受精卵注射CRISPR/Cas9系统制备基因编辑子午岭黑山羊技术体系的建立
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摘要
受精卵注射CRISPR/Cas9系统是制备基因编辑动物的有效方法,其中受精卵收集效率、显微注射胚胎存活率及胚胎移植受体妊娠率是关键影响因素.以子午岭黑山羊为研究对象探讨了情期内不同时间点放置CIDR对超数排卵的影响;分析了胚胎供体和受体发情停止时间差异对胚胎移植妊娠率的影响;设计了3个打靶肌肉生长抑制素基因(MSTN)的小向导RNA(sgRNA),并优化了CRISPR/Cas9系统显微注射参数.结果表明,胚胎供体发情停止后第4天放置阴道栓可有效提高黄体数(15.25±3.69)和受精卵数量(13.875±4.015);利用微量注射泵将Cas9mRNA(60 ng/μL)和打靶MSTN基因的sgRNAs(60 ng/μL)混合液注射到受精卵胞质内,注射参数Pi,Ti和Pc分别为300,0.5和60,注射24 h后胚胎分裂率为76.79%.胚胎移植时受体发情结束时间晚于供体发情结束时间12~16 h妊娠率最高(38.46%).本研究共得到19个存活的个体,经TA克隆测序分析,所有个体MSTN打靶位点上均发现碱基的插入或缺失.综上,本研究以子午岭黑山羊为例,建立了受精卵注射CRISPR/Cas9系统制备基因编辑山羊的技术体系,为利用CRISPR/Cas9技术进行山羊的遗传改造奠定了技术基础.
Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals.Efficiency of zygote collection,embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy.Here,we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation,and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats.We designed three sgRNAs targeting myostatin(MSTN) and also optimized the microinjected parameter of the CRISPR/Cas9 system.The results showed that embryo donor goats effectively increase the corpus luteum amount(15.25±3.69) and zygote amount(13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle.The mixture including Cas9 mRNA(60 ng/μL) and sgRNAs(60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters(Pi,Ti and Pc was 300,0.5 and 60,respectively).Embryo cleavage rate was76.79 % after 24 h of embryo injection.Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest(38.46%).A total of 19 living lambs were obtained,and indels were detected in all the lambs using TA cloning sequencing.Overall,we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats.The established technological system is the basic for generation of gene modified goats.
Zygote injection of CRISPR/Cas9 system is an efficient method for generation of gene modified animals.Efficiency of zygote collection,embryo survival rate after micro-injection and pregnancy rate of surrogate are the key influence factors for the generation of gene modified animals using this strategy.Here,we investigated the influence of CIDR treatment at different time during the estrus cycle to the superovulation,and analyzed the influence of the estrus terminal time between embryo donor and embryo receptor to the pregnancy rate in Ziwuling black goats.We designed three sgRNAs targeting myostatin(MSTN) and also optimized the microinjected parameter of the CRISPR/Cas9 system.The results showed that embryo donor goats effectively increase the corpus luteum amount(15.25±3.69) and zygote amount(13.875±4.015) when they were treated with CIDR at the fourth day of estrus cycle.The mixture including Cas9 mRNA(60 ng/μL) and sgRNAs(60 ng/μL) targeting MSTN was injected into zygote cytoplasm using a micromanipulator at the optimized injection parameters(Pi,Ti and Pc was 300,0.5 and 60,respectively).Embryo cleavage rate was76.79 % after 24 h of embryo injection.Pregnancy rate of embryo receptors whose terminal estrus time was delayed 12–16 h compared with that of embryo donor was the highest(38.46%).A total of 19 living lambs were obtained,and indels were detected in all the lambs using TA cloning sequencing.Overall,we established the technological system based on the zygote injection of CRISPR/Cas9 in Ziwuling black goats.The established technological system is the basic for generation of gene modified goats.
