利用CRISPR/Cas9和piggyBac实现果蝇基因组无缝编辑
|
摘要
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9)是第三代基因组编辑技术。在sgRNA引导下,Cas9核酸内切酶作用于特定基因组序列,产生DNA双链断裂(double-strandedbreaks,DSBs),利用同源定向修复(homology-directedrepair,HDR)可实现对靶基因的特异性基因敲除(knock-out)或敲入(knock-in)。传统的技术方案将CRISPR/Cas9技术与Cre/loxP或FLP/FRT系统联用,可实现高效的基因打靶,也易于移除打靶过程中引入的筛选标记。然而,筛选标记移除过程中会在基因组中残留34个碱基的标签序列。因此,对基因组进行精确编辑的同时不引入无关序列仍有一定难度。在人工诱导多能干细胞(induced pluripotent stem cells, iPSCs)的基因组编辑中,CRISPR/Cas9技术和piggyBac转座酶联用的两步法策略能够实现这一目标:首先运用CRISPR/Cas9技术,利用同源定向修复原理引入基因突变及筛选标记,然后利用piggyBac转座酶将筛选标记精确移除。借鉴该方法的技术原理,本研究对果蝇(Drosophila melanogaster)CG4894基因进行了无缝编辑(seamless genome editing),成功将该基因第18外显子上第21位的酪氨酸(tyrosine,Y)突变为半胱氨酸(cysteine,C),且测序结果显示基因组中除设计位点之外并无其他外源序列残留。CRISPR/Cas9技术和piggyBac转座酶联用策略为果蝇基因组的精确编辑提供了更多选择。
The typeⅡ CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sg RNA, the Cas9 endonuclease generates site-specific double-stranded breaks(DSB) at specific site, which is amenable to repair by homology-directed repair(HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells(i PSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18 th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.
The typeⅡ CRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sg RNA, the Cas9 endonuclease generates site-specific double-stranded breaks(DSB) at specific site, which is amenable to repair by homology-directed repair(HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells(i PSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18 th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.
引文
[1]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/Cas systems.Science,2013,339(6121):819-823.
[2]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna JA,Charpentier E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[3]Mali P,Yang L,Esvelt KM,Aach J,Guell M,DiCarlo JE,Norville JE,Church GM.RNA-guided human genome engineering via Cas9.Science,2013,339(6121):823-826.
[4]Miyaoka Y,Berman JR,Cooper SB,Mayerl SJ,Chan AH,Zhang B,Karlin-Neumann GA,Conklin BR.Systematic quantification of HDR and NHEJ reveals effects of locus,nuclease,and cell type on genome-editing.Sci Rep,2016,6:23549.
[5]van der Weyden L,Adams DJ,Bradley A.Tools for targeted manipulation of the mouse genome.Physiol Genomics,2002,11(3):133-164.
[6]Cary LC,Goebel M,Corsaro BG,Wang HG,Rosen E,Fraser MJ.Transposon mutagenesis of baculoviruses:analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.Virology,1989,172(1):156-169.
[7]Wang HH,Fraser MJ,Cary LC.Transposon mutagenesis of baculoviruses:analysis of TFP3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis viruses.Gene,1989,81(1):97-108.
[8]Handler AM,Harrell RA 2nd.Germline transformation of Drosophila melanogaster with the piggyBac transposon vector.Insect Mol Biol,1999,8(4):449-457.
[9]Lobo N,Li X,Fraser MJ,Jr.Transposition of the piggyBac element in embryos of Drosophila melanogaster,aedes aegypti and trichoplusia ni.Mol Gen Genet,1999,261(4-5):803-810.
[10]Ding S,Wu X,Li G,Han M,Zhuang Y,Xu T.Efficient transposition of the piggyBac(PB)transposon in mammalian cells and mice.Cell,2005,122(3):473-483.
[11]Wu SC,Meir YJ,Coates CJ,Handler AM,Pelczar P,Moisyadi S,Kaminski JM.PiggyBac is a flexible and highly active transposon as compared to sleeping beauty,Tol2,and Mos1 in mammalian cells.Proc Natl Acad Sci USA,2006,103(41):15008-15013.
