CN105255937A - 一种真核细胞III型启动子表达CRISPR sgRNA的方法及其应用 - Google Patents
一种真核细胞III型启动子表达CRISPR sgRNA的方法及其应用 Download PDFInfo
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Abstract
本发明是利用一个真核细胞III型启动子(U6或H1)启动由Drosha切割位点串联的多个发卡结构小RNA的表达,在真核细胞内表达后可以产生多个有生物活性的CRISPR?sgRNA,以U6-sgRNA-shRNA-sgRNA的结构为例,这种U6启动表达的结构在一次转录中产生含有多个sgRNA和shRNA的初级转录本,在真核细胞中这些sgRNA经过加工后可以分别识别各自的靶位点,从而指导Cas9蛋白打靶多个位点,为多基因编辑打下基础。相比于传统的分别表达多个单一gRNA的方法和植物上新近报道的tRNA-gRNA系统,本发明的结构更简单,构建更方便。此外,本发明还可以通过Golden?Gate的方法继续串联shRNA-sgRNA的序列打靶更多的位点或者表达多个shRNA干扰基因的表达。
Description
技术领域
本申请涉及基因启动子表达技术,属于生物技术领域。
背景技术
Drosha:一种III型核糖核酸酶,通过识别mRNAs中类似于miRNAs前体的二级茎环结构来定位和切割mRNAs,这在干细胞中抑制基因表达的机制中有重要作用。
在生物体内,miRNA的成熟较siRNA双链的形成过程要复杂,概括为:首先miRNA的前体pri-miRNA在核内由一种称为Drosha酶处理后成为大约70nt的带有茎环结构的PrecursormiRNAs(pre-miRNAs)(Denlietal.,2004;Gregoryetal.,2004;Hanetal.,2004);这些pre-miRNAs在Exportin-5帮助下转运到细胞核外之后再由胞质Dicer酶进行处理,酶切后成为成熟的miRNAs(Lundetal.,2004;Yietal.,2003)。
CRISPR:规律成簇间隔短回文重复(clusteredregularlyinterspacedshortpalindromicrepeats),细菌的II型CRISPR/Cas系统逐渐成为同时打靶多个基因位点的重要工具。
shRNA:shorthairpinRNA,“短发夹RNA”。shRNA包括两个短反向重复序列,克隆到shRNA表达载体中的shRNA包括两个短反向重复序列,中间由一茎环(loop)序列分隔的,组成发夹结构,由polⅢ启动子控制。随后再连上5-6个T作为RNA聚合酶Ⅲ的转录终止子。在活体中输送“小干扰RNA”(siRNA)的一种办法是,将siRNA序列作为“短发夹”克隆进质粒载体中。当送入动物体内时,该发夹序列被表达出来,形成一个“双链RNA”(shRNA),并被RNAi通道处理。
高等生物通常都有复杂的基因网络来确保细胞内的生物活动有条不紊的进行。因此,能够同时打靶多个位点的分子工具对遗传工程的基础研究和实际应用都有巨大意义。近年来,细菌的II型CRISPR/Cas系统逐渐成为达成这一目的的重要工具。从酿脓链球菌中分离得到的Cas9核酸内切酶通过人工修饰的引导RNA(guideRNA,gRNA)的导向作用可以打靶DNA序列的5’-N20-NGG-3’(N代表任何脱氧核苷酸碱基),N20是与gRNA的5’序列相同的20个碱基,NGG是PAM区(protospacer-adjacentmotif)。Cas9剪切的位点就是PAM附近的区域。这种可以人为修饰的导向RNA和基因组中PAM的高发率使得Cas9-gRNA几乎可以打靶所有的基因元件来实现基因组编辑。正是由于它的简单高效性,基于Cas9的基因编辑工具发展迅速,被用于基因组和表观基因组编辑,转录调控和基因工程的其他应用。
从理论上来讲,多基因编辑可以通过Cas9和靶向不同位点的多个sgRNA一起表达来实现。传统的方法通过显微注射或者表达包含多个单一gRNA(sgRNA)的表达盒来实现。将体外表达的gRNA和Cas9蛋白(或者Cas9的mRNA)注射到细胞或者胚胎只适用于很少的一些系统。因此,最理想的方法就是将多个sgRNA的表达盒压缩到一个载体上。一个典型的sgRNA的表达盒大约是400-500bp,包含RNA聚合酶III(PolIII)的启动子,sgRNA和PolIII终止子。受传递方式和质粒载体承载能力的限制,对于大多数生物来说,用这一sgRNA表达方法同时表达多个sgRNA将是一种挑战。而且,真核生物III型聚合酶转录的RNA需要有一个特定的核苷酸开始,这使得Cas9/gRNA靶位点受限。一个好点的方法就是将多个sgRNA的表达盒压缩到一个合成的基因中,利用一个RNA加工系统从初级转录本中将单个sgRNA分别剪切出来。应用到这一方法的成功案例只有利用Csy4内切核糖核酸酶和tRNA剪切系统。(KabinXie,BastianMinkenberg,YinongYang,BoostingCRISPR/Cas9multiplexeditingcapabilitywiththeendogenoustRNA-processingsystem,PNAS)然而,还需要更有效和精确的方法来同时产生多个sgRNA以提高多基因编辑的能力和充分发挥Cas9系统的应用。
细胞中有大量的各种的RNA。在不同组织中,RNA的合成是高度保守的,并且有各种精细的RNA加工系统来确保其正确的剪切。这一特点启发我们可以用一个有内切酶功能的RNA加工系统来从一个转录本切出多个sgRNA。最近在植物上的一个研究表明,多个sgRNA可以由一个tRNA-gRNA结构的合成基因通过内源的RNase的精确剪切产生。(KabinXie,BastianMinkenberg,YinongYang,BoostingCRISPR/Cas9multiplexeditingcapabilitywiththeendogenoustRNA-processingsystem,PNAS)这一研究说明在植物中不仅可以实现多基因打靶,而且大大提高了CRISPR/Cas9系统用于基因组编辑的效率。然而,类似的方法在动物中未见报道。
III型核糖核酸酶(RNaseIII)一直被认为在生物正常表达小RNA(miRNAs)的过程中有关键作用。多年来,人们一直忽视了它们在其他RNA(例如sgRNA、shRNA等)的产生过程中的重要作用。作为RNaseIII家族的一员,Drosha就可以识别并切割信使RNA(mRNAs),并可能在核糖体RNA(rRNA)的加工过程中起作用。Drosha通过识别mRNAs中类似于miRNAs前体的二级茎环结构来定位和切割mRNAs,这在干细胞中抑制基因表达的机制中有重要作用。Dicer则可以通过调控许多小干扰RNA(smallinterferingRNAs,siRNAs)的产生来调节基因的表达,还可以促使细胞内有害的短RNA片段和病毒的RNA降解来保护和维持细胞生物活动的正常。(TimothyM.Johanson,AndrewM.Lew,MarkM.W.Chong,MicroRNA-independentrolesoftheRNaseIIIenzymesDroshaandDicer,RoyalSocietyPublishging)
