自我杂交链、或这些的任何组合。杂交反应可以构成一个更广泛的过程(如PCR的开始、或经由一种酶的多核苷酸的切割)中的一个步骤。能够与一个给定序列杂交的序列被称为该给定序列的“互补物”。
如本文使用的“表达”是指藉此从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后藉此翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA,表达可以包括真核细胞中mRNA的剪接。
术语“多肽”、“肽”和“蛋白质”在本文可互换地使用,是指具有任何长度的氨基酸的聚合物。该聚合物可以是可以是直链或支链的,它可以包含修饰的氨基酸,并且它可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物;这些修饰例如二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他操纵,如与标记组分的缀合。如本文使用的术语“氨基酸”包括天然的和/或非天然的或者合成的氨基酸,包括甘氨酸以及D和L旋光异构体、以及氨基酸类似物和肽模拟物。
术语“受试者”、“个体”和“患者”在本文中是可互换地使用的,指的是脊椎动物,优选地是哺乳动物,更加优选地是人类。哺乳动物包括但不限于鼠类、猴、人、农畜、体育用动物和宠物。也包括体内获得或体外培养的一种生物实体的组织、细胞及其子代。
术语“治疗剂(therapeutic agent)”、“可用于治疗的试剂(therapeutic capableagent)”或“处理剂(treatment agent)”是可互换地使用的,并且是指在给予受试者时赋予某种有益影响的一种分子或化合物。该有益影响包括诊断确定的实现;改进一种疾病、症状、障碍、或病理学病况;减少或预防一种疾病、症状、障碍或病况的发作;并且总体上对抗一种疾病、症状、障碍或病理学病况。
如此处使用的,“治疗(treatment)”或“进行治疗(treating)”或“减轻”或“改善”是可互换地使用的。这些术语是指如下途径,该途径用于获得有益或希望的结果,包括但不限于一种治疗益处和/或一种预防益处。治疗益处意指治疗中的一种或多种疾病、病况、或症状上的任何治疗上相关的改进或对其的影响。对于预防益处,该组合物可给予至处于发展一种具体的疾病、病况、或症状的风险的受试者,或给予至报告了一种疾病的一个或多个生理学症状的受试者,尽管该疾病、病况、或症状可能还没有体现出来。
术语“有效量”或“治疗有效量”是指一种药剂的足以实现有益或希望的结果的量。治疗有效量可依赖于正治疗的受试者和疾病病状、受试者的重量和年龄、疾病病况的严重度、给药方式等中一项或多个而改变,并可以由本领域普通技术人员容易地确定。该术语也适用通过此处描述的显像方法中的任一项提供一种检测用图像的一个剂量。具体剂量可依赖于以下中一个或多个而变化:所选择的具体药剂、所遵循的给药方案、是否与其他化合物组合给予、给予时间、待显像的组织、以及携带它的物理递送系统。
除非另有说明,本发明的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术,这些在本领域的技能之内。参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis),《分子克隆:实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL),第2次编辑(1989);《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑,(1987));《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司):《PCR 2:实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体:实验室手册》(ANTIBODIES,A LABORATORY MANUAL),以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
本发明的若干方面涉及包括一种或多种载体的载体系统,或载体本身。载体可以被设计为用于在原核或真核细胞中表达CRISPR转录物(例如核酸转录物、蛋白质、或酶)。例如,CRISPR转录物可表达于例如大肠杆菌的细菌细胞、昆虫细胞(使用杆状病毒表达载体)、酵母细胞、或哺乳动物细胞中。适合的宿主细胞进一步讨论于Goeddel(戈德尔),《基因表达技术:酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185,学术出版社(Academic Press),圣地亚哥(San Diego),加利福尼亚州(Calif.)(1990年)中。可替代地,重组表达载体可在体外例如使用T7启动子调节序列和T7聚合酶来转录和翻译。
载体可以被引入原核生物中并且在其中增殖。在一些实施例中,原核生物用来扩增有待引入真核细胞中的载体的多个拷贝,或者作为有待引入真核细胞中的载体的产生中的中间载体(例如,扩增质粒作为病毒载体包装系统的一部分)。在一些实施例中,原核生物用来扩增一个载体的多个拷贝并且表达一种或多种核酸,如提供用于递送到宿主细胞或宿主生物中的一种或多种蛋白质的来源。蛋白质在原核生物中的表达最经常在大肠杆菌中用含有指导融合或非融合蛋白的表达的组成型或诱导型启动子的载体来进行。融合载体将多个氨基酸添加到在其中编码的蛋白质上,如该重组蛋白的氨基端上。这样的融合载体可以用于一个或多个目的,如:(i)增加重组蛋白的表达;(ii)增加重组蛋白的溶解性;以及(iii)通过在亲和纯化中充当配体来辅助重组蛋白的纯化。通常,在融合表达载体中,将蛋白切割位点引入至融合部分与重组蛋白的接合处以使得能够在纯化融合蛋白之后将重组蛋白与融合部分分离。这类酶以及它们的同源识别序列包括因子Xa、凝血酶以及肠激酶。示例性融合表达载体包括pGEX(发玛西亚生物技术有限公司(Pharmacia Biotech Inc);史密斯和约翰逊,1988,《基因》(Gene)67:31-40)、pMAL(纽英伦生物技术公司(New EnglandBiolabs),贝弗利(Beverly),马萨诸塞州(Mass.))以及pRIT5(发玛西亚公司,皮斯卡塔韦(Piscataway),新泽西州(N.J.)),它们分别将谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白或蛋白A融合至靶重组蛋白。
适合的诱导型非融合大肠杆菌表达载体的实例包括pTrc(Amrann(阿姆兰)等人,(1988年)《基因》(Gene)69:301-315)以及pET 11d(Studier(斯图迪尔)等人,《基因表达技术:酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185,学术出版社(Academic Press),圣地亚哥(San Diego),加利福尼亚州(Calif.)(1990年)60-89。
在一些实施例中,载体是酵母表达载体。用于在酵母酿酒中表达的载体的实例包括pYepSec1(巴尔戴利(Baldari)等人,1987.《欧洲分子生物学学会杂志》(EMBO J)6:229-234)、pMFa(库尔坚(Kurjan)和赫斯库伍特兹(Herskowitz),1982.《细胞》(Cell)30:933-943)、pJRY88(舒尔茨(Schultz)等人,1987.《基因》(Gene)54:113-123)、pYES2(InvitrogenCorporation(英杰公司),San Diego(圣地亚哥),Calif(加利福尼亚州))、以及picZ(Invitrogen Corp(英杰公司),San Diego(圣地亚哥),Calif(加利福尼亚州))。
在一些实施例中,使用杆状病毒载体,载体驱动昆虫细胞中的蛋白质表达。可用于在培养的昆虫细胞(例如,SF9细胞)中表达蛋白质的杆状病毒载体包括pAc系列(史密斯(Smith)等人,1983.《分子细胞生物学》(Mol.Cell.Biol.)3:2156-2165)和pVL系列(拉克瑙(Lucklow)和萨莫斯(Summers),1989.《病毒学》(Virology)170:31-39)。
在一些实施例中,使用哺乳动物表达载体,载体能够驱动一种或多种序列在哺乳动物细胞中表达。哺乳动物表达载体的实例包括pCDM8(锡德(Seed),1987.《自然》(Nature)329:840)和pMT2PC(考夫曼(Kaufman)等人,1987.《欧洲分子生物学学会杂志》(EMBO J.)6:187-195)。当用于哺乳动物细胞中时,表达载体的控制功能典型地由一种或多种调节元件提供。例如,常用的启动子来源于多瘤、腺病毒2、巨细胞病毒、猴病毒40、以及本文所述和本领域已知的其他病毒。对于用于原核细胞和真核细胞两者的其他适合的表达系统参见例如萨姆布鲁克(Sambrook)等人的《分子克隆实验指南》(Molecular Cloning:A LaboratoryManual.)(第2版)的第16和第17章,冷泉港实验室(Cold Spring Harbor Laboratory),冷泉港实验室出版社(Cold Spring Harbor Laboratory Press),冷泉港(Cold SpringHarbor),纽约,1989。
在一些实施例中,重组哺乳动物表达载体能够指导核酸优先在特定细胞类型中表达(例如,使用组织特异型调节元件来表达核酸)。组织特异型调节元件是本领域中已知的。适合的组织特异型启动子的非限制性实例包括白蛋白启动子(肝脏特异性的;平克特(Pinkert)等人,1987.《基因发育》(Genes Dev.)1:268-277),淋巴特异性启动子(卡里曼(Calame)和伊顿(Eaton),1988.《免疫学进展》(Adv.Immunol)43:235-275),特别是T细胞受体的启动子(维纳图(Winoto)和巴尔迪莫(Baltimore),1989.《欧洲分子生物学学会杂志》(EMBO J.)8:729-733)和免疫球蛋白(巴纳吉(Baneiji)等人,1983.《细胞》(Cell)33:729-740;奎恩(Queen)和巴尔迪莫(Baltimore),1983.《细胞》(Cell)33:741-748),神经元特异性启动子(例如,神经丝启动子;伯恩(Byrne)和鲁德尔(Ruddle),1989.《美国科学院院刊》(Proc.Natl.Acad.Sci USA)86:5473-5477),胰腺特异性启动子(艾德兰德(Edlund)等人,1985.《科学》(Science)230:912-916),以及乳腺特异性启动子(例如,乳清启动子;美国专利号4,873,316和欧洲申请公开号264,166)。还涵盖发育型调节启动子,例如,鼠科动物同源框蛋白(hox)启动子凯赛尔((Kessel)和格鲁斯(Gruss),1990.《科学》(Science)249:374-379)和α-甲胎蛋白启动子(卡姆皮斯(Campes)和蒂尔曼(Tilghman),1989.《基因发育》(Genes Dev.)3:537-546)。
在一些实施例中,调节元件可操作地连接至CRISPR系统的一个或多个元件,从而驱动该CRISPR系统的该一个或多个元件的表达。一般而言,CRISPR(规律间隔成簇短回文重复),也称为SPIDR(SPacer间隔开的同向重复),构成通常对于特定细菌物种而言特异性的DNA基因座的家族。该CRISPR座位包含在大肠杆菌中被识别的间隔开的短序列重复(SSR)的一个不同类(石野(Ishino)等人,《细菌学杂志》(J.Bacteriol.),169:5429-5433[1987];和中田(Nakata)等人,《细菌学杂志》(J.Bacteriol.),171:3553-3556[1989])、以及相关基因。类似的间隔开的SSR已经鉴定于地中海富盐菌(Haloferax mediterranei)、化脓链球菌、鱼腥藻属、和结核分枝杆菌中(参见,格鲁恩(Groenen)等人,《分子微生物学》(Mol.Microbiol.),10:1057-1065[1993];霍(Hoe)等人,《新发感染性疾病》(Emerg.Infect.Dis.),5:254-263[1999];马斯波尔(Masepohl)等人,《生物化学与生物物理学学报》(Biochim.Biophys.Acta)1307:26-30[1996];以及莫吉卡(Mojica)等人,《分子微生物学》,17:85-93[1995])。这些CRISPR座位典型地不同于其他SSR的重复结构,这些重复已被称为规律间隔的短重复(SRSR)(詹森(Janssen)等人,《OMICS:整合生物学杂志》(OMICS J.Integ.Biol.),6:23-33[2002];以及莫吉卡(Mojica)等人,《分子微生物学》(Mol.Microbiol.),36:244-246[2000])。一般而言,这些重复是以簇存在的短元件,其被具有基本上恒定长度的独特间插序列规律地间隔开(莫吉卡(Mojica)等人,[2000],同上)。虽然重复序列在菌株之间是高度保守的,许多间隔开的重复和这些间隔区的序列一般在菌株与菌株之间不同(冯·埃姆登(van Embden)等人,《细菌学杂志》(J.Bacteriol.),182:2393-2401[2000])。已经在40种以上的原核生物中鉴定出CRISPR座位(参见,例如,詹森(Janssen)等人,《分子微生物学》(Mol.Microbiol.),43:1565-1575[2002];以及莫吉卡(Mojica)等人,[2005]),包括但不限于:气火菌属(Aeropyrum)、热棒菌属(Pyrobaculum)、硫化叶菌属(Sulfolobus)、古球菌属(Archaeoglobus)、盐盒菌属(Halocarcula)、甲烷杆菌属(Methanobacterium)、甲烷球菌属(Methanococcus)、甲烷八叠球菌属(Methanosarcina)、甲烷火菌属(Methanopyrus)、焦球菌属(Pyrococcus)、嗜酸菌属(Picrophilus)、热原体属(Thermoplasma)、棒杆菌属(Corynebacterium)、分枝杆菌属(Mycobacterium)、链霉菌属(Streptomyces)、产水菌属(Aquifex)、紫单胞菌属(Porphyromonas)、绿菌属(Chlorobium)、栖热菌属(Thermus)、芽孢杆菌属(Bacillus)、利斯特菌属(Listeria)、葡萄球菌属(Staphylococcus)、梭菌属(Clostridium)、好热厌氧杆菌属(Thermoanaerobacter)、支原体属(Mycoplasma)、梭杆菌属(Fusobacterium)、固氮弓菌属(Azarcus)、色杆菌属(Chromobacterium)、奈瑟球菌属(Neisseria)、亚硝化单胞菌属(Nitrosomonas)、脱硫弧菌属(Desulfovibrio)、地杆菌属(Geobacter)、粘球菌属(Myxococcus)、弯曲杆菌属(Campylobacter)、类杆菌属(Wolinella)、不动杆菌属(Acinetobacter)、欧文菌属(Erwinia)、埃希菌属(Escherichia)、军团菌属(Legionella)、甲基球菌属(Methylococcus)、巴斯德菌属(Pasteurella)、发光细菌属(Photobacterium)、沙门菌属(Salmonella)、黄单胞菌属(Xanthomonas)、耶尔森菌属(Yersinia)、密螺旋体属(Treponema)、和栖热袍菌属(Thermotoga)。