[12]Wang G,Yang L,Grishin D,Rios X,Ye LY,Hu Y,Li K,Zhang D,Church GM,Pu WT.Efficient,footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.Nat Protoc,2017,12(1):88-103.
[13]Yusa K.Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon.Nat Protoc,2013,8(10):2061-2078.
[14]Zheng W,Feng G,Ren D,Eberl DF,Hannan F,Dubald M,Hall LM.Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform.J Neurosci,1995,15(2):1132-1143.
[15]Kanamori T,Kanai MI,Dairyo Y,Yasunaga K,Morikawa RK,Emoto K.Compartmentalized calcium transients trigger dendrite pruning in Drosophila sensory neurons.Science,2013,340(6139):1475-1478.
[16]Hara Y,Koganezawa M,Yamamoto D.The Dmca1D channel mediates Ca(2+)inward currents in Drosophila embryonic muscles.J Neurogenet,2015,29(2-3):117-123.
[17]Limpitikul WB,Viswanathan MC,O'Rourke B,Yue DT,Cammarato A.Conservation of cardiac L-type Ca(2+)channels and their regulation in Drosophila:a novel genetically-pliable channelopathic model.J Mol Cell Cardiol,2018,119:64-74.
[18]Ren X,Sun J,Housden BE,Hu Y,Roesel C,Lin S,Liu LP,Yang Z,Mao D,Sun L,Wu Q,Ji JY,Xi J,Mohr SE,Xu J,Perrimon N,Ni JQ.Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.Proc Natl Acad Sci USA,2013,110(47):19012-19017.
[19]Huang J,Zhou W,Dong W,Watson AM,Hong Y.Directed,efficient,and versatile modifications of the Drosophila genome by genomic engineering.Proc Natl Acad Sci USA,2009,106(20):8284-8289.
[20]Tang JB,Cao HW,Xu R,Zhang DD,Huang J.Mutant generation of the testis genes and phenotype analyses in Drosophila.Hereditas(Beijing),2018,40(6):478-487.唐浚博,曹浩伟,许蕊,张丹丹,黄娟.果蝇睾丸基因敲除突变体的构建及表型分析.遗传,2018,40(6):478-487.
[21]Ren YX,Xiao RD,Lou XM,Fang XD.Research advance and application in the gene therapy of gene editing technologies.Hereditas(Beijing),2019,41(1):18-27.任云晓,肖茹丹,娄晓敏,方向东.基因编辑技术及其在基因治疗中的应用.遗传,2019,41(1):18-27.
[22]Li X,Bai Y,Cheng X,Kalds PGT,Sun B,Wu Y,Lv H,Xu K,Zhang Z.Efficient SSA-mediated precise genome editing using CRISPR/Cas9.FEBS J,2018,285(18):3362-3375.
[23]Lamb AM,Walker EA,Wittkopp PJ.Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.Fly(Austin),2017,11(1):53-64.
[2]Jinek M,Chylinski K,Fonfara I,Hauer M,Doudna JA,Charpentier E.A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity.Science,2012,337(6096):816-821.
[3]Mali P,Yang L,Esvelt KM,Aach J,Guell M,DiCarlo JE,Norville JE,Church GM.RNA-guided human genome engineering via Cas9.Science,2013,339(6121):823-826.
[4]Miyaoka Y,Berman JR,Cooper SB,Mayerl SJ,Chan AH,Zhang B,Karlin-Neumann GA,Conklin BR.Systematic quantification of HDR and NHEJ reveals effects of locus,nuclease,and cell type on genome-editing.Sci Rep,2016,6:23549.
[5]van der Weyden L,Adams DJ,Bradley A.Tools for targeted manipulation of the mouse genome.Physiol Genomics,2002,11(3):133-164.
[6]Cary LC,Goebel M,Corsaro BG,Wang HG,Rosen E,Fraser MJ.Transposon mutagenesis of baculoviruses:analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.Virology,1989,172(1):156-169.
