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Research Articles

Generation of site-specific mutant mice using the CRISPR/Cas9 system

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  • 1. Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China;
    2. Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China;
    3. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China

Received date: 2015-03-26

  Online published: 2015-10-20

Abstract

The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.

Cite this article

Min Bai, Qi Li, Yanjiao Shao, Yuanhua Huang, Dali Li, Yanlin Ma . Generation of site-specific mutant mice using the CRISPR/Cas9 system[J]. Hereditas(Beijing), 2015 , 37(10) : 1029 -1035 . DOI: 10.16288/j.yczz.15-127

References

[1] 傅继梁, 王铸钢. 基因工程小鼠. 上海: 上海科学技术出版社, 2006: 5-18.
[2] Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell , 1987, 51(3): 503-512.
[3] Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature , 1987, 330(6148): 576-578.
[4] 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.
[5] 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.
[6] Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res , 2014, 24(1): 142-153.
[7] Burgess DJ. Technology: a CRISPR genome-editing tool. Nat Rev Genet , 2013, 14(2): 80-81.
[8] 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.
[9] Li W, Teng F, Li TD, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol , 2013, 31(8): 684-686.
[10] Hou ZG, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis . Proc Natl Acad Sci USA , 2013, 110(39): 15644-15649.
[11] Shen B, Zhang J, Wu HY, Wang JY, Ma K, Li Z, Zhang XG, Zhang PM, Huang XX. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res , 2013, 23(5): 720-723.
[12] Ma YW, Zhang X, Shen B, Lu YD, Chen W, Ma J, Bai L, Huang XX, Zhang LF. Generating rats with conditional alleles using CRISPR/Cas9. Cell Res , 2014, 24(1): 122-125.
[13] Wang HY, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell , 2013, 153(4): 910-918.
[14] Taylor GK, Heiter DF, Pietrokovski S, Stoddard BL. Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family. Nucleic Acids Res , 2011, 39(22): 9705-9719.
[15] Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli . Nucleic Acids Res , 2011, 39(21): 9275-9282.
[16] Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct , 2011, 6: 38.
[17] Platt RJ, Chen SD, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer R, Anderson DG, Hacohen N, Regev A, Feng GP, Sharp PA, Zhang F. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell , 2014, 159(2): 440-455.
[18] Shen B, Zhang WS, Zhang J, Zhou JK, Wang JY, Chen L, Wang L, Hodgkins A, Iyer V, Huang XX, Skarnes WC. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods , 2014, 11(4): 399-402.
[19] Yang H, Wang HY, Shivalila CS, Cheng AW, Shi LY, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell , 2013, 154(6): 1370-1379.
[20] Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y. Histone demethylation by a family of JmjC domain-containing proteins. Nature , 2006, 439(7078): 811-816.
[21] Han ZF, Liu PY, Gu LC, Zhang Y, Li H, Chen S, Chai JJ. Structural basis for histone demethylation by JHDM1. Front Sci , 2007, (1): 52-61.
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