一般而言,“CRISPR系统”统称为转录物和涉及CRISPR相关(“Cas”)基因的表达或指导其活性的其他元件,包括编码Cas基因的序列、tracr(反式激活CRISPR)序列(例如tracrRNA或活性部分tracrRNA)、tracr配对序列(涵盖“同向重复”和在内源CRISPR系统背景下的tracrRNA加工的部分同向重复)、指导序列(在内源CRISPR系统背景下也称为“间隔子(spacer)”)、或来自CRISPR座位的其他序列和转录物。在一些实施例中,CRISPR系统的一个或多个元件来源于I型、II型、或III型CRISPR系统。在一些实施例中,CRISPR系统的一个或多个元件来源于包含内源CRISPR系统的特殊生物,如化脓链球菌。一般而言,CRISPR系统的特征为促进在靶序列的位点处的CRISPR复合物(在内源CRISPR系统的背景下也称为前间区)的形成的元件。在CRISPR复合物形成的背景下,“靶序列”是指指导序列被设计为对其具有互补性的序列,其中在靶序列与指导序列之间的杂交促进CRISPR复合物的形成。完全互补性不是必需的,条件是存在足够互补性以引起杂交并且促进一种CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸,如DNA或RNA多核苷酸。在一些实施例中,靶序列位于细胞的细胞核或细胞质中。在一些实施例中,该靶序列可位于真核细胞的一个细胞器例如线粒体或叶绿体内。可被用于重组到包括该靶序列的靶基因座中的序列或模板被称为“编辑模板”或“编辑多核苷酸”或“编辑序列”。在本发明的方面,外源的模板多核苷酸可被称为编辑模板。在本发明的一个方面,该重组是同源重组。
典型地,在内源CRISPR系统的背景下,CRISPR复合物(包含杂交到靶序列上并且与一种或多种Cas蛋白复合的指导序列)的形成导致在该靶序列中或其附近(例如在1、2、3、4、5、6、7、8、9、10、20、50、或更多个碱基对之内)的一条链或两条链的切割。不希望受到理论的束缚,该tracr序列(其可以包含或其组成为野生型tracr序列的全部或部分(例如野生型tracr序列的约或多于约20、26、32、45、48、54、63、67、85个、或更多个核苷酸))也可以形成CRISPR复合物的一部分,如通过沿着该tracr序列的至少一部分杂交到与该指导序列可操作地连接的tracr配对序列的全部或部分上。在一些实施例中,该tracr序列与一个tracr配对序列具有足够的互补性以进行杂交,并参与一种CRISPR复合物的形成。至于该靶序列,据信不需要完全互补性,只要足以具备功能性。在一些实施例中,当最佳比对时,沿着该tracr配对序列的长度,该tracr序列具有至少50%、60%、70%、80%、90%、95%、或99%序列互补性。在一些实施例中,将驱动CRISPR系统的一个或多个元件的表达的一种或多种载体引入到宿主细胞中,使得该CRISPR系统的这些元件的表达在一个或多个靶位点指导CRISPR复合物的形成。例如,Cas酶、连接到tracr配对序列上的指导序列、以及tracr序列可以各自可操作地连接到在分开的载体上的分开的调节元件上。可替代地,从相同或不同调节元件表达的二个或更多个元件可以结合在单个载体中,其中提供该CRISPR系统的任何组分的一种或多种另外的载体不包括在该第一载体中。结合在单个载体中的CRISPR系统元件可以按照任何适合的取向排列,如一个元件相对于第二元件位于5’(“上游”)或3’(“下游”)。一个元件的编码序列可以位于第二元件的编码序列的同一条链或相对链上,并且取向为相同或相对的方向。在一些实施例中,单个启动子驱动编码CRISPR酶的转录物以及该指导序列、tracr配对序列(任选地可操作地连接到该指导序列上)、和嵌入在一个或多个内含子序列之内(例如,各自在不同的内含子中,两个或更多个在至少一个内含子中,或者全部都在单个内含子中)的tracr序列中的一者或多者的表达。在一些实施例中,该CRISPR酶、指导序列、tracr配对序列、和tracr序列可操作地连接到相同的启动子上并且从其表达。SpCas9的单个载体构建体示于图22中。
在一些实施例中,一个载体包含一个或多个插入位点,如限制性核酸内切酶识别序列(也称为“克隆位点”)。在一些实施例中,一个或多个插入位点(例如,约或多于约1、2、3、4、5、6、7、8、9、10个、或更多个插入位点)位于一种或多种载体的一个或多个序列元件的上游和/或下游。在一些实施例中,一个载体包含在tracr配对序列的上游、并且任选地在可操作地连接到该tracr配对序列上的调节元件的下游的插入位点,使得在将指导序列插入到该插入位点中之后并且在表达时该指导序列引导CRISPR复合物与真核细胞中的靶序列的序列特异性结合。在一些实施例中,一个载体包含一个或多个插入位点,每个插入位点位于两个tracr配对序列之间,从而允许在每个位点插入指导序列。在这样一种安排中,两个或更多个指导序列可以包含单指导序列的两个或更多个拷贝、两个或更多个不同的指导序列、或这些的组合。当使用多个不同的指导序列时,可以使用单个表达构建体使CRISPR活性靶向到细胞内的多个不同的相应靶序列。例如,单个载体可以包含约或多于约1、2、3、4、5、6、7、8、9、10、15、20个、或更多个指导序列。在一些实施例中,可以提供约或多于约1、2、3、4、5、6、7、8、9、10个、或更多个这样的含有靶序列的载体,并且任选地将其递送到细胞中。
在一些实施例中,一个载体包含可操作地连接到编码CRISPR酶(如Cas蛋白)的酶编码序列上的调节元件。Cas蛋白的非限制性实例包括:Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8,Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4、其同系物、或其修饰形式。这些酶是已知的;例如,化脓链球菌Cas9蛋白的氨基酸序列可见于SwissProt数据库登录号Q99ZW2下。在一些实施例中,未修饰的CRISPR酶如Cas9具有DNA切割活性。在一些实施例中,该CRISPR酶是Cas9,并且可以是来自化脓链球菌或肺炎链球菌的Cas9。在一些实施例中,该CRISPR酶指导在靶序列位置处(例如在该靶序列之内和/或在该靶序列的互补物之内)的一条链或两条链的切割。在一些实施例中,该CRISPR酶指导距离靶序列的第一个或最后一个核苷酸约1、2、3、4、5、6、7、8、9、10、15、20、25、50、100、200、500个、或更多个碱基对之内的一条链或两条链的切割。在一些实施例中,一个载体编码相对于相应的野生型酶被突变的CRISPR酶,使得该突变的CRISPR酶缺乏切割含有一个靶序列的靶多核苷酸的一条链和两条链的能力。例如,在来自化脓链球菌的Cas9的RuvC I催化结构域中的天冬氨酸到丙氨酸取代(D10A)将Cas9从切割两条链的核酸酶转化成切口酶(切割单条链)。致使Cas9为切口酶的其他突变实例包括而不限于H840A、N854A、和N863A。在本发明的方面,切口酶可以经由同源重组用于基因组编辑。例如,图21显示了经由同源重组的基因组编辑。图21(a)显示了SpCas9切口酶的示意图,具有在RuvC I催化结构域中的D10A突变。(b)示意图,表示使用有义或反义单链寡核苷酸作为修复模板在人EMX1座位处的同源重组(HR)。(c)由HR修饰的区域的序列。(d)对于野生型(wt)和切口酶(D10A)SpCas9介导的在EMX1靶标1座位处的indel而言的SURVEYOR分析(n=3)。箭头指示预期片段大小的位置。
在一些实施例中,一种Cas9切口酶可与一种或多种指导序列组合使用,例如,两种引导序列,其分别靶向该DNA靶的有义以及反义链。此组合允许两个链被切口并且用以诱导NHEJ。申请人已证明(数据未显示)两种切口酶靶标(即,在相同位置靶向DNA的不同链的sgRNA)在诱导诱变的NHEJ方面的效力。一种单切口酶(具有单个sgRNA的Cas9-D10A)不能够诱导NHEJ并且建立indel,但申请人已经显示,双切口酶(Cas9-D10A以及在相同位置靶向不同链的两个sgRNA)在人胚胎干细胞(hESC)中可以这样做。该效率在hESC中是约50%的核酸酶(即,不具有D10突变的常规Cas9)。
作为一个另外的实例,Cas9的两个或更多个催化结构域(RuvC I、RuvC II、和RuvCIII)可以被突变为产生实质上缺乏所有DNA切割活性的突变的Cas9。在一些实施例中,D10A突变与H840A、N854A、或N863A突变的一者或多者相组合,以便产生实质上缺乏所有DNA切割活性的Cas9酶。在一些实施例中,当该突变的CRISPR酶的DNA切割活性比它的非突变形式低约25%、10%、5%、1%、0.1%、0.01%、或更少时,则该酶被考虑为实质上缺乏所有DNA切割活性。其他突变可能是有用的;其中Cas9或其他CRISPR酶是来自除化脓链球菌外的物种,可产生相应氨基酸的突变以实现类似效果。
在一些实施例中,编码CRISPR酶的酶编码序列经密码子优化,以便在特定的细胞如真核细胞中表达。这些真核细胞可以是特定生物的那些或来源于特定生物,如哺乳动物,包括但不限于人、小鼠、大鼠、兔、狗、或非人类灵长动物。一般而言,密码子优化是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个、或更多个密码子同时维持该天然氨基酸序列而修饰一个核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于具有特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关,而该翻译效率则被认为依赖于(除其他之外)被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此,可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得,例如在密码子使用数据库(“Codon Usage Database”)中,并且这些表可以通过不同的方式调整适用。参见,中村Y.(Nakamura Y.)等人,“从国际DNA序列数据库中制表的密码子使用:2000年的状态”(Codonusage tabulated from the international DNA sequence databases:status for theyear 2000)《核酸研究》(Nucl.Acids Res.)28:292(2000年)。用于密码子优化特定的序列以便在特定的宿主细胞中表达的计算机算法也是可得的,如基因制造(Gene Forge)(Aptagen公司;雅各布斯(Jacobus),PA),也是可得的。在一些实施例中,在编码CRISPR酶的序列中的一个或多个密码子(例如1、2、3、4、5、10、15、20、25、50个、或更多个、或所有密码子)相应于对于特定氨基酸最频繁使用的密码子。
一般而言,指导序列是与靶多核苷酸序列具有足够互补性以便与该靶序列杂交并且指导CRISPR复合物与该靶序列的序列特异性结合的任何多核苷酸序列。在一些实施例中,当使用适合的比对算法进行最佳比对是,在指导序列与其相应的靶序列之间的互补程度是约或多于约50%、60%、75%、80%、85%、90%、95%、97.5%、99%、或更多。可以使用用于比对序列的任何适合的算法来确定最佳比对,其非限制性实例包括史密斯-沃特曼(Smith-Waterman)算法、尼德曼-翁施(Needleman-Wunsch)算法、基于伯罗斯-惠勒变换(Burrows-Wheeler Transform)的算法(例如伯罗斯-惠勒比对工具(Burrows WheelerAligner))、ClustalW、Clustal X、BLAT、Novoalign(Novocraft技术公司)、ELAND(亿明达公司(Illumina),圣地亚哥,加利福尼亚州)、SOAP(在soap.genomics.org.cn可获得)、以及Maq(在maq.sourceforge.net可获得)。在一些实施例中,指导序列在长度上为约或多于约5、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、35、40、45、50、75个、或更多个核苷酸。在一些实施例中,一个指导序列在长度上为少于约75、50、45、40、35、30、25、20、15、12个、或更少的核苷酸。可以通过任何适合的测定法来评估指导序列指导CRISPR复合物与靶序列的序列特异性结合的能力。例如,足以形成CRISPR复合物的CRISPR系统的组分,包括有待测试的指导序列在内,可以例如通过用编码该CRISPR序列的组分的载体进行转染而被提供到具有相应靶序列的宿主细胞中,随后通过如本文所述的测量员测定来评估在该靶序列之内的优先切割。类似地,通过提供该靶序列、包括有待测试的指导序列在内的CRISPR复合物的组分、和不同于该测试指导序列的对照指导序列,并且比较在该测试指导序列与该对照指导序列反应之间的靶序列处的结合或切割率,可以在一个试管中评估靶多核苷酸序列的切割。其他测定法是可能的,并且将由本领域技术人员想到。
指导序列可以被选择为靶向任何靶序列。在一些实施例中,该靶序列是在细胞的基因组内的序列。示例性靶序列包括在靶基因组中为独特的那些。例如,对于化脓链球菌Cas9,在基因组中的独特靶序列可以包括形式MMMMMMMMNNNNNNNNNNNNXGG的Cas9靶位点,其中NNNNNNNNNNNNXGG(N是A、G、T、或C;并且X可以是任何物)在该基因组中单次出现。在基因组中的独特靶序列可以包括形式MMMMMMMMMNNNNNNNNNNNXGG的化脓链球菌Cas9靶位点,其中NNNNNNNNNNNXGG(N是A、G、T、或C;并且X可以是任何物)在该基因组中单次出现。对于嗜热链球菌CRISPR1Cas9,在基因组中的独特靶序列可以包括形式MMMMMMMMNNNNNNNNNNNNXXAGAAW(SEQ ID NO:1)的Cas9靶位点,其中NNNNNNNNNNNNXXAGAAW(SEQ ID NO:2)(N是A、G、T、或C;X可以是任何物;并且W是A或T)在该基因组中单次出现。在基因组中的独特靶序列可以包括形式MMMMMMMMMNNNNNNNNNNNXXAGAAW(SEQ ID NO:3)的嗜热链球菌CRISPR1Cas9靶位点,其中NNNNNNNNNNNXXAGAAW(SEQ ID NO:4)(N是A、G、T、或C;X可以是任何物;并且W是A或T)在该基因组中单次出现。对于化脓链球菌Cas9,在基因组中的独特靶序列可以包括形式MMMMMMMMNNNNNNNNNNNNXGGXG的Cas9靶位点,其中NNNNNNNNNNNNXGGXG(N是A、G、T、或C;并且X可以是任何物)在该基因组中单次出现。在基因组中的独特靶序列可以包括形式MMMMMMMMMNNNNNNNNNNNXGGXG的化脓链球菌Cas9靶位点,其中NNNNNNNNNNNXGGXG(N是A、G、T、或C;并且X可以是任何物)在该基因组中单次出现。在这些序列的每一个中,“M”可以是A、G、T、或C,并且在序列鉴定中不必考虑为是独特的。