发明内容
本发明就是通过利用Drosha的切割位点将多个sgRNA和shRNA的序列串联起来,通过一个真核细胞的III型启动子启动表达。从而实现同时产生多个sgRNA以提高多基因编辑的能力和充分发挥Cas9系统的应用。
本发明是利用一个真核细胞III型启动子(U6或H1)启动由Drosha切割位点串联的多个发卡结构小RNA的表达,在真核细胞内表达后可以产生多个有生物活性的CRISPRsgRNA(本发明的DNA序列图示如下)。以U6-sgRNA-shRNA-sgRNA的结构为例,这种U6启动表达的结构在一次转录中产生含有多个sgRNA和shRNA的初级转录本,在真核细胞中这些sgRNA经过加工后可以分别识别各自的靶位点,从而指导Cas9蛋白打靶多个位点,为多基因编辑打下基础。相比于传统的分别表达多个单一gRNA的方法和植物上新近报道的tRNA-gRNA系统,本发明的结构更简单,构建更方便。此外,本发明还可以通过GoldenGate的方法继续串联shRNA-sgRNA的序列打靶更多的位点或者表达多个shRNA干扰基因的表达。
附图说明
图1:本发明DNA序列结构图;
图2:Cas9表达质粒(pll3.7-SpCas9(Not1))图;
图3:中间载体pcDNA3.1(+)-CMV-3RNA质粒图;
图4:载体msgRNA-2的质粒图;
图5:质粒msgRNA-2酶切检测结果;
图6:双荧光报告载体Re-SSA(CMV).VEGF的质粒图;
图7:双荧光报告载体Re-SSA(EF1a).CCR5a的质粒图;
图8:VEGF位点报告载体检测荧光效果图(实验组和阳性对照组);
图9:CCR5a位点报告载体检测荧光效果图(实验组和阳性对照组);
图10:报告载体流式结果分析图。
具体实施方式
为使本发明的技术方案便于理解,以下结合利用III型启动子U6表达靶向人基因组VEGF基因和CCR5基因的具体试验对本发明作进一步的详细说明,所述是对本发明的解释而不是限定。
实施例1靶位点选择
基于CRISPR/Cas9系统打靶位点的特点,在基因组中寻找含有PAM(NGG/NGGNG)的靶位点。再经过CRISPRDesign网站(http://crispr.mit.edu/)的筛选,选择出以下两个位点作为靶位点:
VEGF靶位点片段:
CTCGGCCACCACAGGGAAGCTGG(SEQIDNO:1)
CCR5a/CCR2靶位点片段:
CACACTTGTCACCACCCCAAAGGTG(SEQIDNO:2)
实施例2Cas9蛋白表达载体
将优化过的酿脓链球菌Cas9序列(hSpCas9)插入到骨架载体pll3.7中。如图2所示。hSpCas9的序列如下(SEQIDNO:3):
GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATC
ACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC
AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCC
ACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTAT
CTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG
GAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAAC
ATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAA
CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATG
ATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTG
GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATC
AACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG
CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTG
ATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGAT
GCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAG
ATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTG
CTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATG
ATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAG
CAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGC
TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAA
AAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAG
CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCC
ATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAG
AAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGA
TTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTG
GTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC
CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTAT
AACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGC
GGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTG
AAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCC
GGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC
AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTG
ACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCAC
CTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGG
CTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGAT
TTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC
CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCAC
GAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTG
AAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATC
GAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG
AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTG
GAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGAT
ATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC
GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGAC
AAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAAC
TACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACC
AAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTG
GTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACT
AAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAG
CTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTAC
CACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTAC
CCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATG
ATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAAC
ATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCT
CTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCC
ACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG
ACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCC
AGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT
TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAA
GAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTT
CTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC
TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG
AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCAC
TATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATC
CTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCC
ATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCT
GCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG
GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGAC
CTGTCTCAGCTGGGAGGCGAC
实施例3串联多个小RNA表达载体(msgRNA-2)
以实验载体为例,该载体设计为U6-sgRNA-shRNA-sgRNA结构,根据选择的两个靶位点(VEGF靶位点和CCR5a靶位点),分别设计对应的sgRNA序列,中间用带有Drosha靶位点的CD40shRNA序列。通过不同的限制性内切酶的酶切位点将各个片段连在一起。本实验使用的两个Drosha靶位点序列如下:
Drosha靶位点片段1:TCCGAGGCAGTAGGCA(SEQIDNO:4)
Drosha靶位点片段2:TGCTGTTGACAGTGAGCG(SEQIDNO:5)
构建该载体需要两步。第一步,将3个RNA序列的基因片段插入到骨架载体pcDNA3.1(+)(Invitrogen)上的多克隆位点NheI和ApaI之间,各个片段之间分别通过限制性内切酶EcoRI、XhoI和ApaI各自识别位点的序列来连接,即NheI-VEGF.sgRNA-EcoRI-CD40.shRNA-XhoI-CCR5a.sgRNA-ApaI的结构。两个sgRNA片段通过PCR引入需要的酶切位点,CD40.shRNA片段通过酶切的方法获得;第二步,用U6启动子替换掉CMV启动子,最终获得串联表达多个小RNA的载体msgRNA-2。
载体的具体构建和检测方法如下:
3.1引物的设计与合成
分别以包含目的片段的载体为模板,利用CloneManagerV7软件进行引物设计,设计能够特异扩增VEGF.sgRNA片段的引物NheI-VEGF.SpsgR.F和EcoRI-SpsgR.R,能够特异扩增CCR5a.sgRNA片段的引物XhoI-CCR5.a.SpsgRNA.F和ApaI-SpsgRNA.R,能够特异扩增U6启动子的引物Primer53(NdeI-U6L27-F)和Primer54(SacI-U6L27-R)。分别在相应的位置引入所需的酶切位点。引物序列设计如表1:
