CN108103586A - 一种CRISPR/Cas9随机文库及其构建和应用 - Google Patents
一种CRISPR/Cas9随机文库及其构建和应用 Download PDFInfo
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Abstract
本发明提供了一种CRISPR/Cas9随机文库及其构建和应用。所述的CRISPR/Cas9随机文库,其特征在于,包括sgRNA,所述的sgRNA含有识别序列,其中该序列的20个核苷酸第1个设定为G,2至20个设定为随机的核苷酸。本发明建立了高通量、通用的、无偏好的sgRNA随机文库,提供功能基因组学高通量功能注释基因组功能元件的有效工具。
Description
技术领域
本发明涉及一种CRISPR/Cas9随机文库及应用。
背景技术
基因组计划完成后,科学家们进行功能基因组研究面临的挑战之一就是高通量功能注释功能基因元件。RNA干扰是一种有效的高通量功能研究的策略[Kaelin,2012]。不过存在脱靶效应、只是敲降而不是敲除基因、只能影响表达基因不能针对非编码功能元件等因素,限制了其应用。CRISPR/Cas9系统是理想的高通量基因敲除工具,不过已有的sgRNA文库通量非常有限,本发明拟研发构建高通量、通用的、无偏好sgRNA随机文库的策略。
基因组学研究面临的挑战之一就是功能注释不同基因组功能元件:人类基因组计划表明,在人类大约3x109bp的基因组上,只有大约2~4%的编码区编码20000-25000个基因[Consortium IHGS.2004]。各种生命过程,包括发育、增殖、分化、衰老等,都是由编码基因的时空表达调控的,而编码基因的表达通过非编码的基因组调控元件实现(图1)。
为此,各国的科学家们成立了ENCODE(Encyclopedia of DNA Elements)联盟,联合开展ENCODE计划,通过Tilling array、Tag sequencing、Genomic sequencing、Promoterassay等技术手段,结合计算分析,以鉴定人类基因组上的功能元件[ENCODE ProjectConsortium.2007]。阶段性研究结果表明,人类基因组的80.4%区域分布了百万级别的功能元件,大部分元件的功能尚未注释[ENCODE Project Consortium.2012]。基因组学研究面临的挑战之一就是高通量功能注释基因组功能元件。
CRISPR/Cas9是识别和注释基因组元件的有力工具:CRISPR/Cas9是2012年研发出的一种新型基因组修饰技术--规律成簇的间隔短回文重复序列(Clustered RegularlyInterspaced Short Palindromic Repeats,CRISPR)。在这一系统中,sgRNA(singleguided RNA)通过靶序列互补引导Cas9蛋白定位剪切双链DNA,形成双链DNA缺口,然后借助同源重组机制(homologous recombination,HR)或者非同源末端连接机制(non-homologous end joining,NHEJ)对断裂的DNA进行修复。引起靶位点的突变、缺失或插入,造成基因敲除效果,或基因敲入效果[Cong et al.,2013;Jinek et al.,2012;Mali etal.,2013]。由于简便、高效、价廉,该技术出现之后立即席卷全球,成为了基因编辑领域最新,但发展最快、应用最广的技术,引发了基因编辑领域的革命。现在CRISPR/Cas9系统已经被成功地用于DNA敲除、DNA敲入、DNA替代、DNA修饰、RNA修饰、DNA标记、基因转录调节等。CRISPR技术被认为将像PCR技术一样影响生命科学的方方面面[Barrangou and Doudna,2016;Hsu et al.,2014;Ledford.2015]。
与其它基因编辑技术不同的是,CRISPR/Cas9由通用的核酸酶Cas9和特异的sgRNA实现靶向,而sgRNA片段小,可以通过同时表达多sgRNA实现多靶点的敲除,使得构建sgRNA文库进行大规模、高通量的功能基因组学研究成为可能[Shalem et al.,2015]。这些特点很快被成功应用于大规模编码基因的功能研究[Koike-Yusaet al.,2014;Shalemet al.,2014;Wang et al.,2014;Zhou et al.,2014]。最近,CRISPR系统的高通量优势也被成功地用到了基因元件的分析上[Korkmazet al.,2016;Rajagopalet al.,2016;Sanjana etal.,2016]。这些研究表明,CRISPR/Cas9系统可用于高通量识别基因功能元件。
构建高通量、通用的、无偏好sgRNA随机文库:不过现有的sgRNA文库针对特定的编码基因[Shalem et al.,2015],或特定基因的调控区[Korkmaz et al.,2016;Rajagopalet al.,2015;Sanjana et al.,2016],都只是针对基因组大约2%的编码区的限量的sgRNA文库或针对特殊的筛选。显而易见,远不能满足人的3x109bp的全基因组的需要。因此,构建高通量、通用的、无偏好sgRNA随机文库势在必行。因此,本发明拟构建sgRNA随机文库。
参考文献
Barrangou R,Doudna JA.Applications of CRISPR technologies in researchand beyond.Nat Biotechnol.2016,34(9):933-941.