在一些实施例中,指导序列被选择为降低在该指导序列内的二级结构程度。可以通过任何适合的多核苷酸折叠算法来确定二级结构。一些算法是基于计算最小吉布斯(Gibbs)自由能。一种这样的算法的实例是mFold,正如祖克(Zuker)和施蒂格勒(Stiegler)《核酸研究》(Nucleic Acids Res.)9(1981),133-148)所描述。折叠算法的另一个实例是使用由维也纳大学(University of Vienna)的理论化学研究所(Institute forTheoretical Chemistry)研发的在线网络服务器RNAfold(参见例如A.R.格鲁伯(A.R.Gruber)等人,2008,《细胞》(Cell)106(1):23-24;以及PA卡尔(PA Carr)和GM丘奇(GMChurch),2009,《自然生物技术》(Nature Biotechnology)27(12):1151-62)。另外的算法可以发现于通过引用结合在此的美国申请序列号61/836,080(代理人案号44790.11.2022;博德参考号BI-2013/004A)中。
一般而言,tracr配对序列包括与tracr序列具有足够互补性以促进下列一项或多项的任何序列:(1)在含有相应tracr序列的细胞中的侧翼为tracr配对序列的指导序列的切除;以及(2)在靶序列处的CRISPR复合物的形成,其中该CRISPR复合物包含杂交到tracr序列上的tracr配对序列。通常,互补程度是就tracr配对序列与tracr序列沿着这两个序列的较短者的长度的最佳比对而言。可以通过任何适合的比对算法来确定最佳比对,并且可以可以进一步对二级结构做出解释,如在该tracr序列或tracr配对序列之内的自我互补性。在一些实施例中,在进行最佳比对时,在该tracr序列与tracr配对序列之间沿着这两者的较短者的长度的互补程度是约或多于约25%、30%、40%、50%、60%、70%、80%、90%、95%、97.5%、99%、或更高。图10B以及11B中提供了一个tracr序列以及一个tracr配对序列之间的最佳比对的实例说明。在一些实施例中,该tracr序列在长度上为约或多于约5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、40、50个、或更多个核苷酸。在一些实施例中,该tracr序列和tracr配对序列被包含在单个转录物中,使得在这两者之间的杂交产生具有二级结构(如发夹)的转录物。用于在发夹结构中使用的优选环形成序列在长度上为四个核苷酸,并且最优选地具有序列GAAA。然而,可以使用更长或更短的环序列,正如可替代的序列。这些序列优选地包括三联体(例如,AAA)、和另外的核苷酸(例如C或G)。环形成序列的实例包括CAAA和AAAG。在本发明的一个实施例中,该转录物或转录的多核苷酸序列具有至少两个或更多个发夹。在优选的实施例中,该转录物具有两个、三个、四个或五个发夹。在本发明的一个另外的实施例中,该转录物具有至多五个发夹。在一些实施例中,该单个转录物进一步包括一种转录终止序列;优选地这是一个polyT序列,例如六个T的核苷酸。图11B中的下面部分中提供了这样的发夹结构的实例说明,其中最终“N”的序列5’的部分以及环的上游对应于该tracr配对序列,并且该环的序列3’的部分对应于该tracr序列。另外的包含指导序列、tracr配对序列、和tracr序列的单个多核苷酸的非限制性实例如下(列出为5’到3’),其中“N”代表指导序列的碱基,小写字体的第一区代表tracr配对序列,且小写字体的第二区代表tracr序列,并且该最后的多聚T序列代表转录终止子:(1)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT(SEQ ID NO:5);(2)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT(SEQ ID NO:6);(3)NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT(SEQ ID NO:7);(4)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT(SEQ ID NO:8);(5)NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT(SEQ ID NO:9);以及(6)NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT(SEQ ID NO:10)。在一些实施例中,序列(1)到(3)与来自嗜热链球菌CRISPR1的Cas9结合使用。在一些实施例中,序列(4)到(6)与来自化脓链球菌CRISPR1的Cas9结合使用。在一些实施例中,该tracr序列是一个与包含该tracr配对序列的转录物分开的转录物(例如在图11B的顶部部分中所示的)。
在一些实施例中,该CRISPR酶是包含一个或多个异源蛋白结构域(例如除了该CRISPR酶之外的约或多于约1、2、3、4、5、6、7、8、9、10个、或更多个结构域)的融合蛋白的一部分。CRISPR酶融合蛋白可以包含任何其他蛋白质,以及任选地在任何两个结构域之间的连接序列。可以融合到CRISPR酶上的蛋白质结构域的实例包括但不限于,表位标签、报告基因序列、以及具有下列活性的一者或多者的蛋白质结构域:甲基酶活性、脱甲基酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。报告基因的实例包括,但不限于,谷胱甘肽-S-转移酶(GST)、辣根过氧化物酶(HRP)、氯霉素乙酰转移酶(CAT)、β-半乳糖苷酶、β-葡糖醛酸糖苷酶、萤光素酶、绿色荧光蛋白(GFP)、HcRed、DsRed、青荧光蛋白(CFP)、黄色荧光蛋白(YFP)、以包括蓝色荧光蛋白(BFP)的自发荧光蛋白。CRISPR酶可以融合到编码一种蛋白质或蛋白质片段的基因序列上,所述蛋白质或蛋白质片段结合DNA分子或结合其他细胞分子,其包括,但不限于,麦芽糖结合蛋白(MBP)、S-tag、Lex A DNA结合结构域(DBD)融合物、GAL4DNA结合结构域融合物、以及单纯疱疹病毒(HSV)BP16蛋白融合物。可以形成包含CRISPR酶的融合蛋白的一部分的另外的结构域描述于US 20110059502中,通过引用将其并入本文。在一些实施例中,使用标记的CRISPR酶来鉴定靶序列的位置。
在本发明的一个方面,可以将一种报告基因引入细胞中,以编码充当测量基因产物的表达的改变或修饰的标记物的基因产物,该报告基因包括但不限于谷胱甘肽-S-转移酶(GST)、辣根过氧化物酶(HRP)、氯霉素乙酰转移酶(CAT)、β-半乳糖苷酶、β-葡萄糖醛酸酶、荧光素酶、绿色荧光蛋白(GFP)、HcRed、DsRed、青色荧光蛋白(CFP)、黄色荧光蛋白(YFP)以及自体荧光蛋白(包括蓝色荧光蛋白(BFP))。在本发明的一个另外的实施例中,可以经由载体将编码该基因产物的DNA分子引入该细胞中。在本发明的一个优选实施例中,该基因产物是荧光素酶。在本发明的一个另外的实施例中,该基因产物的表达被降低。
在一些方面,本发明提供了以下方法,这些方法包括向宿主细胞递送一种或多种多核苷酸,如或如在此描述的一种或多种载体、其一种或多种转录本和/或一种或多种自其转录的蛋白。本发明充当一种使得可以靶向修饰基于DNA的基因组的基础平台。它可以多种递送系统配合,包括但不限于病毒、脂质体、电穿孔、显微注射以及缀合。在一些方面,本发明进一步提供了通过这样的方法产生的细胞以及包括这样的细胞或由这样的细胞产生的生物体(例如动物、植物、或真菌)。在一些实施例中,将与(并且任选地与其复合)指导序列组合的CRISPR酶递送至细胞。可以使用常规的病毒和非病毒基的基因转移方法将核酸引入哺乳动物细胞或靶组织中。可以使用这样的方法向培养物中或宿主生物中的细胞给予编码CRISPR系统的组分的核酸。非病毒载体递送系统包括DNA质粒、RNA(例如在此描述的载体的转录本)、裸核酸以及与递送赋形剂(如脂质体)复合的核酸。病毒载体递送系统包括DNA和RNA病毒,在被递送至细胞后它们具有游离型或整合型基因组。关于基因疗法程序的综述,参见安德森(Anderson),《科学》(Science)256:808-813(1992);纳贝尔(Nabel)&费尔格纳(Felgner),TIBTECH 11:211-217(1993);三谷(Mitani)&卡斯基(Caskey),TIBTECH 11:162-166(1993);狄龙(Dillon),TIBTECH 11:167-175(1993);米勒(Miller),《自然》(Nature)357:455-460(1992);范·布朗特(Van Brunt),《生物技术》(Biotechnology)6(10):1149-1154(1988);维涅(Vigne),《恢复神经学和神经科学》(Restorative Neurologyand Neuroscience)8:35-36(1995);克雷默(Kremer)&佩里科德特(Perricaudet),《英国医学公报》(British Medical Bulletin)51(1):31-44(1995);哈嗒嗒(Haddada)等人,在《微生物学和免疫学当前主题》(Current Topics in Microbiology and Immunology)中多尔夫勒(Doerfler)和博姆(编辑)(1995);以及余(Yu)等人,《基因疗法》(GeneTherapy)1:13-26(1994)。
核酸的非病毒递送方法包括脂转染、核转染、显微注射、基因枪、病毒颗粒、脂质体、免疫脂质体、聚阳离子或脂质:核酸共轭物、裸DNA、人工病毒体以及DNA的试剂增强性摄取。脂转染描述于例如美国专利号5,049,386、4,946,787和4,897,355中并且脂转染试剂是市售的(例如,TransfectamTM和LipofectinTM)。适于多核苷酸的有效的受体识别脂转染的阳离子和中性脂质包括Felgner(费尔格纳),WO 91/17424;WO 91/16024的那些。递送可以针对细胞(例如体外或离体给予)或靶组织(例如体内给予)。
脂质:核酸复合物(包括靶向的脂质体,如免疫脂质复合物)的制备是本领域的技术人员熟知的(参见例如,克丽丝特尔(Crystal),《科学》(Science)270:404-410(1995);布莱泽(Blaese)等人,《癌症基因疗法》(Cancer Gene Ther.)2:291-297(1995);贝尔(Behr)等人,《生物共轭化学》(Bioconjugate Chem.)5:382-389(1994);雷米(Remy)等人,《生物共轭化学》5:647-654(1994);高(Gao)等人,《基因疗法》(Gene Therapy)2:710-722(1995);艾哈迈德(Ahmad)等人,《癌症研究》(Cancer Res.)52:4817-4820(1992);美国专利号4,186,183、4,217,344、4,235,871、4,261,975、4,485,054、4,501,728、4,774,085、4,837,028以及4,946,787)。
使用RNA或DNA病毒基的系统递送核酸利用将病毒靶向体内的特定细胞并将病毒有效负荷(payload)运至细胞核中的高度进化的过程。可以将病毒载体直接给予至患者(体内)或可以使用它们在体外处理细胞,并且任选地,可以将修饰的细胞给予至患者(离体)。常规的病毒基的系统可以包括用于基因转移的逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体以及单纯疱疹病毒载体。用逆转录病毒、慢病毒和腺相关病毒基因转移方法整合进宿主基因组中是可能的,这通常导致插入转基因的长期表达。另外,已经在不同细胞类型和靶组织中观察到高转导效率。
可以通过掺入外源包膜蛋白,扩展靶细胞的潜在靶群而改变逆转录病毒的向性。慢病毒载体是能够转导或感染非分裂细胞并典型地产生较高病毒效价的逆转录病毒载体。因此,逆转录病毒基因转移系统的选择将依赖于靶组织。逆转录病毒载体由顺式作用长末端重复组成,这些长末端重复具有包装多达6-10kb的外源序列的能力。最低量的顺式作用LTR对于载体的复制和包装而言是足够的,然后使用这些载体将治疗基因整合进靶细胞中,以提供永久的转基因表达。广泛使用的逆转录病毒载体包括基于鼠白血病病毒(MuLV)、长臂猿白血病病毒(GaLV)、猴免疫缺陷病毒(SIV)、人类免疫缺陷病毒(HIV)及其组合的那些(参见例如,布赫谢尔(Buchscher)等人,《病毒学杂志》(J.Virol.)66:2731-2739(1992);约翰(Johann)等人,《病毒学杂志》66:1635-1640(1992);佐姆内尔费尔特(Sommnerfelt)等人,《病毒学》(Virol.)176:58-59(1990);威尔逊(Wilson)等人,《病毒学杂志》63:2374-2378(1989);米勒(Miller)等人,《病毒学杂志》65:2220-2224(1991);PCT/US 94/05700)。在瞬时表达是优选的应用中,可以使用腺病毒基系统。腺病毒基载体在许多细胞类型中能够具有非常高的转导效并且无需细胞分裂。用这样的载体,已经获得了较高的效价和表达水平。可以在相对简单的系统中大量地产生此载体。还可以使用腺相关病毒(“AAV”)载体转导具有靶核酸的细胞,例如,在体外产生核酸和肽,以及用于体内和离体基因疗法程序(参见例如,韦斯特(West)等人,《病毒学》(Virology)160:38-47(1987);美国专利号4,797,368;WO 93/24641;科丁(Kotin),《人类基因疗法》(Human Gene Therapy)5:793-801(1994);缪斯科斯卡(Muzyczka),《临床研究杂志》(J.Clin.Invest.)94:1351(1994))。重组AAV载体的构建描述于多个出版物中,包括美国专利号5,173,414;特拉特斯金(Tratschin)等人,《分子与细胞生物学》(Mol.Cell.Biol.)5:3251-3260(1985);特拉特斯金等人,《分子与细胞生物学》4:2072-2081(1984);埃尔莫奈特(Hermonat)&缪斯科斯卡(Muzyczka),《美国国家科学院院刊》(PNAS)81:6466-6470(1984);以及萨穆尔斯基(Samulski)等人,《病毒学杂志》(J.Virol.)63:03822-3828(1989)。
