The Comparison of Three Methods of Monitoring Endogenous Gene Modification

doi:10.13560/j.cnki.biotech.bull.1985.2016.02.010

Abstract

Abstract: In orderto obtain accurate mutation information, 3 methods of monitoring the biological activities of artificial nuclease are preliminary determined excluding direct sequencing.The 3 methods of Surveyor nuclease, T7E1(T7 Endonuclease 1)and HRM(High resolution melting)are all based on the principle of forming the twisted duplex DNA from denaturized annealing mutant and wild type DNA sequence, and whether or not target sites were mutated were determined.The results showed that using the 3 detection methods, the mutations at the target sites of exon 1's CRISPR/Cas9 and exon 3's TALEN in ovine gene MNST were successfully detected.Analyzing and comparingthe results and characteristics of the 3 methods, the advantages and disadvantages of them were obtained, which provided a reference for the analysis and identification of results while the cells uses non-homologous ends to join and repair DNA double strand breaks.In conclusion, the results by Surveyor, T7E1 and HRM show that CRISPR/Cas9 and TALEN’s target sites are mutated.

Key words: genome editing, surveyor nuclease, T7 Endonuclease I, high resolution melting(HRM)

LI Wei-jie, YANG Jiao, HE Gao-ming, WANG Li-min, PI Wen-hui, ZHOU Ping. The Comparison of Three Methods of Monitoring Endogenous Gene Modification[J]. Biotechnology Bulletin, 2016, 32(2): 76-83.

References

[1]Huang MC, Cheong WC, Lim LS, et al.A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis[J].Electrophoresis, 2012, 33(5)：788-796.
[2]Qiu P, Shandilya H, D'Alessio JM, et al.MutationdetectionusingSurveyornuclease[J].Biotechniques, 2004, 36(4)：702-707.
[3]Kim HJ, Lee HJ, Kim H, et al.Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly[J].Genome Res, 2009, 19(7)：1279-1288.
[4]Zhou L, Wang L, Palais R.High-resolutionDNA melting analysis for simultaneous mutation scanning andgenotypinginsolution.Clin Chem,2005, 51(10)：1770-1777.
[5] Cui G, Zhang L, Xu Y, et al .Development of ahigh resolution melt-ing Developmentof a high resolution melting method for genotyping ofrisk HLA-DQA1 and PLA2R1 alleles and ethnicdistribution of theseriskalleles[J].Gene, 2013, 514(2)：125-130.
[6] Er TK, Kan TM, Su YF, et al.High-resolution melting(HRM)analysisas a feasible method for detectingspinalmuscular atrophyviadriedbloodspots.[J]Clin Chim Acta, 2012, 413(21-22)：1781-1785.
[7]Lin Y, Cradick TJ, Bao G.DesigningandtestingtheactivitiesofTALeffectornucleases[J].Methods Mol Biol, 2014, 1114：203-219.
[8]Tricarico R, Crucianelli F, Alvau A.Highresolutionmeltinganalysisfor a rapid identification of heterozygous and homozygous sequencechangesin theMUTYHgene[J].BMC Cancer, 2011, 11：305.
[9]Wittwer CT, Reed GH, Gundry CN.High-resolution genotyping by amplicon melting analysis using LCGreen[J].Clin Chem,2003, 49(6 Pt 1)：853-860.
[10]Zhou Y, Zhu S, Cai C, et al.High-throughputscreeningof a CRISPR/Cas9 library for functional genomics inhumancells[J].Nature, 2014, 509(7501)：487-491.
[11]Shalem O, Sanjana NE, Hartenian E, et al.Genome-scale CRISPR-Cas9 knockout screening in human cells[J].Science, 2014, 343(6166)：84-87.
[12]Tsuji T, Niida Y.Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories[J].Electrophoresis, 2008, 29(7)：1473-1483.
[13]Reed GH, Wittwer CT.Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis[J].Clin Chem, 2004, 50(10)：1748-1754.
[14]Liew M, Pryor R, Palais R.Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons[J].Clin Chem, 2004, 50(7)：1156-1164.
[15]张建佚, 冯洁, 杨泽, 等.高分辨率熔解曲线小扩增子法结合混样法在线粒体13928G > C突变分析中的应用[J].中国医药生物技术, 2009, 6：445-448.