Cong L,Ran FA,Cox D,Lin S,Barretto R,Habib N,Hsu PD,Wu X,Jiang W,Marraffini LA,Zhang F.Multiplex genome engineering using CRISPR/Cassystems.Science 2013,339(6121):819-823.
Consortium IHGS.Finishing the euchromatic sequence of the humangenome.Nature2004,431:931-45.
ENCODE Project Consortium.Identification and analysis of functionalelements in 1%of the human genome by the ENCODE pilot project.Nature 2007,447:799-816.
ENCODE Project Consortium.An integrated encyclopedia of DNA elementsin the human genome.Nature 2012,489:57-74.
Hsu PD,Lander ES,Zhang F.Development and applications of CRISPR-Cas9for genome engineering.Cell 2014,157(6):1262-1278.
Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna JA,Charpentier E.Aprogrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.Science 2012,337(6096):816-821.
Kaelin WG Jr.Molecular biology.Use and abuse of RNAi to studymammalian gene function.Science 2012,337(6093):421-422.
Kleinstiver BP,Prew MS,Tsai SQ,Topkar VV,Nguyen NT,Zheng Z,GonzalesAP,Li Z,Peterson RT,Yeh JR,Aryee MJ,Joung JK.Engineered CRISPR-Cas9 nucleaseswith altered PAM specificities.Nature 2015,523(7561):481-485.
Koike-Yusa H,Li Y,Tan EP,Velasco-Herrera Mdel C,Yusa K.Genome-widerecessive genetic sereening in mammalian cells with a lentiviral CRISPR-guideRNA library.Nature Biotechnology 2014,32(3):267-273.
Korkmaz G,Lopes R,Ugalde AP,Nevedomskaya E,Han R,Myacheva K,Zwart W,Elkon R,Agami R.Functional genetic screens for enhancer elements in the humangenomeusing CRISPR-Cas9.Nature Biotechnology 2016,34(2):192-198.LedfordH.CRISPR,the_disruptor.Nature2015,522(7554):20-24.
Mali P,Yang L,Esvelt KM,Aach J,Guell M,DiCarlo JE,Norville JE,ChurchGM.RNA-guided human genome engineering viaCas9.Science 2013,339(6121):823-826.Maston GA,Evans SK,and Green MR.Transcriptional regulatory elements inthe human genome.Annual Review of Genomics and Human Genetics2006,7:29-59.
Rajagopal N,Srinivasan S,Kooshesh K,Guo Y,Edwards MD,Banerjee B,SyedT,Emons BJ,Gifford DK,Sherwood RI.High-throughput mapping of regulatoryDNA.Nature Biotechnology 2016,34(2):167-174.
Sanjana NE,Wright J,Zheng K,Shalem O,Fontanillas P,Joung J,Cheng C,Regev AZhang F.High-resolution interrogation of functional elements in thenoncoding genome.Science 2016,353(6307):1545-1549.
Shalem O,Sanjana NE,Hartenian E,Shi X,Scott DA,Mikkelsen TS,Heckl D,Ebert BL,Root DE,Doench JG,Zhang F.Genome-scale CRISPR-Cas9 knockoutscreening in human cells.Science 2014,343(6166):84-87.
Shalem O,Sanjana NE,Zhang F.High-throughput functional genomics usingCRISPR-Cas9.Nat Rev Genet.2015,16(5):299-311.
Wang T,Wei JJ,Sabatini DM,Lander ES.Genetic screens in human cellsusing the CRISPR-Cas9 system.Science 2014,343(6166):80-84.
Zhou Y,Zhu S,Cai C,Yuan P,Li C,Huang Y,Wei W.High-throughputscreening of a CRISPR/Cas9 library for functional genomics in humancells.Nature 2014,509(7501):487-991.