典型地使用包装细胞形成能够感染宿主细胞的病毒粒子。这样的细胞包括包装腺病毒的293细胞和包含逆转录病毒的ψ2细胞或PA317细胞。在基因疗法中使用的病毒载体通常由将核酸载体包装进病毒粒子的细胞系产生。这些载体典型地包含包装并且随后整合进宿主所需的最低量序列,其他病毒序列由用于有待表达的一个或多个多核苷酸的表达盒替换。失去的病毒功能典型地由包装细胞系反式地提供。例如,在基因疗法中使用的AAV载体典型地仅具有来自AAV基因组的ITR序列,这些序列为包装并整合进宿主基因组所需。将病毒DNA包装进以下细胞系中,该细胞系包含编码辅助质粒的其他AAV基因,即rep和cap,但缺少ITR序列。该细胞系还可以被作为辅助者的腺病毒感染。该辅助病毒促进AAV载体的复制和从辅助质粒表达AAV基因。由于缺少ITR序列,未以显著的量包装该辅助质粒。可以通过例如与AAV相比腺病毒更加敏感的热处理减少腺病毒的污染。用于将核酸递送至细胞的另外的方法是本领域的技术人员已知的。参见例如通过引用结合在此的US 20030087817。
在一些实施例中,用一种或多种在此描述的载体瞬时地或非瞬时地转染宿主细胞。在一些实施例中,当细胞天然地出现在受试者体内时将其转染。在一些实施例中,被转染的细胞取自受试者。在一些实施例中,该细胞来源于取自受试者的细胞,如细胞系。用于组织培养的多种多样的细胞系在本领域是已知的。细胞系的实例包括但不限于,C8161、CCRF-CEM、MOLT、mIMCD-3、NHDF、HeLa-S3、Huh1、Huh4、Huh7、HUVEC、HASMC、HEKn、HEKa、MiaPaCell、Panc1、PC-3、TF1、CTLL-2、C1R、Rat6、CV1、RPTE、A10、T24、J82、A375、ARH-77、Calu1、SW480、SW620、SKOV3、SK-UT、CaCo2、P388D1、SEM-K2、WEHI-231、HB56、TIB55、Jurkat、J45.01、LRMB、Bcl-1、BC-3、IC21、DLD2、Raw264.7、NRK、NRK-52E、MRC5、MEF、Hep G2、海拉B、海拉T4、COS、COS-1、COS-6、COS-M6A、BS-C-1猴肾上皮细胞、BALB/3T3小鼠胚胎成纤维细胞、3T3Swiss、3T3-L1、132-d5人类胎儿成纤维细胞;10.1小鼠成纤维细胞、293-T、3T3、721、9L、A2780、A2780ADR、A2780cis、A172、A20、A253、A431、A-549、ALC、B16、B35、BCP-1细胞、BEAS-2B、bEnd.3、BHK-21、BR 293、BxPC3、C3H-10T1/2、C6/36、Cal-27、CHO、CHO-7、CHO-IR、CHO-K1、CHO-K2、CHO-T、CHO Dhfr-/-、COR-L23、COR-L23/CPR、COR-L23/5010、COR-L23/R23、COS-7、COV-434、CML T1、CMT、CT26、D17、DH82、DU145、DuCaP、EL4、EM2、EM3、EMT6/AR1、EMT6/AR10.0、FM3、H1299、H69、HB54、HB55、HCA2、HEK-293、HeLa、Hepa1c1c7、HL-60、HMEC、HT-29、Jurkat、JY细胞、K562细胞、Ku812、KCL22、KG1、KYO1、LNCap、Ma-Mel 1-48、MC-38、MCF-7、MCF-10A、MDA-MB-231、MDA-MB-468、MDA-MB-435、MDCK II、MDCK II、MOR/0.2R、MONO-MAC 6、MTD-1A、MyEnd、NCI-H69/CPR、NCI-H69/LX10、NCI-H69/LX20、NCI-H69/LX4、NIH-3T3、NALM-1、NW-145、OPCN/OPCT细胞系、Peer、PNT-1A/PNT 2、RenCa、RIN-5F、RMA/RMAS、Saos-2细胞、Sf-9、SkBr3、T2、T-47D、T84、THP1细胞系、U373、U87、U937、VCaP、维洛(Vero)细胞、WM39、WT-49、X63、YAC-1、YAR及其转基因变种。细胞系可获得自本领域的技术人员已知的多种来源(参见例如,美国典型培养物保藏中心(American Type Culture Collection)(ATCC)(马纳萨斯(Manassus),弗吉尼亚州))。在一些实施例中,使用用一种或多种在此描述的载体转染的细胞建立新的细胞系,该新的细胞系包括一种或多种载体来源的序列。在一些实施例中,使用用如在此描述的CRISPR系统的组分转染(如通过用一种或多种载体进行瞬时转染或用RNA进行转染)且通过CRISPR复合物的活性修饰的细胞建立新的细胞系,该新的细胞系包括以下细胞,这些细胞包含吸水但是缺少任何其他外源序列。在一些实施例中,在评估一种或多种测试化合物中使用用一种或多种在此描述的载体瞬时或非瞬时转染的细胞或来源于这样的细胞的细胞系。
本发明的方面涉及产生用于研究疾病的遗传变异的哺乳动物细胞的等基因系。本发明的一个另外的方面涉及产生转基因的或病毒介导的递送的基因修饰的动物模型。本发明还包括用于产生在生物医学、农业和工业上有用的产品的微生物、细胞、植物、动物或合成的生物的基因组修饰。在仍另一个方面,本发明包括基因疗法。可以将本发明用作生物学研究工具,用于理解基因组,例如基因敲除研究。本发明涉及许多其他方法和组合物,这些方法和组合物依赖于基础编辑能力和重写基因组的DNA成分以及基于DNA的生物的靶向失活。本发明还可以用作治疗,用于靶向细菌感染、病毒感染等的具体菌株。
在一些实施例中,使用一种或多种在此描述的载体产生非人转基因动物或转基因植物。在一些实施例中,该转基因动物是一种哺乳动物,如小鼠、大鼠或兔。在某些实施例中,该生物体或受试者是植物。在某些实施例中,该生物体或受试者或植物是藻类。用于产生转基因植物和动物的方法在本领域是已知的,并且通常以如在此描述的细胞转染方法开始。还提供了转基因动物,正如转基因植物一样,尤其是作物和藻类。转基因动物或植物可用于在除了提供疾病模型之外的应用中。通过表达例如比通常在野生型中可见的更高的蛋白质、碳水化合物、营养素或维生素水平,这些应用可包括食物或饲料生产。在这方面,转基因植物,尤其是豆类和块茎类,以及动物,尤其是哺乳动物,如家畜(奶牛、绵羊、山羊和猪),而且还有禽类和食用昆虫,是优选的。
转基因藻类或其他植物,如油菜,可以在植物油或生物燃料例如像醇类(尤其是甲醇和乙醇)的生产中特别有用。这些可以被工程化为表达或过表达高水平的油或醇类,以供在油或生物燃料行业中使用。
在一个方面,本发明提供了修饰真核细胞中的靶多核苷酸的方法,这些方法可以在体内、离体或在体外。在一些实施例中,该方法包括从人或非人动物或植物(包括微藻)取样细胞或细胞群,并且修饰该细胞或这些细胞。培养可以发生在离体的任何阶段。该细胞或这些细胞甚至可以被重新引入该非人动物或植物(包括微藻)中。
在一个方面,本发明提供了用于修饰在一种真核细胞中的靶多核苷酸的方法。在一些实施例中,该方法包括允许一种CRISPR复合物结合到该靶多核苷酸上以实施所述靶多核苷酸的切割,由此修饰该靶多核苷酸,其中该CRISPR复合物包括与杂交到在所述靶多核苷酸内的一个靶序列上的指导序列复合的CRISPR酶,其中所述指导序列连接到一种tracr配对序列上,该tracr配对序列进而杂交到一种tracr序列上。
在一个方面,本发明提供了修饰一种多核苷酸在真核细胞中的表达的方法。在一些实施例中,该方法包括允许一种CRISPR复合物结合到该多核苷酸上,这样使得所述结合导致所述多核苷酸的表达增加或降低;其中该CRISPR复合物包括与杂交到在所述多核苷酸内的一个靶序列上的指导序列复合的CRISPR酶,其中所述指导序列连接到一种tracr配对序列上,该tracr配对序列进而杂交到一种tracr序列上。
随着作物基因组学的最新进展,使用CRISPR-Cas系统进行有效且符合成本效益的基因编辑和操纵的能力将允许快速选择并比较单个和多元遗传操作,以转化这样的基因组,以便改善生产并增强性状。在此方面,参考美国专利和出版物:美国专利号6,603,061-农杆菌介导的植物转化法(Agrobacterium-Mediated Plant Transformation Method);美国专利号7,868,149-植物基因组序列及其用途(Plant Genome Sequences and UsesThereof)以及US 2009/0100536-转具有增强的农艺性状的基因植物(Transgenic Plantswith Enhanced Agronomic Traits),将每者的所有内容和披露通过引用以其全文结合在此。在本发明的实践中,莫雷尔(Morrell)等人“作物基因组学:进展与应用(Cropgenomics:advances and applications)”《遗传学自然评论》(Nat Rev Genet.)2011年12月29日;13(2):85-96的内容和披露也通过引用以其全文结合在此。
在植物中,病原体常常是宿主特异性的。例如,番茄尖镰孢菌番茄专化型(Fusarium oxysporum f.sp.lycopersici)引起番茄枯萎病,而且只攻击番茄,并且香石竹尖镰孢禾柄锈菌小麦专化型(F.oxysporum f.dianthii Puccinia graminisf.sp.tritici)只攻击小麦。植物具有抵抗大多数病原体的现有的和诱导性的防御。跨植物代的突变和重组事件导致产生敏感性的遗传变异性,特别是当病原体以比植物更高的频率繁殖时。在植物中可以存在非宿主抗性,例如,该宿主和病原体是不相容的。还可以存在水平抗性,例如典型地由许多基因控制的针对所有种的病原体的部分抗性,以及垂直抗性,例如典型地由很少的基因控制的针对病原体的某些种而不是其他种的完全抗性。在基因对基因水平中,植物和病原体一起演化,并且在一者中的遗传变化使在另一者中的变化平衡。因此,使用自然变异,育种者针对产率、质量、均匀性、耐性、抗性将最有用的基因进行结合。抗性基因的来源包括天然或外来品种、传家宝品种(Heirloom Varieties)、近缘野生植物、以及诱导的突变,例如用诱变剂处理植物材料。利用本发明,向植物育种者提供了一种新的诱导突变的工具。因此,本领域的技术人员可以分析抗性基因的来源的基因组,并且在具有所希望的特征或性状的品种方面,采用本发明来诱导抗性基因的发生,这样具有比先前的诱变剂更好的精确性,并且因此加速并改良植物育种计划。
在一个方面,本发明提供了以下试剂盒,这些试剂盒包含披露于以上方法和组合物中的元件中的任何一个或多个。在一些实施例中,该试剂盒包括一种载体系统以及用于使用该试剂盒的说明书。在一些实施例中,该载体系统包括:(a)一种第一调节元件,该第一调节元件可操作地连接到一种tracr配对序列以及用于在该tracr配对序列的上游插入一种指导序列的一个或多个插入位点,其中当表达时,该指导序列引导CRISPR复合物在真核细胞中与一个靶序列的序列特异性结合,其中该CRISPR复合物包括与以下各项复合的一种CRISPR酶:(1)杂交到该靶序列的指导序列,以及(2)杂交到该tracr序列的tracr配对序列;和/或(b)一种第二调节元件,该第二调节元件可操作地连接到编码所述CRISPR酶的酶编码序列,所述CRISPR酶包括核定位序列的。元件可以单独地或组合地提供,并且可以被提供于任何适合的容器中,如小瓶、瓶子或管。在一些实施例中,该试剂盒包括一种或多种语言,例如多于一种语言的说明书。
在一些实施例中,试剂盒包括一种或多种用于在利用在此描述的元件中的一种或多种的方法中使用的试剂。试剂可以被提供于任何适合的容器中。例如,试剂盒可以提供一种或多种反应或存储缓冲液。可以按在具体测定中可用的形式或按在使用之前需要添加一种或多种其他组分的形式(例如按浓缩或冻干形式)提供试剂。缓冲液可以是任何缓冲液,包括但不限于碳酸钠缓冲液、碳酸氢钠缓冲液、硼酸盐缓冲液、Tris缓冲液、MOPS缓冲液、HEPES缓冲液及其组合。在一些实施例中,该缓冲液是碱性的。在一些实施例中,该缓冲液具有从约7至约10的pH。在一些实施例中,该试剂盒包括一个或多个寡核苷酸,该一个或多个寡核苷酸对应于一个用于插入进载体中的指导序列,以便可操作地连接该指导序列和一个调节元件。在一些实施例中,该试剂盒包括一个同源重组模板多核苷酸。
在一个方面,本发明提供了用于使用CRISPR系统的一个或多个元件的方法。本发明的CRISPR复合物提供了用于修饰靶多核苷酸的有效手段。本发明的CRISPR复合物具有多种多样的实用性,包括修饰(例如,缺失、插入、转位、失活、激活)多种细胞类型中的靶多核苷酸。正因为如此,本发明的CRISPR复合物在例如基因疗法、药物筛选、疾病诊断以及预后中具有广阔的应用谱。示例性CRISPR复合物包括与一种指导序列复合的CRISPR酶,该指导序列与靶多核苷酸内的靶序列杂交。该指导序列与一种tracr配对序列连接,该tracr配对序列进而与一种tracr序列杂交。
CRISPR复合物的靶多核苷酸可以是对真核细胞而言内源或外源的任何多核苷酸。例如,该靶多核苷酸可以是一种驻留在真核细胞的细胞核中的多核苷酸。该靶多核苷酸可以是一个编码基因产物(例如,蛋白质)的序列或一个非编码序列(例如,调节多核苷酸或无用DNA)。不希望被理论所束缚,据信该靶序列应该与PAM(原型间隔子邻近基序)相关;也就是说,与由CRISPR复合物识别的短序列相关。对PAM的精确序列和长度要求取决于使用的CRISPR酶而不同,但是PAM典型地是临近原型间隔子(也就是说,靶序列)的2-5个碱基对序列。在以下实例部分中给出PAM序列的实例,并且熟练人员将能够鉴定与给定的CRISPR酶一起使用的另外的PAM序列。
CRISPR复合物的靶多核苷酸可以包括多个疾病相关基因和多核苷酸以及信号传导生化途径相基因和多核苷酸,如分别提交于2012年12月12日和2013年1月2日、标题均为用于序列操纵的系统方法和组合物(SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION)的分别具有博德(Broad)参考号BI-2011/008/WSGR案卷号44063-701.101和BI-2011/008/WSGR案卷号44063-701.102的美国临时专利申请61/736,527和61/748,427中所列举,将所有这些申请的内容通过引用以其全文结合在此。
靶多核苷酸的实例包括与信号传导生化途径相关的序列,例如信号传导生化途径相关基因或多核苷酸。靶多核苷酸的实例包括疾病相关基因或多核苷酸。“疾病相关”基因或多核苷酸是指与非疾病对照的组织或细胞相比,在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因或多核苷酸。在改变的表达与疾病的出现和/或进展相关的情况下,它可以是一个以异常高的水平被表达的基因;它可以是一个以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的,并且可以处于正常或异常水平。
疾病相关基因和多核苷酸的实例可获得自约翰斯·霍普金斯大学的麦考斯克-纳森遗传医学研究所(McKusick-Nathans Institute of Genetic Medicine,Johns HopkinsUniversity)(巴尔的摩,马里兰州)和国立医学图书馆的国家生物技术信息中心(NationalCenter for Biotechnology Information,National Library of Medicine)(贝塞斯达,马里兰州)。
