基于SSA修复机制和特异性核酸酶活性检测的双荧光报告载体系统的开发及应用

doi:10.16288/j.yczz.15-246

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HEREDITAS(Beijing) ›› 2015, Vol. 37 ›› Issue (10): 1053-1060.doi: 10.16288/j.yczz.15-246

• Techniques and Methods • Previous Articles Next Articles

Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism

Furong Han¹， Ling Wang^1，2， Kun Xu¹， Zhiying Zhang¹， Xin Wang¹

1. College of Animal Science & Technology, Northwest A&F University, Yangling 712100, China;
2. School of Biological Science and Engineering, Shaanxi University of technology, Hanzhong 723000, China

Received:2015-06-01 Online:2015-10-20 Published:2015-10-20

Abstract

Abstract: Reporter vector system has become an important method for measuring activity of specific nucleases because of its fast construction, simple modification, easy operation, economic effectiveness as well as its role in enriching positive cells with genomic modification through mediating screen of specific nucleases positive cells. After introducing double strand breaks (DSBs), a reporter system based on non-homologous end joining (NHEJ)-mediated repair can only repair maximally two thirds of reporter genes after optimization, while single strand annealing (SSA)-mediated repair can repair all reporter genes theoretically which has higher sensitivity and facilitates the detection of specific nuclease with low activity and provides an effective way to detect specific nuclease activity in genome modification studies. In this study, we designed and constructed three sets of dual-fluorescence reporter systems based on SSA repair mechanism and applied the mRFP-eGFP system in measuring the effective activity of three pairs of ZFNs, which was 8.9%, 9.3% and 5.0%, respectively. Our study provides an effective way to detect the activity of nucleases.

Key words: SSA, dual-fluorescence reporter system, specific nucleases

Furong Han， Ling Wang， Kun Xu， Zhiying Zhang， Xin Wang. Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism[J]. HEREDITAS(Beijing), 2015, 37(10): 1053-1060.

References

[1] Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran S. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res , 2005, 33(18): 5978-5990.
[2] Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol , 2001, 21(1): 289-297.
[3] Choulika A, Perrin A, Dujon B, Nicolas JF. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol , 1995, 15(4): 1968-1973.
[4] Plessis A, Perrin A, Haber JE, Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics , 1992, 130(3): 451-460.
[5] Bellaiche Y, Mogila V, Perrimon N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics , 1999, 152(3): 1037-1044.
[6] Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA , 1996, 93(3): 1156-1160.
[7] Choo Y, Isalan M. Advances in zinc finger engineering. Curr Opin Struct Biol , 2000, 10(4): 411-416.Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanov AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics , 2010, 186(2): 757-761.
[8] Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM. TALEN and CRISPR/Cas genome editing systems: tools of discovery. Acta Naturae , 2014, 6(3): 19-40.
[9] Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science , 2012, 337(6096): 816-821.
[10] Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu XB, Jiang WY, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science , 2013, 339(6121): 819-823.
[11] Mali P, Yang LH, 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.
[12] 吴金青, 梅瑰, 刘志国, 陈瑶生, 丛佩清, 何祖勇. 应用SSA报告载体提高ZFN和CRISPR/Cas9对猪 IGF2 基因的打靶效率. 遗传, 2015, 37(1): 55-62.
[13] Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science , 2003, 300(5620): 763.
[14] Kim H, Um E, Cho SR, Jung C, Kim H, Kim JS. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods , 2011, 8(11): 941-943.
[15] Ramakrishna S, Cho SW, Kim S, Song M, Gopalappa R, Kim JS, Kim H. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun , 2014, 5: 3378.
[16] Wang L, Lin J, Zhang TT, Xu K, Ren CH, Zhang ZY. Simultaneous screening and validation of effective zinc finger nucleases in yeast. PLoS One , 2013, 8(5): e64687.
[17] Ren CH, Xu K, Liu ZT, Shen JC, Han FR, Chen ZL, Zhang ZY. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol Life Sci , 2015, 72(14): 2763-2772.
[18] Xu K, Ren CH, Liu ZT, Zhang TT, Li D, Wang L, Yan Q, Guo LJ, Shen JC, Zhang ZY. Efficient genome engineering in eukaryotes using Cas9 from Streptococcus thermophilus . Cell Mol Life Sci , 2015, 72(2): 383-399.
[19] 王令. 锌指核酸酶的构建及其在动物基因组编辑中的应用[学位论文]. 杨凌: 西北农林科技大学, 2013.
[20] Kuhar R, Gwiazda KS, Humbert O, Mandt T, Pangallo J, Brault M, Khan I, Maizel N, Rawlings DJ, Scharenberg AM, Certo MT. Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks. Nucleic Acids Res , 2014, 42(1): e4.

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Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism

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