表1:实验引物设计表
其中:引物5’端的黑体斜体表示的碱基为酶切位点,前面的黑色字体为保护碱基,带下划线的部分为与模板匹配的区域,剩下的黑色字体部分为引入的打靶序列。
3.2中间载体pcDNA3.1(+)-CMV-3RNA的构建
(1)PCR获得VEGF.sgRNA片段:以含有该VEGF.sgRNA片段的载体pX330-U6-Chimeric_BB-CBh-hSpCas9(从ADDGENE购得)为模板,用引物NheI-VEGF.SpsgR.F和EcoRI-SpsgR.R通过TouchdownPCR的方法扩增。PCR反应体系如表2所述,反应条件为:95℃预变性5min;95℃变性30s,68℃退火30s,72℃延伸20s,18个循环,每个循环的退火温度降1℃;95℃变性30s,50℃退火30s,72℃延伸20s,25个循环;最后72℃延伸10min。产物:117bp的VEGF.sgRNA片段
表2:VEGF.sgRNA片段PCR体系
(2)利用酶切的方法获得CD40.shRNA片段:用EcoRI和XhoI从载体JMB84-U6-5'miR-3'miR-CD40shRNA1656(LongZhang,TingtingZhang,LingWang,ZhilongChenandZhiyingZhangInvivotargeteddeliveryofCD40shRNAtomouseintestinaldendriticcellsbyoraladministrationofrecombinantSacchromycesCerevisiae,GeneTherapy)中酶切获得。酶切体系见表3,37℃消化4h。酶切产物胶回收,目的片段大小为109bp。
表3:CD40.shRNA片段酶切体系
(3)PCR获得CCR5a.sgRNA片段:以含有该CCR5a.sgRNA的载体pX330-U6-Chimeric_BB-CBh-hSpCas9(从ADDGENE购得)为模板,用引物XhoI-CCR5.a.SpsgRNA.F和ApaI-SpsgRNA.R通过TouchdownPCR的方法扩增。PCR反应体系为如表4所述,反应条件为:95℃预变性5min;95℃变性30s,68℃退火30s,72℃延伸20s,18个循环,每个循环的退火温度降1℃;95℃变性30s,50℃退火30s,72℃延伸20s,25个循环;最后72℃延伸10min。产物:120bp的CCR5a.sgRNA片段
表4:CCR5a.sgRNA片段PCR体系
(4)用NheI和ApaI酶切pcDNA3.1(+)得到的5322bp片段作为骨架,用NheI和EcoRI切VEGF.sgRNA片段、用XhoI和ApaI切CCR5a.sgRNA片段,酶切体系见表5、表6和表7,胶回收目的片段。将骨架和酶切回收的VEGF.sgRNA片段、CCR5a.sgRNA片段和CD40.shRNA片段连接,连接体系见表8。经过16℃过夜后转化大肠杆菌DH5α感受态细胞,涂LB/Amp平板,挑取单克隆并在LB/Amp液体培养基中37℃培养8h。
表5:骨架载体pcDNA3.1(+)酶切体系
表6:VEGF.sgRNA片段酶切体系
表7:CCR5a.sgRNA片段酶切体系
表8:中间载体pcDNA3.1(+)-CMV-3RNA连接体系
pcDNA3.1(+)-CMV-3RNA载体的质粒图谱如图3所示,包括ampicillin和neomycin抗性基因,CMV启动子和构建该质粒时用到的几个酶切位点等。
3.3多个小RNA串联载体msgRNA-2的构建
以载体JMB84-U6-5'miR-3'miR-CD40shRNA1656(LongZhang,TingtingZhang,LingWang,ZhilongChenandZhiyingZhangInvivotargeteddeliveryofCD40shRNAtomouseintestinaldendriticcellsbyoraladministrationofrecombinantSacchromycesCerevisiae,GeneTherapy)为模板,Primer53(NdeI-U6L27-F)和Primer54(SacI-U6L27-R)为引物通过TouchdownPCR的方法扩增得到511bp的带有酶切位点的U6启动子序列。PCR反应体系为如表9所述,反应条件为:95℃预变性5min;95℃变性30s,68℃退火30s,72℃延伸65s,18个循环,每个循环的退火温度降1℃;95℃变性30s,50℃退火30s,72℃延伸65s,25个循环;最后72℃延伸10min。以中间载体pcDNA3.1(+)-CMV-3RNA为骨架(酶切体系见表10),PCR扩增得到的U6启动子为插入片段(酶切体系见表11),经过NdeI和SacI酶切后连接得到串联表达多个小RNA的载体msgRNA-2,连接体系见表12。
表9:扩增U6启动子的PCR体系
表10:中间载体酶切体系
表11:U6启动子片段酶切体系
表12:载体msgRNA-2的连接体系
上面几个过程的各种反应的条件与3.2中的相同,此处不再赘述。构建获得的msgRNA-2的质粒图谱如图4。
提取质粒msgRNA-2,并经NdeI酶切鉴定,结果如图5所示,其中泳道M为Trans2KPlusDNAMarkerII;泳道1-6为为挑单克隆提取得到的质粒经NdeI酶切的产物。送阳性质粒到南京金斯瑞生物科技有限公司进行测序分析,保存测序正确的质粒备用。
实施例4报告载体
选用实验室已经构建好的包含所选的靶位点双荧光报告载体Re-SSA(CMV).VEGF和Re-SSA(EF1a).CCR5a(ChonghuaRen,KunXu,ZhongtianLiu,JuncenShen,FurongHan,ZhilongChen,ZhiyingZhang,Dual-reportersurrogatesystemsforefficientenrichmentofgeneticallymodifiedcells,CellularandMolecularLifeSciences)。两个报告载体的质粒图谱如图6和图7。
实施例5.双荧光报告载体系统检测msgRNA-2在HEK293T细胞中的表达多个sgRNA的工作效率
质粒测序正确后,将该质粒、cas9表达质粒和双荧光报告载体一起转染HEK293T细胞检测它是否能够正确表达出有导向作用的sgRNAs。利用报告载体的红色荧光可以看转染效率,绿色荧光可以反应工作效率。
(1)载体系统转染HEK293T细胞