[7]Wang HH,Fraser MJ,Cary LC.Transposon mutagenesis of baculoviruses:analysis of TFP3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis viruses.Gene,1989,81(1):97-108.
[8]Handler AM,Harrell RA 2nd.Germline transformation of Drosophila melanogaster with the piggyBac transposon vector.Insect Mol Biol,1999,8(4):449-457.
[9]Lobo N,Li X,Fraser MJ,Jr.Transposition of the piggyBac element in embryos of Drosophila melanogaster,aedes aegypti and trichoplusia ni.Mol Gen Genet,1999,261(4-5):803-810.
[10]Ding S,Wu X,Li G,Han M,Zhuang Y,Xu T.Efficient transposition of the piggyBac(PB)transposon in mammalian cells and mice.Cell,2005,122(3):473-483.
[11]Wu SC,Meir YJ,Coates CJ,Handler AM,Pelczar P,Moisyadi S,Kaminski JM.PiggyBac is a flexible and highly active transposon as compared to sleeping beauty,Tol2,and Mos1 in mammalian cells.Proc Natl Acad Sci USA,2006,103(41):15008-15013.
[12]Wang G,Yang L,Grishin D,Rios X,Ye LY,Hu Y,Li K,Zhang D,Church GM,Pu WT.Efficient,footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.Nat Protoc,2017,12(1):88-103.
[13]Yusa K.Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon.Nat Protoc,2013,8(10):2061-2078.
[14]Zheng W,Feng G,Ren D,Eberl DF,Hannan F,Dubald M,Hall LM.Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform.J Neurosci,1995,15(2):1132-1143.
[15]Kanamori T,Kanai MI,Dairyo Y,Yasunaga K,Morikawa RK,Emoto K.Compartmentalized calcium transients trigger dendrite pruning in Drosophila sensory neurons.Science,2013,340(6139):1475-1478.
[16]Hara Y,Koganezawa M,Yamamoto D.The Dmca1D channel mediates Ca(2+)inward currents in Drosophila embryonic muscles.J Neurogenet,2015,29(2-3):117-123.
[17]Limpitikul WB,Viswanathan MC,O'Rourke B,Yue DT,Cammarato A.Conservation of cardiac L-type Ca(2+)channels and their regulation in Drosophila:a novel genetically-pliable channelopathic model.J Mol Cell Cardiol,2018,119:64-74.
[18]Ren X,Sun J,Housden BE,Hu Y,Roesel C,Lin S,Liu LP,Yang Z,Mao D,Sun L,Wu Q,Ji JY,Xi J,Mohr SE,Xu J,Perrimon N,Ni JQ.Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.Proc Natl Acad Sci USA,2013,110(47):19012-19017.
[19]Huang J,Zhou W,Dong W,Watson AM,Hong Y.Directed,efficient,and versatile modifications of the Drosophila genome by genomic engineering.Proc Natl Acad Sci USA,2009,106(20):8284-8289.
[20]Tang JB,Cao HW,Xu R,Zhang DD,Huang J.Mutant generation of the testis genes and phenotype analyses in Drosophila.Hereditas(Beijing),2018,40(6):478-487.唐浚博,曹浩伟,许蕊,张丹丹,黄娟.果蝇睾丸基因敲除突变体的构建及表型分析.遗传,2018,40(6):478-487.
[21]Ren YX,Xiao RD,Lou XM,Fang XD.Research advance and application in the gene therapy of gene editing technologies.Hereditas(Beijing),2019,41(1):18-27.任云晓,肖茹丹,娄晓敏,方向东.基因编辑技术及其在基因治疗中的应用.遗传,2019,41(1):18-27.
[22]Li X,Bai Y,Cheng X,Kalds PGT,Sun B,Wu Y,Lv H,Xu K,Zhang Z.Efficient SSA-mediated precise genome editing using CRISPR/Cas9.FEBS J,2018,285(18):3362-3375.
[23]Lamb AM,Walker EA,Wittkopp PJ.Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.Fly(Austin),2017,11(1):53-64.