发明内容
本发明的目的是提供构建具有较高通量和通用性的sgRNA随机文库。
为了达到上述目的,本发明提供了一种CRISPR/Cas9随机文库,其特征在于,包括sgRNA,所述的sgRNA含有识别序列,其中该序列的20个核苷酸第1个为G,2至20个为随机的核苷酸N。
优选地,所述的识别序列为随机识别序列。
优选地,所述的sgRNA包含骨架部分和随机识别序列。
优选地,所述的sgRNA还包含T7启动子序列。
优选地,所述的sgRNA的序列为SEQ ID NO:1或SEQ ID NO:3。
本发明还提供了上述的CRISPR/Cas9随机文库的构建方法,其特征在于,包括:采用将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机核苷酸N的策略,通过随机引物合成、退火、补齐双链后,进行纯化后得到随机体外转录模板;对sgRNA进行体外转录,纯化后,得到sgRNA随机文库。
优选地,所述的随机体外转录模板的序列包含SEQ ID NO:3。
本发明还提供了上述的CRISPR/Cas9随机文库在基因组突变中的应用。
本发明的策略是:将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机的核苷酸N,N随机地为A或T或C或G(图2)。1、理论上,不考虑PAM的情况下,419种sgRNA分子将会覆盖全基因组,并且没有种系特异性;2、同时使用spCas9的野生型和VQR突变体[Kleinstiveret al.,2015]。spCas9的PAM序列为NGG,分布于1/8基因组,VQRspCas9的PAM序列为NGA及NGCG,分布于1/8加1/32基因组,两种spCas9可以覆盖至少1/4基因组。显而易见,这类sgRNA随机文库可以匹配多种Cas9和它们的突变体,将极大地提高sgRNA随机文库的通量和通用性。这一策略还可以推广到其它Cas9类似物,如saCas9、Cpf1等。将进一步提高sgRNA随机文库的通量和通用性。
在此基础上,本发明通过深度测序验证随机文库的sgRNA靶向的随机性和覆盖率;利用dCas9 ChIP-Seq检测sgRNA随机文库的dCas9/sgRNA与基因组DNA结合的随机性和覆盖率;利用斑马鱼胚胎注射结合基因组深度测序检测Cas9/sgRNA介导的基因组突变的随机性和覆盖率。
为了对随机文库构建进行概念验证(proof-of-principle),本发明进行了本项目的前期研究,构建了小规模的sgRNA随机文库并通过质检和前期测试证明可以制备高质量的sgRNA随机文库,并用于基因组的高通量、随机突变,取得了预期的结果。本发明的研究将开发构建随机文库的策略,并构建、验证高通量、通用的、无偏好的sgRNA文库,提供功能基因组学高通量功能注释基因组功能元件的有效工具。
与传统sgRNA文库相比,本发明的区别点如下:
与现有技术相比,本发明的有益效果是:
本发明针对以往研究的限制,制备的文库体现出的特点包括:(1)高通量(针对全基因组而不是特定区域);(2)通用的(针对所有而不是特定物种的基因组);(3)无偏好(针对所有而不是预测的基因元件)。
由于这是一种全新的文库,不仅有助于本发明分析编码基因,更重要的是将极大地推动对基因组上未知的功能元件的识别和注释。可能发现一些新的功能基因组元件,为本发明的生物医药研究提供新的靶点。从而从技术上和学术上推动本领域的发展,具有深远的社会和经济价值,体现出世界领先的研究水平。
功能基因组研究面临的挑战之一就是高通量功能注释功能基因元件。CRISPR/Cas9系统是理想的高通量基因敲除工具,不过已有的sgRNA文库通量非常有限,不能满足哺乳动物全基因组的需要。为此,本发明拟采用将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机核苷酸N的策略,制备sgRNA随机文库,在此基础上,本发明将验证随机文库的sgRNA靶向的随机性和覆盖率、dCas9/sgRNA与基因组DNA结合的随机性和覆盖率、Cas9/sgRNA介导基因组突变的随机性和覆盖率。从而建立构建、验证高通量、通用的、无偏好的sgRNA随机文库,提供功能基因组学高通量功能注释基因组功能元件的有效工具。
附图说明
图1为基因表达调控模式图。在非编码区分布着调控基因表达的promoter核心、promoter元件、enhancer、silencer、insulator、locus control region(LCR)等。(MastonGA,Evans SK,and Green MR.2006)
图2为随机文库构建示意图;
图3为退火产物示意图;
图4随机sgRNA转录模板示意图;
图5随机sgRNA模板与退火引物的琼脂糖凝胶电泳图;
图6随机sgRNA转录模板的测序结果图;