HEK293T细胞系置于含DMEM,10%胎牛血清,100μg/mL青链霉素的培养基中,37℃,5%的CO2培养箱培养。HEK293T细胞系的转染:以24孔板为例,接种HEK293T细胞至24孔板中,待细胞密度接近70%时,换新鲜培养基。吸出24孔中的培养基,每孔加入37℃预热的新鲜培养基500μL,2-4小时后开始转染。取两个1.5mL灭菌EP管,一个加入约1.8μg的质粒,然后加入Opti-MEM至总体积30μL;另一个EP管内加入2μLSo-Fast转染试剂和Opti-MEM至30μL。轻轻混匀两个EP管内的混合物,然后将含转染试剂的Opti-MEM缓慢加入含质粒的EP管中,边加边轻轻震荡,使其充分混匀。加完混匀后,将混合物置于室温20min,然后将一个转染混合体系滴加至一个24孔中,轻轻晃动几下,将培养板放回培养箱,12小时后换新鲜培养基。转染所用的体系如表13,表中各组分下面的数字显示的是添加的体积量,单位为uL。
表13:双荧光报告载体检验工作效率的转染体系
(2)转染48h后,利用荧光显微镜观察分别观察每个孔的红色荧光和绿色荧光。图8和图9展示了两个位点实验组和阳性对照组的荧光照片。
(3)转染48h后,取各组细胞过流式细胞仪,统计发红光和发绿光的细胞占细胞总数的百分比。结果如图10所示。
图中红光反应转染效率,绿光反应工作效率。由图可见,实验组的效率不低于阳性对照组,即在该水平上串联表达sgRNA的系统效率不低于单独表达sgRNA的系统。
本申请发明人承诺,在本申请中所利用的由本实验室构建的载体如JMB84-U6-5'miR-3'miR-CD40shRNA1656以及双荧光报告载体Re-SSA(CMV).VEGF和Re-SSA(EF1a).CCR5a已经由本实验室的非专利文献公开,将在专利有效期内免费为公众提供。
序列表
<110>申请人姓名张智英、闫强、徐坤、邢佳妮、郭杨、任充华
<120>发明名称一种真核细胞III型启动子(U6或H1)表达多个CRISPRsgRNA
的方法及其在基因组编辑中的应用
<130>2015
<160>9
<170>patentinversion3.3
<210>1
<211>24
<213>人工序列
<400>1
CTCGGCCACCACAGGGAAGCTGGT
<210>2
<211>25
<213>人工序列
<400>1
CACACTTGTCACCACCCCAAAGGTG
<210>3
<211>4101
<213>人工序列
<400>1
GACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATC60
ACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC120
AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCC180
ACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTAT240
CTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTG300
GAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAAC360
ATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAA420
CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATG480
ATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTG540
GACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATC600
AACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG660
CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTG720
ATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGAT780
GCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAG840
ATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTG900
CTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATG960
ATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAG1020
CAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGC1080
TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAA1140
AAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAG1200
CAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCC1260
ATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAG1320
AAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGA1380
TTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTG1440
GTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC1500
CTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTAT1560
AACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGC1620
GGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTG1680
AAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCC1740
GGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATC1800
AAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTG1860
ACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCAC1920
CTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGG1980
CTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGAT2040
TTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGC2100
CTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCAC2160
GAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTG2220
AAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATC2280
GAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATG2340
AAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTG2400
GAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGAT2460
ATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATC2520
GTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGAC2580
AAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAAC2640
TACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACC2700
AAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTG2760
GTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACT2820
AAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAG2880
CTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTAC2940
CACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTAC3000
CCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATG3060
ATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAAC3120
ATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCT3180
CTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCC3240
ACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAG3300
ACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCC3360
AGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAT3420
TCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAA3480
GAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTT3540
CTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTAC3600
TCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAG3660
AAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCAC3720
TATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG3780
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATC3840
CTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCC3900
ATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCT3960
GCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG4020
GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGAC4080
CTGTCTCAGCTGGGAGGCGAC
<210>4
<211>16
<213>人工序列
<400>1
TCCGAGGCAGTAGGCA
<210>5
<211>18
<213>人工序列
<400>1
TGCTGTTGACAGTGAGCG
<210>6
<211>49
<213>人工序列
<400>1
CACGCTAGCCTCGGCCACCACAGGGAAGCGTTTTAGAGCTAGAAATAGC
<210>7
<211>27
<213>人工序列
<400>1
CCGGAATTCAAAGCACCGACTCGGTGC
<210>8
<211>49
<213>人工序列
<400>1
CTGCTCGAGCACACTTGTCACCACCCCAAGTTTTAGAGCTAGAAATAGC
<210>9
<211>27
<213>人工序列
<400>1
GATGGGCCCAAAAAAGCACCGACTCGG
<210>10
<211>27
<213>人工序列
<400>1
CGCCATATGCCCGAGTCCAACACCCGT
<210>11
<211>29
<213>人工序列
<400>1
GCCGAGCTCTAGTATATGTGCTGCCGAAG
Claims (8)
1.基于Drosha靶序列的特点和III型启动子实现CRISPR-Cas9系统sgRNA(又称gRNA)和/或RNA干扰shRNA(或miRNA)等多个小RNA共表达的方法,所述的方法通过III型启动子同时启动多个串联的sgRNA和/或shRNA表达,通过Drosha靶序列和细胞中RNA剪切机制实现多个功能性sgRNA和/或shRNA的表达并各自发挥作用。
2.权利要求1中提及的Drosha靶序列以及其优化和改进序列分别如SEQIDNO:4和SEQIDNO:5所示。
3.根据权利要求1所述的方法设计和构建的sgRNA1-shRNA1-sgRNA2-shRNA2-…shRNAn-sgRNAn多个小RNA表达盒或表达载体,其特征在于多个sgRNANA通过Drosha靶序列互相间隔串联,其中shRNA也可以是miRNA等其他形式的小发夹RNA。