疾病相关基因和多核苷酸的实例列于表A和B中。在万维网上可获得的疾病具体信息可获得自约翰斯·霍普金斯大学的麦考斯克-纳森遗传医学研究所(McKusick-NathansInstitute of Genetic Medicine,Johns Hopkins University)(巴尔的摩,马里兰州)和国立医学图书馆的国家生物技术信息中心(National Center for BiotechnologyInformation,National Library of Medicine)(贝塞斯达,马里兰州)。信号传导生化途径相关基因和多核苷酸的实例列于表C中。
这些基因和途径的突变可以导致产生不当的蛋白质或以不当的量影响功能的蛋白质。通过引用而特此结合来自2012年12月12日提交的美国临时申请61/736,527和2013年1月2日提交的61/748,427的基因、疾病和蛋白质的另外的实例。这样的基因、蛋白质和途径可以是CRISPR复合物的靶多核苷酸。
表A
表B:
表C:
本发明的实施例还涉及与敲除基因、扩增基因以及修复与DNA重复不稳定性和神经障碍相关的具体突变有关的方法和组合物(罗伯特D.·威尔斯(Robert D.Wells)、芦沢哲夫(Tetsuo Ashizawa),遗传不稳定性与神经疾病(Genetic Instabilities andNeurological Diseases),第二版,学术出版社(Academic Press),2011年10月13日-《医学》(Medical))。已经发现串联重复序列的特定方面对超过二十种人类疾病负责(重复不稳定新见:RNA·DNA杂交体的作用(New insights into repeat instability:role ofRNA·DNA hybrids),麦基弗EI(McIvor EI)、波拉克U(Polak U)、纳皮尔拉拉M(NapieralaM),《RNA生物学》(RNA Biol.)2010年9月-10月;7(5):551-8)。可以利用CRISPR-Cas系统修正基因组不稳定性缺陷。
本发明的一个另外的方面涉及利用CRISPR-Cas系统修正EMP2A和EMP2B基因缺陷,这些基因已经被鉴定为与拉福拉病(Lafora disease)相关。拉福拉病是一种由可以作为青年期的癫痫发作而开始的进行性肌阵挛性癫痫表征的常染色体隐性病症。该疾病的少数病可以由尚未鉴定的基因的突变引起。该疾病引起发作、肌肉痉挛、行走困难、痴呆,并且最终引起死亡。当前没有疗法被证明有效对抗疾病进展。与癫痫相关的其他遗传异常还可以靶向CRISPR-Cas系统并且基础遗传学进一步描述于由朱利亚诺阿文济尼(GiulianoAvanzini)、杰弗里L.诺贝尔斯(Jeffrey L.Noebels)编辑的《癫痫与遗传癫痫遗传学》(Genetics of Epilepsy and Genetic Epilepsies),马里亚尼儿科神经学基金会(Mariani Foundation Paediatric Neurology):20;2009)中。
在本发明的又另一个方面,该CRISPR-Cas系统可以用来矫正几种基因突变引起的眼部缺陷,其进一步描述于《眼的遗传疾病》(Genetic Diseases of the Eye),第二次编辑,由伊莱亚斯(Elias)I.特拉布勒西(Traboulsi)编辑,牛津大学出版社,2012年。
本发明的若干另外的方面涉及修正与范围广泛的遗传性疾病相关的缺陷,这些遗传性疾病在专题小节遗传性障碍(Genetic Disorders)下被进一步描述于国立卫生研究院的网站(网址为health.nih.gov/topic/GeneticDisorders)。遗传性脑病可以包括但不限于,肾上腺脑白质营养不良、胼胝体发育不全、艾卡尔迪综合征(Aicardi Syndrome)、阿尔佩斯病(Alpers'Disease)、阿尔茨海默病、巴特综合征(Barth Syndrome)、巴藤病(BattenDisease)、CADASIL、小脑变性、费波瑞病(Fabry's Disease)、格斯特曼-施特劳斯纳病(Gerstmann-Straussler-Scheinker Disease)、亨廷顿病以及其他三联体重复障碍、莱氏病(Leigh's Disease)、莱施-奈恩综合征、门克斯病、线粒体肌病以及NINDS空洞脑(Colpocephaly)。这些疾病在小节遗传性脑部障碍(Genetic Brain Disorders)下被进一步描述于国立卫生研究院的网站。
在一些实施例中,该病症可以是瘤形成。在一些实施例中,在该病症是瘤形成的情况下,有待被靶向的基因是列于表A中的那些基因中的任一种(在这种情况下是PTEN等)。在一些实施例中,该病症可以是年龄相关性黄斑变性。在一些实施例中,该病症可以是一种精神分裂症障碍。在一些实施例中,该病症可以是一种三核苷酸重复障碍。在一些实施例中,该病症可以是脆性X综合征。在一些实施例中,该病症可以是一种分泌酶相关障碍。在一些实施例中,该病症可以是一种朊病毒相关障碍。在一些实施例中,该病症可以是ALS。在一些实施例中,该病症可以是一种药物成瘾。在一些实施例中,该病症可以是自闭症。在一些实施例中,该病症可以是阿尔茨海默病。在一些实施例中,该病症可以是炎症。在一些实施例中,该病症可以是帕金森病。
与帕金森病相关的蛋白质的实例包括但不限于α-突触核蛋白、DJ-1、LRRK2、PINK1、帕金蛋白、UCHL1、Synphilin-1以及NURR1。
成瘾相关的蛋白质的实例可以包括例如ABAT。
炎症相关蛋白的实例可以包括例如由Ccr2基因编码的单核细胞趋化蛋白-1(MCP1)、由Ccr5基因编码的C-C趋化因子受体类型5(CCR5)、由Fcgr2b基因编码的IgG受体IIB(FCGR2b,亦称CD32)或由Fcer1g基因编码的FcεR1g(FCER1g)蛋白。
心血管疾病相关蛋白的实例可以包括例如IL1B(白介素1,β)、XDH(黄嘌呤脱氢酶)、TP53(肿瘤蛋白p53)、PTGIS(前列腺素I2(前列腺环素)合酶)、MB(肌红蛋白)、IL4(白介素4)、ANGPT1(血管生成素1)、ABCG8(ATP-结合盒,亚家族G(WHITE),成员8)或CTSK(组织蛋白酶K)。
阿尔茨海默病相关蛋白的实例可以包括例如由VLDLR基因编码的极低密度脂蛋白受体蛋白(VLDLR)、由UBA1基因编码的泛素样改性剂激活酶1(UBA1)或由UBA3基因编码的NEDD8-激活酶E1催化亚基蛋白(UBE1C)。
自闭症谱系障碍相关蛋白的实例可以包括例如由BZRAP1基因编码的苯二氮卓受体(外周)相关蛋白1(BZRAP1)、由AFF2基因编码的AF4/FMR2家族成员2蛋白(AFF2)(亦称MFR2)、由FXR1基因编码的脆性X智力迟钝常染色体同系物1蛋白(FXR1)或由FXR2基因编码的脆性X智力迟钝常染色体同系物2蛋白(FXR2)。
黄斑变性相关蛋白的实例可以包括例如由ABCR基因编码的ATP-结合盒,亚家族A(ABC1)成员4蛋白(ABCA4)、由APOE基因编码的载脂蛋白E蛋白(APOE)或由CCL2基因编码的趋化因子(C-C基序)配体2蛋白(CCL2)。
精神分裂症相关蛋白的实例可以包括NRG1、ErbB4、CPLX1、TPH1、TPH2、NRXN1、GSK3A、BDNF、DISC1、GSK3B及其组合。
涉及肿瘤抑制的蛋白质的实例可以包括例如ATM(共济失调性毛细血管扩张突变的)、ATR(共济失调性毛细血管扩张和Rad3相关的)、EGFR(表皮生长因子受体)、ERBB2(v-erb-b2红白血病病毒癌基因同系物2)、ERBB3(v-erb-b2红白血病病毒癌基因同系物3)、ERBB4(v-erb-b2红白血病病毒癌基因同系物4)、Notch 1、Notch 2、Notch 3或Notch 4。
与分泌酶障碍相关的蛋白质的实例可以包括例如PSENEN(早老素增强子2同系物(秀丽隐杆线虫))、CTSB(组织蛋白酶B)、PSEN1(早老素1)、APP(淀粉样蛋白β(A4)前体蛋白)、APH1B(前咽缺陷1同系物B(秀丽隐杆线虫))、PSEN2(早老素2(阿尔茨海默病4))或BACE1(β-位点APP-切割酶1)。
与肌萎缩性侧索硬化相关的蛋白质的实例可以包括SOD1(超氧化物歧化酶1)、ALS2(肌萎缩性侧索硬化2)、FUS(融合在肉瘤中)、TARDBP(TAR DNA结合蛋白)、VAGFA(血管内皮生长因子A)、VAGFB(血管内皮生长因子B)以及VAGFC(血管内皮生长因子C)及其任何组合。
与朊病毒病相关的蛋白质的实例可以包括SOD1(超氧化物歧化酶1)、ALS2(肌萎缩性侧索硬化2)、FUS(融合在肉瘤中)、TARDBP(TAR DNA结合蛋白)、VAGFA(血管内皮生长因子A)、VAGFB(血管内皮生长因子B)以及VAGFC(血管内皮生长因子C)及其任何组合。
与朊病毒病症中的神经退行性疾病相关的蛋白质的实例可以包括例如A2M(α-2-巨球蛋白)、AATF(凋亡拮抗转录因子)、ACPP(前列腺的酸性磷酸酶)、ACTA2(肌动蛋白α2平滑肌主动脉)、ADAM22(ADAM金属肽酶结构域)、ADORA3(腺苷A3受体)或ADRA1D(α-1D肾上腺素能受体(Alpha-1D adrenergic receptor或Alpha-1D adrenoreceptor))。
与免疫缺陷相关的蛋白质的实例可以包括例如A2M[α-2-巨球蛋白];AANAT[芳烷基胺N-乙酰转移酶];ABCA1[ATP-结合盒,亚家族A(ABC1),成员1];ABCA2[ATP-结合盒,亚家族A(ABC1),成员2];或ABCA3[ATP-结合盒,亚家族A(ABC1),成员3]。
与三核苷酸重复障碍相关的蛋白质的实例包括例如AR(雄激素受体)、FMR1(脆性X智力迟钝1)、HTT(亨廷顿蛋白)或DMPK(强直性肌营养不良蛋白激酶)、FXN(弗氏共济失调蛋白(frataxin))、ATXN2(共济失调蛋白2)。
与神经传递障碍相关的蛋白质的实例包括例如SST(生长抑素)、NOS1(一氧化氮合酶1(神经元的))、ADRA2A(肾上腺素能的,α-2A-,受体)、ADRA2C(肾上腺素能的,α-2C-,受体)、TACR1(速激肽受体1)或HTR2c(5-羟色胺(血清素)受体2C)。
神经发育相关序列的实例包括例如A2BP1[共济失调蛋白2-结合蛋白1]、AADAT[氨基己二酸氨基转移酶],AANAT[芳烷基胺N-乙酰转移酶]、ABAT[4-氨基丁酸氨基转移酶]、ABCA1[ATP-结合盒,亚家族A(ABC1),成员1]或ABCA13[ATP-结合盒,亚家族A(ABC1),成员13]。
可用本发明系统治疗的优选病症的另外的实例可以选自:艾卡迪-古铁雷斯综合征(Aicardi-Goutières Syndrome);亚历山大病;阿伦-赫恩登-达德利综合征(Allan-Herndon-Dudley Syndrome);POLG相关障碍;α-甘露糖苷贮积症(II和III型);阿尔斯特伦综合征( Syndrome);安格曼;综合征;共济失调-毛细血管扩张;神经元蜡样-脂褐质沉积;β-地中海贫血;双侧视神经萎缩和1型(婴儿)视神经萎缩;视网膜母细胞瘤(双侧的);卡纳万病(Canavan Disease);脑-眼-面-骨骼综合征(CerebrooculofacioskeletalSyndrome)1[COFS1];脑腱黄瘤病;科尼利亚迪兰吉综合征(Cornelia de LangeSyndrome);MAPT相关障碍;遗传性朊病毒病;德拉韦综合征(Dravet Syndrome);早期发病家族性阿尔茨海默病;弗里德赖希共济失调[FRDA];多发性畸形;岩藻糖苷贮积症;福山(Fukuyama)先天性肌营养不良;半乳糖唾液酸贮积症;戈谢病(Gaucher Disease);有机酸血症;噬血细胞淋巴组织细胞增生症;早衰症;粘脂贮积症II;婴儿游离唾液酸贮积病;PLA2G6相关神经变性;耶韦尔和朗格-尼尔森综合征(Jervell and Lange-NielsenSyndrome);结合性大疱性表皮松解;亨廷顿病;克拉伯病(Krabbe Disease)(婴儿的);线粒体DNA相关雷吉综合征(Mitochondrial DNA-Associated Leigh Syndrome)和NARP;莱施-奈恩综合征;LIS1相关无脑回;洛氏综合征(Lowe Syndrome);槭糖尿病;MECP2复制综合征;ATP7A相关铜转运障碍;LAMA2相关肌营养不良;芳基硫酸酯酶A缺乏;I、II或III型粘多糖贮积症;过氧化物酶体生物发生障碍,齐薇格谱系综合征(Zellweger Syndrome Spectrum);伴随脑铁累积障碍的神经变性;酸性鞘磷脂酶缺乏;C型尼曼-皮克病;甘氨酸脑病;ARX相关障碍;尿素循环障碍;COL1A1/2相关成骨不全;线粒体DNA缺失综合征;PLP1相关障碍;佩里综合征(Perry Syndrome);费伦-麦克德米德综合征(Phelan-McDermid Syndrome);II型糖原贮积症(蓬佩病(Pompe Disease))(婴儿的);MAPT相关障碍;MECP2相关障碍;1型肢近端型点状软骨发育不全(Rhizomelic Chondrodysplasia Punctata Type 1);罗伯茨综合征(Roberts Syndrome);桑德霍夫病(Sandhoff Disease);辛德勒病(Schindler Disease)-1型;腺苷脱氨酶缺乏;史-伦-奥三氏综合征;脊髓性肌萎缩;婴儿发病脊髓小脑性共济失调;己糖胺酶A缺乏;1型致死性发育不良;胶原VI型相关障碍;I型乌谢尔综合征(UsherSyndrome);先天性肌营养不良;沃尔夫-赫奇霍恩综合征(Wolf-Hirschhorn Syndrome);溶酶体酸脂酶缺乏;以及着色性干皮病。
如将是显而易见的,设想的是可以使用本发明系统靶向任何感兴趣的多核苷酸序列。使用本发明系统进行治疗可能是有用的病症或疾病的一些实例被包括在上表中并且当前与那些病症相关的基因的实例也被提供于此。然而,示例的基因并不是穷尽性的。
实例
以下实例出于说明本发明的不同实施例的目的而给出并且并不意在以任何方式限制本发明。本发明实例连同在此描述的方法目前代表优选的实施例,是示例性的,并且并不旨在限制本发明的范围。涵盖在如由权利要求书的范围所定义的本发明的精神内的在此的变化以及其他用途是本领域普通技术人员可以想到的。
实例1:真核细胞的细胞核中的CRISPR复合物活性
一个示例性II型CRISPR系统是来自化脓链球菌SF370的II型CRISPR座位,该座位包含四个基因Cas9、Cas1、Cas2和Csn1的聚簇以及两个非编码RNA元件tracrRNA和由非重复序列的短段(间隔子,每个约30bp)间隔开的重复序列(同向重复)的特征性阵列。在此系统中,以四个连续步骤产生靶向的DNA双链断裂(DSB)(图2A)。第一步,从CRISPR座位转录两个非编码RNA前-crRNA阵列和tracrRNA。第二步,将tracrRNA杂交到前-crRNA的同向重复上,然后将其加工成含有单独的间隔子序列的成熟crRNA。第三步,该成熟crRNA:tracrRNA复合物经由在crRNA的间隔子区与原型间隔子DNA之间形成异源双链体而将Cas9指向由原型间隔子和对应的PAM组成的DNA靶标。最后,Cas9介导PAM上游的靶DNA的切割,以在原型间隔子内产生一个DSB(图2A)。此实例描述了一种用于将此RNA可编程的核酸酶系统适配成指导真核细胞的细胞核中的CRISPR复合物活性的示例性过程。