图7随机sgRNA琼脂糖凝胶电泳图;
图8为随机sgRNA文库的基因组分布分析图;
(A)随机文库构建示意图;
(B)随机文库中的靶向序列的碱基分布;
(C)随机sgRNA文库的靶向序列(带PAM与否)在斑马鱼基因组上的分布;
(D)随机sgRNA文库的靶向序列(带PAM与否)在斑马鱼编码基因上的分布;
图9为随机sgRNA文库在小鼠和人基因组上的分布分析图;
(A)随机sgRNA转录模板制备;
(B)序列长度的分布;
(C)随机sgRNA文库的靶向序列(带PAM与否)在小鼠基因组上的分布;
(D)随机sgRNA文库的靶向序列(带PAM与否)在小鼠编码基因上的分布;
(E)随机sgRNA文库的靶向序列(带PAM与否)在人基因组上的分布;
(F)随机sgRNA文库的靶向序列(带PAM与否)在人编码基因上的分布;
图10为随机sgRNA文库和斑马鱼基因组结合点的分布分析图;
(A)dCas9/随机sgRNA与斑马鱼全基因组的体内结合分布;
(B)与斑马鱼基因的体内结合分布;
图11随机sgRNA文库介导斑马鱼基因组突变造成的发育表型;
图12随机sgRNA文库介导斑马鱼基因组突变的分布分析图。
(A)在斑马鱼基因组上的发生的基因组编码数;
(B)基因组编码长度的序列分布;
(C)基因组编码位置相对于编码基因的分布;
具体实施方式
下面将结合实施例对本发明的实施方案进行清楚、完整的描述,显然,所描述的实施例仅用于说明本发明的一部分实施例,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,皆为可以通过市售购买获得的常规产品。
实施例
1、sgRNA随机文库的设计和制备
采用将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机核苷酸N的策略,利用17bp的T7启动子(taatacgactcactata,SEQ ID NO:2)驱动sgRNA表达。sgRNA由83bp的骨架部分和20bp的识别区组成。为提高T7启动子的转录效率,设定识别区的第1个核苷酸为G,第2至20个为随机核苷酸N,全长120bp,(SEQ ID NO:3,
taatacgactcactatagnnnnnnnnnnnnnnnnnngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttt)。sgRNA模板由随机上游引物和下游引物合成。取合成的sgRNA引物进行体外退火补齐后作为随机转录模板,测序验证后进行体外转录,具体步骤如下:
通过常规方法合成随机上游引物(SEQ ID NO:4,ttaataatacgactcactatannnnnnnnnnnnnnnnnngttttagagctagaaatagcaagtt)和下游引物(SEQ ID NO:5,aaaaaaagcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac);
体外退火:混合5μL上游引物(100μM)和5μL下游引物(100μM),用95℃金属浴加热5min,并自然冷却至室温(图3);
制备随机sgRNA的随机转录模板:使用高保真PrimeSTAR HS DNA Polymerase(Takara,DR010)补齐双链(图4),反应体系如下:
在热循环仪中68℃反应1h,4℃保持;切胶纯化得到随机sgRNA体外转录模板后,并进行琼脂糖凝胶电泳鉴定随机转录模板的纯度(图5),进一步Sanger测序(图6)确定随机转录模板的高质量;
使用试剂MEGAshortscript Kit(Ambion,Life Technologies,AM1354)对sgRNA进行体外转录,纯化后,得到sgRNA随机文库,琼脂糖凝胶电泳鉴定显示得到的随机sgRNA产量大、纯度高(图7)。结果表明,本发明成功制备了高质量的sgRNA随机文库。
2、sgRNA随机文库的验证
(1)sgRNA靶向的随机性和覆盖率的验证
文库的高通量和无偏好首先体现在sgRNA靶向序列的高通量和无偏好。将得到的sgRNA随机文库抽样做深度测序,进行生物信息学分析,验证合成sgRNA文库的随机性和全基因组覆盖率;
取sgRNA随机文库进行(HiSeq)深度测序分析sgRNA靶点在斑马鱼、小鼠和人基因组上的分布。结果显示,核苷酸种类在sgRNA随机文库的靶序列上随机分布,显示出文库有很好的随机性和覆盖率。sgRNA靶点随机分布在斑马鱼的全基因组上(图8)。随机文库的sgRNA长度一致;sgRNA靶点随机分布在小鼠和人的全基因组上,sgRNA靶点随机分布于小鼠和人基因组的不同区域(图9)。
上述结果表明,采用将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机核苷酸N的策略,制备的sgRNA随机文库高质量,体现出均匀分布、无偏好。表明本发明的设计和合成策略可行性非常好。