4.根据权利要求3所述的表达载体,其中所述的shRNA被miRNA或其他形式的小发夹RNA取代。
5.根据权利要求1所述的方法设计和构建的shRNA1-shRNA2-…shRNAn多小RNA表达盒或表达载体,其特征在于多个shRNA通过Drosha靶序列依次串联,其中shRNA也可以是miRNA等其他形式的小发夹RNA。
6.根据权利要求1所述的方法设计和构建的Promotor-sgRNA1-shRNA1-sgRNA2-shRNA2-…shRNAn-sgRNAn多个小RNA表达盒或表达载体,其中Promotor代表权利要求1中提及的III型启动子以及其他任何可用于该串联结构表达的启动子;其中shRNA也可以是miRNA等其他形式的小发夹RNA,优选得,所述的启动子为人U6启动子、鼠U6启动子、人H1启动子。
7.基于权利要求3~6所述的多个sgRNA和/或shRNA串联表达的任何形式及其在基因组打靶、编辑和基因表达调控中的应用。
8.权利要求7所述的应用范围为真核细胞或真核生物。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
CN106446600A (zh) * | 2016-05-20 | 2017-02-22 | 同济大学 | 一种基于CRISPR/Cas9的sgRNA的设计方法 |
CN106868031A (zh) * | 2017-02-24 | 2017-06-20 | 北京大学 | 一种基于分级组装的多个sgRNA串联并行表达的克隆方法及应用 |
CN107937432A (zh) * | 2017-11-24 | 2018-04-20 | 华中农业大学 | 一种基于crispr系统的基因组编辑方法及其应用 |
US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
WO2019184655A1 (zh) * | 2018-03-27 | 2019-10-03 | 苏州克睿基因生物科技有限公司 | CRISPR/Cas系统在基因编辑中的应用 |
US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
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US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
CN112442513A (zh) * | 2019-09-02 | 2021-03-05 | 南京启真基因工程有限公司 | Cas9过表达载体及其构建方法和应用 |
US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150037297A1 (en) * | 1999-08-30 | 2015-02-05 | David S Terman | Sickled Erythrocytes and Progenitors Target Cytotoxics to Tumors |
CN105002214A (zh) * | 2015-04-08 | 2015-10-28 | 许中伟 | 用于载体表达的复合多联gRNA和RNAi的表达框架 |
-
2015
- 2015-08-14 CN CN201510500937.XA patent/CN105255937A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150037297A1 (en) * | 1999-08-30 | 2015-02-05 | David S Terman | Sickled Erythrocytes and Progenitors Target Cytotoxics to Tumors |
CN105002214A (zh) * | 2015-04-08 | 2015-10-28 | 许中伟 | 用于载体表达的复合多联gRNA和RNAi的表达框架 |
Non-Patent Citations (4)
Title |
---|
JIAQIANG WANG ET AL.: "Generation of cell-type-specific gene mutations by expressing the sgRNA of the CRISPR system from the RNA polymerase II promoters", 《PROTEIN CELL》 * |
KUN XU ET AL.: "Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus", 《CELL. MOL. LIFE SCI》 * |
LIOR NISSIM ET AL.: "An integrated RNA and CRISPR/Cas toolkit for multiplexed synthetic circuits and endogenous gene regulation in human cells", 《BIORXIV》 * |
QIANGYAN ET AL.: "Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure", 《SCIENTIFIC REPORTS》 * |
Cited By (53)
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US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
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WO2019184655A1 (zh) * | 2018-03-27 | 2019-10-03 | 苏州克睿基因生物科技有限公司 | CRISPR/Cas系统在基因编辑中的应用 |
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US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
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CN112442513A (zh) * | 2019-09-02 | 2021-03-05 | 南京启真基因工程有限公司 | Cas9过表达载体及其构建方法和应用 |
US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
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