引文
1 Li W,Teng F,Li T,et al.Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems.Nat Biotechnol,2013,31:684–686
2 Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,2013,153:910–918
3 Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339:819–823
4 Mali P,Yang L,Esvelt K M,et al.RNA-guided human genome engineering via Cas9.Science,2013,339:823–826
5 Cho S W,Kim S,Kim J M,et al.Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol,2013,31 :230–232
6 Yang H,Wang H,Shivalila C S,et al.One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell,2013,154:1370–1379
7 Hwang W Y,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol,2013,31:227–229
8 Niu Y,Shen B,Cui Y,et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.Cell,2014,156:836–843
9 康难难,陈红全,高基民.利用猪单个胚胎建立高效快速的CRISPR/Cas9打靶位点检测方法.中国科学:生命科学,2016,46:304–313
10 严芳,周焕斌.CRISPR/Cas9技术在植物基因功能研究和新种质创制中的应用与展望.中国科学:生命科学,2016,46:498–513
11 陈晨,郝莎,白杨,等.CRISPR/Cas9-sg RNA全基因组文库筛选人单核细胞白血病功能性促癌/抑癌基因体系的建立与优化.中国科学:生命科学,2016,46:839–850
12 Mc Pherron A C,Lee S J.Double muscling in cattle due to mutations in the myostatin gene.Proc Natl Acad Sci USA,1997,94:12457–12461
13 Mosher D S,Quignon P,Bustamante C D,et al.A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs.PLo S Genet,2007,3:e79
14 Shen B,Zhang W,Zhang J,et al.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.Nat Meth,2014,11:399 –402
15 Mc Pherron A C,Lawler A M,Lee S J.Regulation of skeletal muscle mass in mice by a new TGF-p superfamily member.Nature,1997,387:83–90
16 Boman I A,Klemetsdal G,Blichfeldt T,et al.A frameshift mutation in the coding region of the myostatin gene(MSTN)affects carcass conformation and fatness in Norwegian White Sheep(Ovis aries).Animal Genet,2009,40:418–422
17 Schuelke M,Wagner K R,Stolz L E,et al.Myostatin mutation associated with gross muscle hypertrophy in a child.N Engl J Med,2004,350:2682–2688
18 Kambadur R,Sharma M,Smith T P L,et al.Mutations in myostatin(GDF8)in double-muscled belgian blue and piedmontese cattle.Genome Res,1997,7:910–915
19 Wang X,Niu Y,Zhou J,et al.Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.Sci Rep,2016,6:32271
20 Wang X,Yu H,Lei A,et al.Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system.Sci Rep,2015,5:13878
21 皮文辉,石国庆,刘守仁.羊用氟孕酮阴道海绵栓的制作及使用效果.中国畜牧杂志,2004,1:41
22 张效生,冯建忠,张金龙,等.羊用硅橡胶孕酮栓的制作及使用效果.黑龙江畜牧兽医,2010,9:54–55
23 聂竞舟,安磊,田见晖.绵羊胚胎移植受体同期发情方法的比较.黑龙江动物繁殖,2013,1:25–28
24 毛杨毅,罗惠娣,贺东昌,等.不同季节绒山羊同期发情效果研究.家畜生态学报,2012,5:42–45
2 Wang H,Yang H,Shivalila C S,et al.One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell,2013,153:910–918
3 Cong L,Ran F A,Cox D,et al.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339:819–823
4 Mali P,Yang L,Esvelt K M,et al.RNA-guided human genome engineering via Cas9.Science,2013,339:823–826
5 Cho S W,Kim S,Kim J M,et al.Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol,2013,31 :230–232
6 Yang H,Wang H,Shivalila C S,et al.One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell,2013,154:1370–1379
7 Hwang W Y,Fu Y,Reyon D,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat Biotechnol,2013,31:227–229
8 Niu Y,Shen B,Cui Y,et al.Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.Cell,2014,156:836–843
9 康难难,陈红全,高基民.利用猪单个胚胎建立高效快速的CRISPR/Cas9打靶位点检测方法.中国科学:生命科学,2016,46:304–313
10 严芳,周焕斌.CRISPR/Cas9技术在植物基因功能研究和新种质创制中的应用与展望.中国科学:生命科学,2016,46:498–513
11 陈晨,郝莎,白杨,等.CRISPR/Cas9-sg RNA全基因组文库筛选人单核细胞白血病功能性促癌/抑癌基因体系的建立与优化.中国科学:生命科学,2016,46:839–850
12 Mc Pherron A C,Lee S J.Double muscling in cattle due to mutations in the myostatin gene.Proc Natl Acad Sci USA,1997,94:12457–12461
13 Mosher D S,Quignon P,Bustamante C D,et al.A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs.PLo S Genet,2007,3:e79
14 Shen B,Zhang W,Zhang J,et al.Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects.Nat Meth,2014,11:399 –402
15 Mc Pherron A C,Lawler A M,Lee S J.Regulation of skeletal muscle mass in mice by a new TGF-p superfamily member.Nature,1997,387:83–90
16 Boman I A,Klemetsdal G,Blichfeldt T,et al.A frameshift mutation in the coding region of the myostatin gene(MSTN)affects carcass conformation and fatness in Norwegian White Sheep(Ovis aries).Animal Genet,2009,40:418–422
17 Schuelke M,Wagner K R,Stolz L E,et al.Myostatin mutation associated with gross muscle hypertrophy in a child.N Engl J Med,2004,350:2682–2688
18 Kambadur R,Sharma M,Smith T P L,et al.Mutations in myostatin(GDF8)in double-muscled belgian blue and piedmontese cattle.Genome Res,1997,7:910–915
19 Wang X,Niu Y,Zhou J,et al.Multiplex gene editing via CRISPR/Cas9 exhibits desirable muscle hypertrophy without detectable off-target effects in sheep.Sci Rep,2016,6:32271
20 Wang X,Yu H,Lei A,et al.Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system.Sci Rep,2015,5:13878
21 皮文辉,石国庆,刘守仁.羊用氟孕酮阴道海绵栓的制作及使用效果.中国畜牧杂志,2004,1:41
22 张效生,冯建忠,张金龙,等.羊用硅橡胶孕酮栓的制作及使用效果.黑龙江畜牧兽医,2010,9:54–55
23 聂竞舟,安磊,田见晖.绵羊胚胎移植受体同期发情方法的比较.黑龙江动物繁殖,2013,1:25–28
24 毛杨毅,罗惠娣,贺东昌,等.不同季节绒山羊同期发情效果研究.家畜生态学报,2012,5:42–45