细胞培养和转染
在37℃下,伴随5%CO2孵育,将人类胚肾(HEK)细胞系HEK 293FT(生命技术公司)维持在补充有10%胎牛血清(海可隆公司(HyClone))、2mM GlutaMAX(生命技术公司)、100U/mL青霉素及100μg/mL链霉素的杜尔贝科改良伊格尔培养基(Dulbecco’s modifiedEagle’s Medium)(DMEM)中。在37℃下,在5%CO2下,用补充有5%胎牛血清(海可隆公司)、2mM GlutaMAX(生命技术公司)、100U/mL青霉素及100μg/mL链霉素的DMEM维持小鼠neuro2A(N2A)细胞系(ATCC)。
在转染前一天,将HEK 293FT或N2A细胞以200,000个细胞/孔的密度接种到24孔板(康宁公司(Corning))中。遵循制造商推荐的方案,使用Lipofectamine 2000(生命技术公司)转染细胞。对于24孔板的每个孔,使用总共800ng的质粒。
基因组修饰的Surveyor测定和测序分析
如上所述,用质粒DNA转染HEK 293FT或N2A细胞。转染后,在基因组DNA提取之前,将细胞在37℃下孵育72小时。遵循制造商的方案,使用QuickExtract DNA提取试剂盒(Epicentre)提取基因组DNA。简言之,将细胞重悬于QuickExtract溶液中并在65℃下孵育15分钟并且在98℃下孵育10分钟。将提取的基因组DNA立即加工或存储在-20℃下。
对于每个基因而言,PCR扩增围绕CRISPR靶位点的基因组区域,并且遵循制造商的方案,使用QiaQuick Spin柱(凯杰公司(Qiagen))纯化产物。将总共400ng的纯化的PCR产物与2μl 10X Taq聚合酶PCR缓冲液(Enzymatics公司)和超纯水混合,至终体积为20μl,并使其经受重退火过程,以使得可以形成异源双链体:95℃持续10min,以-2℃/s 95℃降至85℃,以-0.25℃/s 85℃降至25℃,并且在25℃保持1分钟。重退火后,遵循制造商推荐的方案,将产物用Surveyor核酸酶和Surveyor增强子S(转基因组学公司(Transgenomics))处理,并且在4%-20%Novex TBE聚丙烯酰胺凝胶(生命技术公司)上进行分析。将凝胶用SYBRGold DNA染色剂(生命技术公司)染色30分钟并用Gel Doc凝胶成像系统(伯乐公司(Bio-rad))成像。定量是基于作为切割的DNA的部分的量度的相对条带强度。图7提供了此Surveyor测定的一个示意图。
用于检测同源重组的限制性片段长度多态性测定。
将HEK 293FT和N2A细胞用质粒DNA转染,并且如上所述,在基因组DNA提取之前,将其在37℃下孵育72小时。使用引物在同源重组(HR)模板的同源臂外PCR扩增靶基因组区域。将PCR产物分离在1%琼脂糖凝胶上并用MinElute GelExtraction试剂盒(凯杰公司)提取。将纯化产物用HindIII(费尔芒斯公司(Fermentas))消化并在6%Novex TBE聚丙烯酰胺凝胶(生命技术公司)上进行分析。
RNA二级结构预测和分析
使用质心结构预测算法,使用由维也纳大(University of Vienna)的理论化学研究所(Institute for Theoretical Chemistry)研发的在线网络服务器RNAfold进行RNA二级结构预测(参见例如A.R.格鲁伯(A.R.Gruber)等人,2008,《细胞》(Cell)106(1):23-24;以及PA卡尔(PA Carr)和GM丘奇(GM Church),2009,《自然生物技术》(NatureBiotechnology)27(12):1151-62)。
RNA纯化
将HEK 293FT细胞如上所述地维持和转染。通过胰酶消化收获细胞,随后在磷酸盐缓冲生理盐水(PBS)中洗涤。遵循制造商的方案,用TRI试剂(西格玛公司)提取总细胞RNA。使用Naonodrop(赛默飞世尔科技公司(Thermo Scientific))对提取的总RNA进行定量并标准化至同一浓度。
哺乳动物细胞中的crRNA和tracrRNA表达的Northern印迹分析
将RNA与等体积的2X加样缓冲液(Ambion)混合,加热至95℃持续5min,在冰上冷冻1min,并且然后在将凝胶预跑胶至少30分钟后,加样到8%变性聚丙烯酰胺凝胶(SequaGel,国家诊断(National Diagnostics))上。将样品以40W极限电泳1.5小时。之后,在室温下,将RNA转移到半干转移设备(伯乐公司(Bio-rad))中的300mA的Hybond N+膜(通用电气医疗公司(GE Healthcare))上,持续1.5小时。使用Stratagene UV Crosslinker theStratalinker(Stratagene)上的自动交联按钮将RNA交联到该膜上。在42℃下,伴随旋转,将该膜在ULTRAhyb-Oligo杂交缓冲液(Ambion)中预杂交30min,并且然后添加探针并杂交过夜。探针订购自IDT并将其用具有T4多核苷酸激酶(新英格兰生物实验室(New EnglandBiolabs)))的[γ-32P]ATP(珀金埃尔默公司)标记。将该膜用预加温的(42℃)2xSSC、0.5%SDS洗涤一次,持续1min,随后在42℃下洗涤两次,持续30分钟。将该膜在室温下向荧光屏暴露一小时或过夜并且然后用感光成像仪(Typhoon)扫描。
细菌CRISPR系统构建和评价
从具有用于吉布森拼接(Gibson Assembly)的侧翼同源臂的化脓链球菌SF370基因组DNA PCR扩增CRISPR座位元件(包括tracrRNA、Cas9和前导子)。将两个BsaI类型IIS位点引入在两个同向重复之间,以促进间隔子的方便插入(图8)。使用Gibson AssemblyMaster Mix(NEB)将PCR产物克隆进EcoRV-消化的pACYC184中,在tet启动子的下游。省略其他内源CRISPR系统元件,Csn2的最后50bp除外。将编码具有互补突出端的间隔子的寡聚物(整合DNA技术公司(Integrated DNA Technology))克隆进BsaI-消化的载体pDC000(NEB)中并且然后用T7连接酶(Enzymatics公司)连接,以产生pCRISPR质粒。包含具有在哺乳动物细胞中的PAM表达的间隔子的激发质粒(图6A中所示出的表达构建体,其中功能性是如显示于图6B中的Surveyor测定的结果所确定的)。将转录起始位点标为+1,并且还指示了转录终止子和通过Northern印迹探测的序列。还通过Northern印迹确认了加工的tracrRNA的表达。图6C显示了提取自293FT细胞的总RNA的Northern印迹分析的结果,用携带长或短tracrRNA以及SpCas9和DR-EMX1(1)-DR的U6表达构建体转染这些细胞。左图和右图分别是来自用或没用SpRNA酶III转染的293FT细胞。U6指示用靶向人类U6 snRNA的探针进行印迹的加样对照。短tracrRNA表达构建体的转染产生丰富水平的加工形式的tracrRNA(~75bp)。在Northern印迹上检测极低量的长tracrRNA。
为了促进转录精确起始,选择基于RNA聚合酶III的U6启动子,以驱动tracrRNA的表达(图2C)。类似地,研发基于U6启动子的构建体,以表达由单个间隔子组成的前-crRNA阵列,该单个间隔子侧翼为两个同向重复(DR,还被术语“tracr配对序列”所涵盖;图2C)。将起始间隔子设计成靶向人EMX1座位(图2C)中的33-碱基对(bp)靶位点(满足Cas9的NGG识别基序的30-bp原型间隔子加3-bp CRISPR基序(PAM)序列),该座位是大脑皮层的发育中的一个关键基因。
为了测试CRISPR系统(SpCas9、SpRNA酶III、tracrRNA以及前-crRNA)在哺乳动物细胞中的异源表达是否可以实现哺乳动物染色体的靶向切割,用CRISPR组分的组合转染HEK 293FT细胞。由于哺乳动物细胞核中的DSB通过导致形成indel的非同源末端连接(NHEJ)途径而部分地修复,使用Surveyor测定检测靶EMX1座位处的潜在的切割活性(图7)(参见例如格斯钦(Guschin)等人,2010,《分子生物学方法》(Methods Mol Biol)649:247)。所有四种CRISPR组分的共转染能够在原型间隔子中诱导高达5.0%切割(参见图2D)。所有CRISPR组分(减去SpRNA酶III)的共转染在原型间隔子中也诱导高达4.7%indel,表明可能存在能够辅助crRNA成熟的内源性哺乳动物RNA酶,如例如相关的切丁酶(Dicer)和Drosha酶。除去剩余的三种组分中的任何组分消除CRISPR系统的基因组切割活性(图2D)。包含已证实切割活性的靶座位的扩增子的桑格测序:在43个测序的克隆中,发现5个突变的等位基因(11.6%)。使用多种指导序列的类似实验产生了高达29%的indel百分比(参见图3-6、10和11)。这些结果定义了一种在哺乳动物细胞中的有效的CRISPR介导的基因组修饰的三组分系统。为了优化切割效率,申请人还测试了tracrRNA的不同亚型是否影响切割效率并且发现,在实例系统中,仅仅短(89-bp)转录本形式能够介导人类EMX1基因组座位的切割(图6B)。
图12提供了哺乳动物细胞中的crRNA加工的另外的Northern印迹分析。图12A示出了一个示意图,显示了侧翼为两个同向重复(DR-EMX1(1)-DR)的单个间隔子的表达载体。靶向人EMX1座位原型间隔子1(参见图6)的30bp间隔子和同向重复序列显示于图12A下方的序列中。直线指示其反向互补系列用于产生供EMX1(1)crRNA检测用的Northern印迹探针的区域。图12B显示了提取自293FT细胞的总RNA的Northern印迹分析,用携带DR-EMX1(1)-DR的U6表达构建体转染这些细胞。左图和右图分别是来自用或没用SpRNA酶III转染的293FT细胞。DR-EMX1(1)-DR仅在存在SpCas9和短tracrRNA的情况下被加工为成熟crRNA并且不依赖于SpRNA酶III的存在。检测的来自转染的293FT总RNA的成熟crRNA是约33bp并且比来自化脓链球菌的39-42bp成熟crRNA短。这些结果证明,CRISPR系统可以被移植进真核细胞中并被重新编程,以促进内源哺乳动物靶多核苷酸的切割。
图2示出了描述于此实例中的细菌CRISPR系统。图2A示出了一个示意图,显示了来自化脓链球菌SF370的CRISPR座位和通过此系统CRISPR介导的DNA切割的建议机制。加工自同向重复-间隔子阵列的成熟crRNA指导Cas9到以下基因组靶标,这些靶标由互补原型间隔子和原型间隔子相邻基序(PAM)组成。靶标-间隔子碱基配对后,Cas9介导靶DNA中的双链断裂。图2B示出了具有使得导入到哺乳动物细胞核中成为可能的核定位信号(NLS)的化脓链球菌Cas9(SpCas9)和RNA酶III(SpRNA酶III)的工程化。图2C示出了由组成型EF1a启动子驱动的SpCas9和SpRNA酶III以及由RNA Pol3启动子U6驱动的tracrRNA和前-crRNA阵列(DR-间隔子-DR)的哺乳动物表达,上述启动子促进转录精确起始和终止。将来自具有令人满意的PAM序列的人EMX1座位的原型间隔子用作前-crRNA阵列中的间隔子。图2D示出了SpCas9介导的次要插入和缺失的Surveyor核酸酶测定。使用和不使用SpRNA酶III、tracrRNA以及携带EMX1-靶间隔子的前-crRNA阵列表达SpCas9。图2E示出了靶座位与EMX1-靶向crRNA之间的碱基配对的一个示意表示以及一个显示了与SpCas9切割位点邻近的微小缺失的示例性色谱图。图2F示出了从43个克隆扩增子的测序分析鉴定出的突变的等位基因,这些扩增子显示出多种微小插入和缺失。破折号指示缺失的碱基,并且非比对或错配碱基指示插入或突变。比例尺=10μm。
为了进一步简化该三组分系统,改编嵌合的crRNA-tracrRNA杂交体设计,其中成熟crRNA(包括一种指导序列)可以经由茎-环而融合到部分tracrRNA上,以模拟天然的crRNA:tracrRNA双链体。为了增加共递送效率,产生双顺反子表达载体,以驱动嵌合的RNA和SpCas9在转染细胞中的共表达。平行地,使用该双顺反子载体表达前-crRNA(DR-指导序列-DR)与SpCas9,以诱导用分开表达的tracrRNA加工为crRNA(比较图11B的顶图和底图)。图8提供了前-crRNA阵列(图8A)或嵌合的crRNA(由图8B中的指导序列插入位点的下游和EF1α启动子的上游的短线表示)与hSpCas9的双顺反子表达载体的示意图,显示了各种元件的位置和指导序列插入点。图8B中的围绕指导序列插入位点的位置的展开的序列还显示了部分DR序列(GTTTTAGAGCTA)(SEQ ID NO:11)和部分tracrRNA序列(TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT)(SEQ ID NO:12)。可以使用退火的寡核苷酸将指导序列插入在BbsI位点之间。寡核苷酸的序列设计显示在图8中的示意图的下方,其中指示了适当的连接适配子。WPRE表示土拨鼠肝炎病毒转录后调节元件。通过靶向与上述相同的EMX1座位而测试嵌合RNA介导的切割效率。使用扩增子的Surveyor测定和桑格测序两者,申请人确认嵌合RNA设计以大约4.7%的修饰率促进人EMX1座位的切割(图3)。
通过设计靶向人EMX1和PVALB以及小鼠Th座位中的多个位点的嵌合RNA,通过靶向人类和小鼠细胞两者中的另外的基因组座位而测试CRISPR介导的切割在真核细胞中的普遍性。图13示出了人类PVALB(图13A)和小鼠Th(图13B)座位中的一些另外的靶向原型间隔子的筛选。提供了各自的最后的外显子内的基因座位位和三个原型间隔子的位置的示意图。加下划线的序列包括30bp的原型间隔子序列和对应于PAM序列的3’端处的3bp。分别在DNA序列的上方和下方指示了有义链和反义链上的原型间隔子。人类PVALB和小鼠Th座位分别达到了6.3%和0.75%的修饰率,证明了CRISPR系统跨多种生物在修饰不同座位方面的广阔适用性(图5)。虽然对于每个座位而言,使用嵌合的构建体仅伴随三分之一的间隔子检测切割,但是当使用共表达的前-crRNA安排时,所有靶序列都被切割,伴随达到27%的有效的indel产生(图6和13)。
图11提供了可以被重新编程为靶向哺乳动物细胞中的多个基因组座位的SpCas9的另外的图解。图11A提供了人EMX1座位的示意图,显示了五个原型间隔子的位置,由加下划线的序列指示。图11B提供了前-crRNA/trcrRNA复合物的示意图,显示了前-crRNA和tracrRNA的同向重复区之间的杂交(顶部),以及嵌合RNA设计的示意图,包括一个20bp指导序列和由部分同向重复组成的tracr配对和tracr序列以及以发夹结构杂交的tracrRNA序列(底部)。比较人EMX1座位中的五个原型间隔子处的Cas9介导的切割的效率的Surveyor测定的结果示于图11C中。使用加工的前-crRNA/tracrRNA复合物(crRNA)或嵌合的RNA(chiRNA)靶向每个原型间隔子。
