三种CRISPR/Cas9基因敲除变异检测方法的比较分析
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摘要
比较三种常用的CRISPR/Cas9基因敲除检测变异方法,从准确性、耗时、成本和应用限制等方面进行评估。采用T7核酸内切酶I、限制性内切酶和非变性聚丙烯酰胺凝胶电泳法分别对利用CRISPR/Cas9方法敲除的fibinb和ngs基因的50尾斑马鱼(Danio rerio)突变纯合个体和突变杂合个体进行检测,用野生型个体作对照;同时用非变性聚丙烯酰胺凝胶电泳对CRISPR/Cas9方法敲除的ogn和ddb1基因的突变杂合个体进行了检测。结果表明:三种方法均能够区分突变杂合个体,其中T7核酸内切酶I检测法耗时最短,但该方法不能用于鉴定突变纯合个体;限制性内切酶检测法能够用于鉴定纯合个体,但需要有特异性的识别位点;非变性聚丙烯酰胺凝胶电泳法能够区分纯合和杂合个体,对序列无依赖性,能够检测出序列中存在的所有变异。成本上,非变性聚丙烯酰胺凝胶电泳法最经济;限制性内切酶的价格存在较大的差异,因此检测成本依赖于酶的价格。三种检测方法各有优缺点,在应用实践中可根据试验需要选择合适的方法。
The accuracy, time consuming and limitations on use of 3 methods were evaluated in this study. to compare three different detecting methods for gene mutation using the conventional CRISPR/Cas9 system. CRISPR/Cas9 method was used to knockdown the fibinb and ngs gene in Danio rerio and the gene mutation of 50 homozygous and 50 heterozygous individuals was detected using T7 endonuclease I method, restriction enzyme method and non-denaturing PAGE method, respectively. The wild type individuals were used as the control. The results indicated that all of the three methods could distinguish the heterozygous from homozygous individuals. T7 endonuclease I method had the shortest time-consuming but could not be applied into the identification of mutant homozygous individuals. Restriction enzyme method could be used to identify the homozygous individuals depending on the specific recognition sites. However, parts of sites could not be detected using the restriction enzymes. Non-denaturing PAGE could distinguish the homozygous from heterozygous individuals, and this method was sequence-independent and could detect all mutations in the sequences. Non-denaturing PAGE was the most economical in three methods based on the cost standpoint. There were great differences on the costs of restriction enzyme method depending on the price of enzymes. The three detection methods all have their own advantages and disadvantages so that the appropriate method should be chosen according to the experimental requirements in practical application.
The accuracy, time consuming and limitations on use of 3 methods were evaluated in this study. to compare three different detecting methods for gene mutation using the conventional CRISPR/Cas9 system. CRISPR/Cas9 method was used to knockdown the fibinb and ngs gene in Danio rerio and the gene mutation of 50 homozygous and 50 heterozygous individuals was detected using T7 endonuclease I method, restriction enzyme method and non-denaturing PAGE method, respectively. The wild type individuals were used as the control. The results indicated that all of the three methods could distinguish the heterozygous from homozygous individuals. T7 endonuclease I method had the shortest time-consuming but could not be applied into the identification of mutant homozygous individuals. Restriction enzyme method could be used to identify the homozygous individuals depending on the specific recognition sites. However, parts of sites could not be detected using the restriction enzymes. Non-denaturing PAGE could distinguish the homozygous from heterozygous individuals, and this method was sequence-independent and could detect all mutations in the sequences. Non-denaturing PAGE was the most economical in three methods based on the cost standpoint. There were great differences on the costs of restriction enzyme method depending on the price of enzymes. The three detection methods all have their own advantages and disadvantages so that the appropriate method should be chosen according to the experimental requirements in practical application.
引文
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[16]王晓通,连林生,赵春江,等.非变性聚丙烯酰胺凝胶电泳中与杂合子相伴产生的非目的条带的鉴定[J].农业生物技术学报,2011,18(3):616-622.
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[3]Barrangou R.Cas9 targeting and the CRISPR revolution [J].Science,2014,344(6185):707-708.
[4] Matthew H L,Lei S Q,Luke A G,et al.CRISPR interference (CRISPRi) for sequence-specific control of gene expression [J].Nat Prot,2013,8(11):2180-2196.
[5] Carlson D F,Walton M W,Fahrenkrug S C.Precision editing of large animal genomes [J].Adv Genet,2012,80:37-97.
[6] Woong Y H,Yan F F,Deepak R,et al.Efficient genome editing in zebrafish using a CRISPR-Cas system [J].Nat Biotechnol,2013,31(3):227-229.
[7] Seung W C,Sojung K,Jong M K,et al.Targeted genome engineering in human cells with the cas9 RNA-guided endonuclease [J].Nat Biotechnol,2013,31(3):230-232.
[8] Doudna J A,Charpentier E.Genome editing.The new frontier of genome engineering with CRISPR-Cas9 [J].Science,2014,346(6213):1258096.
[9] 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 [J].Cell,2013,153:910-918.
[10] Crispo M,Mulet A P,Tesson L,et al.Efficient generation of myostatin knock-out sheep using CRISPR/Cas9technology and microinjection into zygotes [J].PLoS One,2015,10(8):366-375.
[11] Wang X,Yu H,Lei A,et al.Generation of gene-modified goats targeting MSTN and FGF5via zygote injection of CRISPR/Cas9 system [J].Sci Rep,2015,(5):13878.
[12]潘洪杏,刘侠,万秀清,等.利用CRISPR-Cas9基因组编辑技术定向敲除烟草eIF4E-6基因[J].分子植物育种,2017,15(2):538-544.
[13]李国玲,钟翠丽,倪生,等.利用CRISPR/Cas9系统建立Xist基因敲除猪模型[J].遗传,2016,38(12):1081-1089.
[14]董文洁,杨远勤,方先龙,等.利用CRISPR/Cas9基因编辑系统构建TP53基因敲除HeLa细胞系[J].中国细胞生物学学报,2016,38(9):1-9.
[15]黄振玉,竹个个,邹华锋,等.利用CRISPR/Cas9敲除斑马鱼细胞周期蛋白M2(CNNM2)基因[J].上海海洋大学学报,2015,24(6):810-816.
[16]王晓通,连林生,赵春江,等.非变性聚丙烯酰胺凝胶电泳中与杂合子相伴产生的非目的条带的鉴定[J].农业生物技术学报,2011,18(3):616-622.
[17]徐湛宁,李福贵,陈杰,等.草鱼鳃组织蛋白双向电泳技术的建立及优化[J].淡水渔业,2017,47(5):14-20,46.
[18]向小华,焦禹顺,吴新儒,等.一种从非变性聚丙烯凝胶中回收小片段DNA的方法[J].生物技术通报,2015,31(5):68-72.