(2)sgRNA结合的随机性和覆盖率的验证
Cas9/sgRNA发挥作用的第一步是和靶点的结合,文库和全基因组的结合的随机性和覆盖率可以反应文库的随机性和覆盖率。利用dCas9 ChIP-Seq检测sgRNA随机文库的dCas9/sgRNA与基因组DNA结合的效率和随机性;
利用dCas9 ChIP-Seq检测sgRNA随机文库的dCas9/sgRNA与基因组DNA结合的情形。取注射dCas9-FLAG mRNA(使用Ambion,Life Technologies,AM1354制备,200pg/受精卵)和sgRNA随机文库(250pg/受精卵)的斑马鱼胚胎,经过甲醛(Sigma,F8775)室温交联10分钟、微球菌核酸酶(CST,#9003)37℃裂解20分钟、超声(30秒/30秒间歇,10个循环;Bioruptorpico)之后取上清,留取部分作为内参(input-dCas9,高通量测序时做为对照),其余用于ChIP:1,5-10μg裂解好的染色质片段加入1μgFLAG抗体(Sigma,F1804)或者10μg正常的IgG(CST,#9003),在4℃孵育过夜;2,加入30μLProtein G beads(CST,#9003)4℃孵育2h;3,离心后对beads进行洗涤,然后用酚氯仿抽提再用酒精沉淀DNA,最后溶于水中;4,将所得到的DNA样品送到测序平台建库,建库后进行高通量测序。结果表明,dCas9/sgRNA结合位点随机分布在斑马鱼的全基因组上(图10),肯定了CRISPR/Cas9系统高效,并进一步肯定了sgRNA随机文库的高通量和无偏好。
(3)文库切割的随机性和覆盖率的验证
本发明拟利用斑马鱼胚胎注射,进行基因组深度测序检测,分析Cas9/sgRNA介导的基因组突变的随机性和覆盖率。
本发明实施了试验性筛选。胚胎注射50pg上述的sgRNA得到F0(首建鱼)后,通过采用斑马鱼遗传筛选常用的两代配繁策略,在F3(第三子代)胚胎中通过形态学观察筛选影响胚胎发育的隐性突变。本发明一共繁殖得到了453个F2(第二子代)家族,代表867套实施sgRNA随机文库注射以诱导突变的基因组。在这些家族中有380个成功实现自交,对收集到的F3胚胎,本发明在5个不同的发育阶段进行观察以分析从原肠运动到器官发生多个发育事件中的异常(图11)。证明sgRNA随机文库注射可以在斑马鱼体内有效产生突变。随后,本发明随机选择三条F1(第一子代)斑马鱼进行基因组全测序,结果如图12所示,随机文库在斑马鱼基因组引起大量、广泛突变(图12A),不同个体发生的突变相近(图12B),并且突变均匀分布在全基因组(图12C)。表明,sgRNA随机文库可以高效、随机介导基因组突变。
已有的实验结果表明,本发明的策略以用于成功构建sgRNA随机文库,提供功能基因组学研究急需的高通量、通用的、无偏好的有效工具。
序列表
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<120> 一种CRISPR/Cas9随机文库及其构建和应用
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Claims (8)
1.一种CRISPR/Cas9随机文库,其特征在于,包括sgRNA,所述的sgRNA含有识别序列,其中该序列的20个核苷酸第1个为G,2至20个为随机的核苷酸N。
2.如权利要求1所述的CRISPR/Cas9随机文库,其特征在于,所述的识别序列为随机识别序列。
3.如权利要求1所述的CRISPR/Cas9随机文库,其特征在于,所述的sgRNA包含骨架部分和随机识别序列组成。
4.如权利要求1所述的CRISPR/Cas9随机文库,其特征在于,所述的随机sgRNA还包含T7启动子序列。
5.如权利要求1所述的CRISPR/Cas9随机文库,其特征在于,所述的随机sgRNA的序列为SEQ ID NO:1或SEQ ID NO:3。
6.权利要求1-4中任一项所述的CRISPR/Cas9随机文库的构建方法,其特征在于,包括:采用将sgRNA识别序列的20个核苷酸第1个设定为G,2至20个设定为随机N的策略,通过随机引物合成、退火、补齐双链后,进行纯化后得到随机体外转录模板;对sgRNA进行体外转录,纯化后,得到sgRNA随机文库。
7.如权利要求6所述的CRISPR/Cas9随机文库的构建方法,其特征在于,所述的随机体外转录模板的序列包含SEQ ID NO:3。
8.权利要求1-4中任一项所述的CRISPR/Cas9随机文库在基因组突变中的应用。
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