由于RNA的二级结构对于分子间相互作用可以是决定性的,使用基于最小自由能和玻尔兹曼(Boltzmann)加权结构集合的结构预测算法比较基因组靶向实验中使用的所有指导序列的假定的二级结构(参见例如格鲁贝尔(Gruber)等人,2008,《核酸研究》(NucleicAcids Research),36:W70)。分析揭示到在大多数情况下,在嵌合的crRNA背景下有效的指导序列基本上不含二级结构基序,而无效的指导序列更可能形成可以阻止与靶原型间隔子DNA进行碱基配对的内部二级结构。因此,可能的是,当使用嵌合的crRNA时,间隔子二级结构的变异性可能影响CRISPR介导的干扰的效率。
SpCas9的另外的载体设计显示于图22中,示出了掺入连接到指导寡核苷酸的插入位点的U6启动子和连接到SpCas9编码序列上的Cbh启动子的单个表达载体。显示于图22b中的载体包括一个连接到H1启动子上的tracrRNA编码序列。
在细菌测定中,所有的间隔子均促进有效的CRISPR干扰(图3C)。这些结果表明,可能存在影响哺乳动物细胞中的CRISPR活性效率的另外的因素。
为了研究CRISPR介导的切割的特异性,使用一系列具有单点突变的EMX1-靶向嵌合的crRNA分析指导序列中的单核苷酸突变对哺乳动物基因组中的原型间隔子切割的影响(图3A)。图3B示出了比较当与不同突变的嵌合的RNA配对时Cas9的切割效率的Surveyor核酸酶测定的结果。PAM的5’的高达12-bp的单碱基错配基本上消除了由SpCas9进行的基因组切割,而在离上游位置更远处具有突变的间隔子保留针对原始原型间隔子靶标的活性(图3B)。除PAM之外,SpCas9在间隔子的最后12-bp内具有单碱基特异性。此外,CRISPR能够像一对靶向同一EMX1原型间隔子的TALE核酸酶(TALEN)一样有效地介导基因组切割。图3C提供了一个示意图,显示了靶向EMX1的TALEN的设计并且图3D显示了比较TALEN和Cas9的效率的Surveyor凝胶(n=3)。
已经通过易错NHEJ机制建立了一套用于在哺乳动物细胞中实现CRISPR介导的基因编辑的组分,测试CRISPR刺激同源重组(HR)的能力,同源重组是一种用于使得基因组中的编辑精确的高保真基因修复途径。野生型SpCas9能够介导位点特异的DSB,这些DSB可以通过NHEJ和HR两者进行修复。另外,将SpCas9的RuvC I催化结构域中的天冬氨酸至丙氨酸取代(D10A)工程化,以将核酸酶转化为切口酶(SpCas9n;示于图4A中)(参见例如萨普拉诺萨克斯(Sapranausaks)等人,2011,《核酸研究》(Nucleic Acids Research),39:9275;加索纳斯(Gasiunas)等人,2012,《美国国家科学院院刊》(Proc.Natl.Acad.Sci.USA),109:E2579),这样使得带切口的基因组DNA经历高保真同源定向修复(HDR)。Surveyor测定确认到,SpCas9n在EMX1原型间隔子靶标处不产生indel。如图4B所示,EMX1-靶向嵌合的crRNA与SpCas9的共表达在靶位点中产生indel,而与SpCas9n的共表达不产生indel(n=3)。此外,327个扩增子的测序未检测到由SpCas9n诱导的任何indel。选择相同的座位,以通过用靶向EMX1、hSpCas9或hSpCas9n的嵌合RNA以及用于在原型间隔子附近引入一对限制酶切位点(HindIII和NheI)的HR模板共转染HEK 293FT细胞而测试CRISPR介导的HR。图4C提供了HR策略的示意图,包含重组点的相对位置和引物退火序列(箭头)。SpCas9和SpCas9n的确催化将HR模板整合进EMX1座位中。PCR扩增靶区域,随后用HindIII进行限制酶切消化揭示了对应于期望片段尺寸的切割产物(显示于图4D中的限制性片段长度多态性分析中的箭头),其中SpCas9和SpCas9n介导类似水平的HR效率。申请人使用基因组扩增子的桑格测序进一步证实了HR(图4E)。这些结果证明了CRISPR促进哺乳动物基因组中的靶向基因插入的实用性。鉴于14-bp(来自间隔子的12-bp和来自PAM的2-bp)特异地靶向野生型SpCas9,切口酶的可获得性可以显著减少脱靶修饰的可能性,因为单链断裂不是易错NHEJ途径的底物。
构建模拟具有阵列的间隔子的CRISPR座位的天然构造的表达构建体(图2A),以测试多元序列靶向的可能性。使用编码一对EMX1-和PVALB-靶向间隔子的单CRISPR阵列,在两个座位处都检测到了有效的切割(图4F,显示了crRNA阵列的示意设计和显示出切割的有效修饰的Surveyor印迹两者)。使用针对由119bp间隔开的EMX1内的两个靶标的间隔子通过共存DSB还检测到了较大基因组区的靶向缺失,并且检测到了1.6%缺失效率(182个扩增子中的3个;图4G)。这证明,CRISPR系统可以介导单个基因组中的多元编辑。
实例2:CRISPR系统修饰和替代方案
使用RNA编程序列特异的DNA切割的能力定义了一个新类别的针对多种研究和工业应用的基因组工程化工具。可以进一步改善CRISPR系统的若干方面,以增加CRISPR靶向的效率和通用性。优化的Cas9活性可以依赖于处于比存在于哺乳动物细胞核中的游离Mg2+高的水平的游离Mg2+的可获得性(参见例如季聂克(Jinek)等人,2012,《科学》(Science),337:816),并且对恰好位于原型间隔子的下游的NGG基序的偏好限制靶向人类基因组中的平均每12-bp的能力(图9,评价了人类染色体序列的正链和负链两者)。这些约束中的一些可以通过探索CRISPR座位跨微生物宏基因组的多样性而克服(参见例如马卡洛娃(Makarova)等人,2011,《自然微生物学综述》(Nat Rev Microbiol),9:467)。可以通过类似于实例1中描述的方法将其他CRISPR座位移植进哺乳动物细胞环境中。例如,图10示出了来自嗜热链球菌LMD-9的CRISPR 1的用于在哺乳动物细胞中进行异源表达的II型CRISPR系统的适应,以实现CRISPR介导的基因组编辑。图10A提供了来自嗜热链球菌LMD-9的CRISPR 1的示意图。图10B示出了嗜热链球菌CRISPR系统的表达系统的设计。使用组成型EF1α启动子表达人类密码子优化的hStCas9。使用促进转录精确起始的U6启动子表达tracrRNA和crRNA的成熟版本。示出了来自成熟crRNA和tracrRNA的序列。crRNA序列中的由小写字母“a”指示的单个碱基用于除去作为RNA polIII转录终止子的polyU序列。图10C提供了一个示意图,显示了靶向人EMX1座位的指导序列。图10D显示了使用Surveyor测定的靶座位中的hStCas9介导的切割的结果。RNA指导间隔子1和2分别诱导了14%和6.4%。在这两个原型间隔子位点处跨生物复制物的切割活性的统计分析还提供于图5中的。图14提供了嗜热链球菌CRISPR系统的另外的原型间隔子和在人EMX1座位中的对应的PAM序列靶标示意图。两个原型间隔子序列被凸显并且通过相对于对应的凸显序列为3’加下划线而指示满足NNAGAAW基序的对应的PAM序列。两个原型间隔子均靶向反义链。
实例3:样品靶序列选择算法
设计一个软件程序,以基于指定的CRISPR酶的希望的指导序列长度和CRISPR基序序列(PAM),在输入DNA序列的两条链上鉴定候选CRISPR靶序列。例如,可以通过在输入序列和该输入序列的反向互补体两者上检索5’-Nx-NGG-3’而鉴定来自化脓链球菌的Cas9的具有PAM序列NGG的靶位点。同样地,可以通过在输入序列和该输入序列的反向互补体两者上检索5’-Nx-NNAGAAW-3’而鉴定化脓链球菌CRISPR1的Cas9的具有PAM序列NNAGAAW的靶位点。同样地,可以通过在输入序列和该输入序列的反向互补体两者上检索5’-Nx-NGGNG-3’而鉴定化脓链球菌CRISPR3的Cas9的具有PAM序列NGGNG的靶位点。可以通过程序固定或由使用者指定Nx中的值“x”,如20。
由于在DNA靶位点的基因组中出现多次可以导致非特异的基因组编辑,因此在鉴定所有潜在的位点后,基于序列在相关参比基因组中出现的次数,该程序将其滤出。对于其序列特异性是通过‘种子(seed)’序列(如来自PAM序列的11-12bp 5’,包括PAM序列本身)确定的那些CRISPR酶,过滤步骤可以基于该种子序列。因此,为了避免另外的基因组座位处的编辑,基于种子:PAM序列在相关基因组中出现的次数过滤结果。可以允许使用者选择种子序列的长度。处于通过过滤器的目的,还可以允许使用者指定种子:PAM序列在基因组中出现的次数。默认筛选独特序列。通过改变种子序列的长度和该序列在基因组中出现的次数两者而改变过滤水平。该程序可以另外或可替代地通过提供鉴定的一个或多个靶序列的反向互补体而提供与报告的一个或多个靶序列互补的指导序列的序列。一些靶位点在人类基因组中的示例性可视化提供于图18中。
用于优化序列选择的方法和算法的另外的细节可以发现于通过引用结合在此的美国申请序列号61/064,798(代理人案号44790.11.2022;博德参考号BI-2012/084)中。
实例4:多种嵌合的crRNA-tracrRNA杂交体的评价
此实例描述了针对具有掺入不同长度的野生型tracrRNA序列的tracr序列的嵌合的RNA(chiRNAs;在单个转录本中包括指导序列、tracr配对序列以及tracr序列)而获得的结果。图16a示出了嵌合RNA和Cas9的双顺反子表达载体的示意图。Cas9由CBh启动子驱动并且嵌合的RNA由U6启动子驱动。嵌合的指导RNA由接合到tracr序列的20bp指导序列(N)组成(从下链的第一个“U”到该转录本的结尾),其在如指示的各点被截短。指导和tracr序列由tracr-配对序列GUUUUAGAGCUA(SEQ ID NO:13),随后是环序列GAAA隔开。人EMX1和PVALB座位处的Cas9介导的indel的SURVEYOR测定的结果分别示于图16b和16c中。箭头指示预期的SURVEYOR片段。ChiRNA由其“+n”标志指示,并且crRNA是指指导和tracr序列被表达为分开的转录本的杂交体RNA。一式三份地进行的这些结果的定量示于图17a和17b的直方图中,分别对应于图16b和16c(“N.D.”指示未检测到indel)。原型间隔子ID及其对应的基因组靶标、原型间隔子、PAM序列以及链位置提供于表D中。将指导序列设计成与整个原型间隔子序列互补(在杂交体系统中的分开的转录本的情况下),或仅为部分加下划线(在嵌合的RNA的情况下)。
表D:
用于优化指导序列的另外的细节可以发现于通过引用结合在此的美国申请序列号61/836,127(代理人案号44790.08.2022;博德参考号BI-2013/004G)中。
初始,靶向人HEK 293FT细胞中的EMX1座位内的三个位点。使用SURVEYOR核酸酶测定评估每种chiRNA的基因组修饰效率,该测定检测由DNA双链断裂(DSB)及其通过非同源末端连接(NHEJ)DNA损伤修复途径进行的随后修复造成的突变。被指定为chiRNA(+n)的构建体指示,野生型tracrRNA的直达第+n个核苷酸被包括在嵌合的RNA构建体中,其中48、54、67和85的值用于n。嵌合的RNA在所有三个EMX1靶位点处都包含长片段的野生型tracrRNA(chiRNA(+67)和chiRNA(+85))介导的DNA切割,其中chiRNA(+85)尤其展示出比以分开的转录本形式表达指导序列和tracr序列的对应的crRNA/tracrRNA杂交体显著更高水平的DNA切割(图16b和17a)。还使用chiRNA靶向PVALB座位中的两个位点,其不产生使用杂交体系统(作为分开的转录本而表达的指导序列和tracr序列)的可检测的切割。chiRNA(+67)和chiRNA(+85)能够在两个PVALB原型间隔子处介导显著的切割(图16c和17b)。
对于EMX1和PVALB座位中的所有五个靶标,伴随增加的tracr序列长度观察到一致增加的基因组修饰效率。不希望受任何理论的束缚,由tracrRNA的3’端形成的二级结构可以在增强CRISPR复合物形成率方面发挥一定作用。
实例5:Cas9多样性
CRISPR-Cas系统是一种由跨细菌和古生菌的相异物种利用的针对侵入型外源DNA的获得性免疫机制。II型CRISPR-Cas9系统由一套编码负责将外源DNA“捕获(acquisition)”进CRISPR座位中的蛋白质的基因以及一套编码负责“执行(execution)”DNA切割机制的蛋白质的基因组成;这些包括DNA核酸酶(Cas9)、非编码反式激活cr-RNA(tracrRNA)以及侧翼为同向重复的外源DNA衍生的间隔子阵列(crRNA)。当被Cas9成熟后,tracRNA和crRNA双链体指导Cas9核酸酶到由间隔子指导序列规定的靶DNA序列,并且在靶DNA中介导短序列基序附近的DNA中的双链断裂,该导短序列基序为切割所需并且对于每种CRISPR-Cas系统而言是特异的。II型CRISPR-Cas系统发现在整个细菌界中并且在用于靶标切割的Cas9蛋白序列和尺寸、tracrRNA和crRNA同向重复序列、这些元件的基因组组织以及基序要求方面高度相异。一个物种可以具有多种相异的CRISPR-Cas系统。
申请人评价了来自基于与已知Cas9的序列同源性和与已知亚结构域直向同源的结构鉴定的细菌种类的207个假定的Cas9,这些亚结构域包括HNH内切核酸酶结构域和RuvC内切核酸酶结构域[信息来自尤金库宁(Eugene Koonin)和基拉马卡洛娃(KiraMakarova)]。基于此套的蛋白质序列保守的系统发生分析揭示了五个Cas9家族,包括三组大的Cas9(~1400个氨基酸)和两组小的Cas9(~1100个氨基酸)(参见图19和20A-F)。
Cas9和Cas9酶转化为切口酶或DNA结合蛋白的突变及其具有的改变的功能性的用途的另外的细节可以发现于通过引用结合在此的美国申请序列号61/836,101和61/835,936(代理人案号分别为44790.09.2022和4790.07.2022并且博德参考号分别为BI-2013/004E和BI-2013/004F)中。
实例6:Cas9直向同源物
申请人分析了Cas9直向同源物,以鉴定相关的PAM序列和对应的嵌合的指导RNA。具有一套展开的PAM提供了跨基因组的更广泛的靶向并且还显著增加独特靶位点的数目并且提供了在基因组中鉴定具有增加水平的特异性的新颖的Cas9的潜力。
可以通过测试每种Cas9容忍指导RNA及其DNA靶标之间的错配的能力评价Cas9直向同源物的特异性。例如,已经通过测试指导RNA中的突变对切割效率的影响表征了SpCas9的特异性。用指导序列与靶DNA之间的单个或多个错配建立指导RNA文库。基于这些发现,可以基于以下准则选择SpCas9的靶位点:
为了最大化SpCas9编辑具体基因的特异性,应该在感兴趣的座位内选择一个靶位点,这样使得潜在的‘脱靶’基因组序列遵守以下四个约束:首先,它们的后面不应该是具有的5’-NGG或NAG序列的PAM。其次,它们与靶序列的整体序列相似性应该是最小化的。第三,最大数目的错配应该位于脱靶位点的PAM-近侧区内。最后,最大数目的错配应该是连续的或由少于四个碱基隔开。
可以使用类似的方法评价其他Cas9直向同源物的特异性和建立在靶标种类的基因组内选择特定靶位点的标准。如前所述,基于此套的蛋白质序列保守的系统发生分析揭示了五个Cas9家族,包括三组大的Cas9(~1400个氨基酸)和两组小的Cas9(~1100个氨基酸)(参见图19和20A-F)。Cas直向同源物的另外的细节可以发现于通过引用结合在此的美国申请序列号61/836,101和61/835,936(代理人案号分别为44790.09.2022和4790.07.2022并且博德参考号分别为BI-2013/004E和BI-2013/004F)中。
实例7:使用Cas9靶向并操纵植物基因而对植物(微藻)进行工程化
递送Cas9的方法
方法1:申请人使用在组成型启动子(如Hsp70A-Rbc S2或β2-微管蛋白)的控制之下表达Cas9的载体递送Cas9和指导RNA。
方法2:申请人使用在组成型启动子(如Hsp70A-Rbc S2或β2-微管蛋白)的控制之下表达Cas9和T7聚合酶的载体递送Cas9和T7聚合酶。将使用包含驱动指导RNA的T7启动子的载体递送该指导RNA。
方法3:申请人将Cas9 mRNA和体外转录的指导RNA递送到藻类细胞中。RNA可以在体外转录。Cas9 mRNA将由Cas9的编码区以及来自的Cop1的3’UTR组成,以保证Cas9 mRNA的稳定。
用于同源重组,申请人提供了一个另外的同源定向修复模板。
在位于Cop1的3’UTR之后的β-2微管蛋白启动子的控制之下驱动Cas9的表达的盒的序列。
TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT(SEQ ID NO:19)
在位于Cop1的3’UTR之后的β-2微管蛋白启动子的控制之下驱动T7聚合酶的表达的盒的序列:
TCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACatgcctaagaagaagaggaaggttaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgaccattacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctcgcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgacaaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtacgattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgcgttcgcgtaaGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT(SEQ IDNO:20)
由T7启动子驱动的指导RNA的序列(T7启动子,N表示靶向序列):
gaaatTAATACGACTCACTATANNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt(SEQ ID NO:21)
基因递送:
来自衣藻资源中心(Chlamydomonas Resource Center)的莱茵衣藻株CC-124和CC-125将被用于电穿孔。电穿孔方案遵循来自GeneArt Chlamydomonas Engineering试剂盒的标准推荐方案。
而且,申请人产生组成性地表达Cas9的莱茵衣藻系。这可以通过使用pChlamy1(使用PvuI线性化)并选择潮霉素抗性菌落而完成。以下是包含Cas9的pChlamy1的序列。以此方式实现基因敲除,简单地需要递送指导RNA的RNA。对于同源重组,申请人递送了指导RNA以及线性化同源重组模板。
pChlamy1-Cas9:
TGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGTTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGTCGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATGAGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGCTGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGAGCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAGCTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCACTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGCAGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACTTTTTTACAAGAGAAGTCACTCAACATCTTAAAATGGCCAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGGAGATTCGAGGTACCATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCTAACATATGATTCGAATGTCTTTCTTGCGCTATGACACTTCCAGCAAAAGGTAGGGCGGGCTGCGAGACGGCTTCCCGGCGCTGCATGCAACACCGATGATGCTTCGACCCCCCGAAGCTCCTTCGGGGCTGCATGGGCGCTCCGATGCCGCTCCAGGGCGAGCGCTGTTTAAATAGCCAGGCCCCCGATTGCAAAGACATTATAGCGAGCTACCAAAGCCATATTCAAACACCTAGATCACTACCACTTCTACACAGGCCACTCGAGCTTGTGATCGCACTCCGCTAAGGGGGCGCCTCTTCCTCTTCGTTTCAGTCACAACCCGCAAACATGACACAAGAATCCCTGTTACTTCTCGACCGTATTGATTCGGATGATTCCTACGCGAGCCTGCGGAACGACCAGGAATTCTGGGAGGTGAGTCGACGAGCAAGCCCGGCGGATCAGGCAGCGTGCTTGCAGATTTGACTTGCAACGCCCGCATTGTGTCGACGAAGGCTTTTGGCTCCTCTGTCGCTGTCTCAAGCAGCATCTAACCCTGCGTCGCCGTTTCCATTTGCAGCCGCTGGCCCGCCGAGCCCTGGAGGAGCTCGGGCTGCCGGTGCCGCCGGTGCTGCGGGTGCCCGGCGAGAGCACCAACCCCGTACTGGTCGGCGAGCCCGGCCCGGTGATCAAGCTGTTCGGCGAGCACTGGTGCGGTCCGGAGAGCCTCGCGTCGGAGTCGGAGGCGTACGCGGTCCTGGCGGACGCCCCGGTGCCGGTGCCCCGCCTCCTCGGCCGCGGCGAGCTGCGGCCCGGCACCGGAGCCTGGCCGTGGCCCTACCTGGTGATGAGCCGGATGACCGGCACCACCTGGCGGTCCGCGATGGACGGCACGACCGACCGGAACGCGCTGCTCGCCCTGGCCCGCGAACTCGGCCGGGTGCTCGGCCGGCTGCACAGGGTGCCGCTGACCGGGAACACCGTGCTCACCCCCCATTCCGAGGTCTTCCCGGAACTGCTGCGGGAACGCCGCGCGGCGACCGTCGAGGACCACCGCGGGTGGGGCTACCTCTCGCCCCGGCTGCTGGACCGCCTGGAGGACTGGCTGCCGGACGTGGACACGCTGCTGGCCGGCCGCGAACCCCGGTTCGTCCACGGCGACCTGCACGGGACCAACATCTTCGTGGACCTGGCCGCGACCGAGGTCACCGGGATCGTCGACTTCACCGACGTCTATGCGGGAGACTCCCGCTACAGCCTGGTGCAACTGCATCTCAACGCCTTCCGGGGCGACCGCGAGATCCTGGCCGCGCTGCTCGACGGGGCGCAGTGGAAGCGGACCGAGGACTTCGCCCGCGAACTGCTCGCCTTCACCTTCCTGCACGACTTCGAGGTGTTCGAGGAGACCCCGCTGGATCTCTCCGGCTTCACCGATCCGGAGGAACTGGCGCAGTTCCTCTGGGGGCCGCCGGACACCGCCCCCGGCGCCTGATAAGGATCCGGCAAGACTGGCCCCGCTTGGCAACGCAACAGTGAGCCCCTCCCTAGTGTGTTTGGGGATGTGACTATGTATTCGTGTGTTGGCCAACGGGTCAACCCGAACAGATTGATACCCGCCTTGGCATTTCCTGTCAGAATGTAACGTCAGTTGATGGTACT(SEQ ID NO:22)。
对于所有修饰的莱茵衣藻细胞而言,申请人使用了PCR、SURVEYOR核酸酶测定和DNA测序,以验证成功的修饰。
虽然已经在此示出并描述了本发明的优选实施例,但是对本领域的普通技术人员而言应该显而易见是这样的实施例仅以举例方式提供。在不偏离本发明的情况下众多变化、改变和取代现在将被本领域的普通技术人员想到。应该理解的是,在实践本发明中可以采用在此描述的本发明的实施例的不同替代方案。旨在按照以下权利要求限定本发明的范围,并且在这些权利要求范围内的方法和结构及其等同物也包括在本发明的范围内。
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Claims (27)
1.一种改变一种或多种基因产物的表达的方法,该方法包括向包含并表达编码该一种或多种基因产物的DNA分子的细胞中引入一种工程化的非天然存在的CRISPR-Cas系统,该系统包括一种Cas蛋白和一种或多种指导RNA,该一种或多种指导RNA靶向这些DNA分子,由此该一种或多种指导RNA靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的基因组座位,由此该一种或多种基因产物的表达被改变;并且,其中该Cas蛋白和该指导RNA并不共同天然存在。
2.一种改变一种或多种基因产物的表达的方法,该方法包括向包含并表达编码该一种或多种基因产物的DNA分子的细胞中引入一种工程化的非天然存在的载体系统,该载体系统包括一种或多种载体,该一种或多种载体包括:
a)一种第一调节元件,该第一调节元件可操作地连接到一种或多种CRISPR-Cas系统指导RNA上,该一种或多种CRISPR-Cas系统指导RNA与编码该一种或多种基因产物的DNA分子的基因组座位中的靶序列杂交,
b)一种第二调节元件,该第二调节元件可操作地连接至一种Cas蛋白,
其中组分(a)和(b)位于该系统的相同或不同载体上,
由此这些指导RNA靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的基因组座位,由此该一种或多种基因产物的表达被改变;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。
3.如权利要求1或权利要求2所述的方法,其中两种或更多种基因产物的表达被改变。
4.如任何以上权利要求所述的方法,其中该系统的载体或Cas蛋白进一步包括一个或多个NLS。
5.如任何以上权利要求所述的方法,其中这些指导RNA包括一种融合到tracr序列上的指导序列。
6.如任何以上权利要求所述的方法,其中该细胞是一种真核细胞。
7.如权利要求6所述的方法,其中该真核细胞是一种哺乳动物细胞。
8.如权利要求7所述的方法,其中该哺乳动物细胞是一种人类细胞。
9.如任何以上权利要求所述的方法,其中该Cas蛋白是一种Cas9蛋白。
10.如任何以上权利要求所述的方法,其中该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。
11.如任何以上权利要求所述的方法,其中该基因产物的表达被降低。
12.如任何以上权利要求所述的方法,其中该基因产物是一种蛋白质。
13.如任何以上权利要求所述的方法,其中引入该细胞中是经由一种递送系统,该递送系统包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
14.一种工程化的非天然存在的载体系统,该载体系统包括一种或多种载体,该一种或多种载体包括:
a)一种第一调节元件,该第一调节元件可操作地连接到一种或多种CRISPR-Cas系统指导RNA上,该一种或多种CRISPR-Cas系统指导RNA与编码一种或多种基因产物的DNA分子的基因组座位中的靶序列杂交,
b)一种第二调节元件,该第二调节元件可操作地连接至一种Cas蛋白,
其中组分(a)和(b)位于该系统的相同或不同载体上,
由此这些指导RNA在细胞中靶向编码该一种或多种基因产物的DNA分子的基因组座位并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的基因组座位,由此该一种或多种基因产物的表达被改变;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。
15.一种工程化的、可编程的、非天然存在的CRISPR-Cas系统,该系统包括一种Cas蛋白以及一种或多种指导RNA,该一种或多种指导RNA在细胞中靶向编码一种或多种基因产物的DNA分子的基因组座位,并且该Cas蛋白切割编码该一种或多种基因产物的DNA分子的基因组座位,由此该一种或多种基因产物的表达被改变;并且,其中该Cas蛋白和这些指导RNA并不共同天然存在。
16.如权利要求14或15所述的系统,其中两种或更多种基因产物的表达被改变。
17.如权利要求14至16中任一项所述的系统,其中该系统进一步包括一个或多个NLS。
18.如权利要求14至17中任一项所述的系统,其中这些指导RNA包括一种融合到tracr序列上的指导序列。
19.如权利要求14至18中任一项所述的系统,其中该细胞是一种真核细胞。
20.如权利要求19所述的系统,其中该真核细胞是一种哺乳动物细胞。
21.如权利要求20所述的系统,其中该哺乳动物细胞是一种人类细胞。
22.如权利要求14至21中任一项所述的系统,其中该CRISPR-Cas系统是一种II型CRISPR-Cas系统。
23.如权利要求14至22中任一项所述的系统,其中该Cas蛋白是一种Cas9蛋白。
24.如权利要求14至23中任一项所述的系统,其中该Cas蛋白是密码子优化的,用于在真核细胞中进行表达。
25.如权利要求14至24中任一项所述的系统,其中该基因产物的表达被降低。
26.如权利要求14至25中任一项所述的系统,其中该基因产物是一种蛋白质。
27.如权利要求14至26中任一项所述的系统,其中经由一种递送系统将该系统引入该细胞中,该递送系统包括病毒粒子、脂质体、电穿孔、显微注射或缀合。
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