Abstract：Abstract Myostatin (MSTN) is a negative regulator to muscle cells growth and differentiation. MSTN gene's mutation will lost its function, which will make animals have significantly more muscle mass. The purposes of this study are to find the best sgRNA which could edit sheep's (Ovis aries) MSTN gene efficiently and build EGFP and sgRNA co-expression vectors, with which CRISPR/Cas9 system could improve sheep MSTN gene's editing efficiency. First using Gibson Assembly method to incorporate the 2A+ enhanced green fluorescent protein (EGFP) into pX330, the study got the pX330-EGFP vector. Then 12 sgRNAs were designed and using Golden Gate method separately, inserted these single-guide RNA (sgRNA) oligonucleotides into pX330-EGFP plasmid, and 12 pX330-EGFP-sgRNA expression plasmids were got. The 12 pX330-EGFP-sgRNA vectors were transferred into sheep fibroblasts by electroporation. After 48 h, using SURVEROR analysis, it was be found that 3 groups (T2, T9, Q2) of cell's DNA were edited, which were groups of sgRNA. Then 100 green cells of each group were collected and extracted the DNA, after amplification of the MSTN gene by PCR, the productions were send for sequencing analysis. The results showed that the targeting efficiency of sgRNA-T2, sgRNA-T9 and sgRNA-Q2 were 40%, 40% and 60% respectively. In this study, we build EGFP and sgRNA co-expression vetors and selected the best sgRNA to sheep MSTN gene which was 60%. This protocol will be helpful to find more sgRNAs to different genes. These results provide a scientific basic for the production of MSTN gene editing sheep.
[1] Mcpherron A C, Lawler A M, Lee S J. Regulation of skeletal muscle mass in mice by a new TGF-psuperfamily member[J]. Nature, 1997, 387(6628)：83.[2] 潘英树, 张永宏, 高妍,等. 朗德鹅MSTN基因外显子3多态性的研究[J]. 东北农业科学, 2008, 33(6):30-31. [3] Thomas M, Langley B, Berry C, et al. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation.[J]. Journal of Biological Chemistry, 2000, 275(51):40235-43.[4] Mcpherron A C,Lee S J. Double muscling in cattle due to mutations in the myostatin?gene[J]. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(23)：12457-12461. [5] Clop A, Marcq F, Takeda H, et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.[J]. Nature genetics, 2006, 38(7)：813.[6] Grobet L, Martin LJ, Poncelet D, Pirottin D, Brouwers B, et al. (1997) A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet 17：71–74.[7] Marchitelli C, Savarese MC, Crisa A, Nardone A, Marsan PA, et al. (2003)Double muscling in Marchigiana beef breed is caused by a stop codon in the third exon of myostatin gene. Mamm Genome 14：392–395.[8] Grobet L, Poncelet D, Royo LJ, Brouwers B, Pirottin D, et al. (1998)Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle. Mamm Genome 9：210–213.[9] Karim L, Coppieters W, Grobet L, Valentini A, Georges M (2000) Convenient genotyping of six myostatin mutations causing double muscling cattle using a multiplex oligo nucleotide ligation assay. Anim Genet 31：396–399.[10] 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[J]. Plos Genetics, 2007, 3(5)： e79.[11] Daopin, S., Piez, K. A., Ogawa, Y. & Davies, D. R. (1992) Science257,369–373.[12] Schlunegger, M. P.&Gru¨tter, M. G. (1992) Nature (London) 358,430–434.[13] Griffith, D. L., Keck, P. C., Sampath, T. K., Rueger, D. C. &Carlson, W. D. (1996) Proc. Natl. Acad. Sci. USA 93, 878–883.[14] Mittl, P. R., Priestle, J. P., Cox, D. A., McMaster, G. Cerletti, N.& Gru¨tter, M. G. (1996) Protein Sci. 5, 1261–1271.[15] Mason, A. J. (1994) Mol. Endocrinol. 8, 325–332.[16] Mojica FJ,Diez-Villasenor C,Garcia-Martinez J,et al.Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements[J]. J Mol Evol,2005,60(2)：174-82[17] Jinek M, Chylinski K, Fonfara I, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science, 2012, 337(60 96)：816-821.[18] Auer TO, Duroure K, De Cian A, et al.Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homolog y independent DNA repair[J].Genome Res,2013,24(1)：142-153.[19] Hou Z, Zhang Y, Propson NE, et al. Efficient genome engineering in hum an pluri potent stem cells using Cas9 from Neisseria meningitidis [J].Proc N atl Acad Sci U S A, 2013,110(39)：15644-15649.[20] Li W, Teng F, Li T, et al. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems[J].Nat Bi otechnol, 2013, 31(8)：684-686.[21] Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol, 2011, 9(6)：467–477. [22] Cong L, Ran FA, Cox D, Lin SL, Barretto R, Habib N, Hsu PD, Wu X B, Jiang WY, Marraffini LA, Zhang F. Multiplex genome engineering usi ng CRISPR/Cas systems. Science, 2013, 339(6121)：819–823. [23] 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. [24] Wang GC, Ma M, Ye YZ, Xi JZ. High-throughput functional screening using CRISPR/Cas9 system. Here-ditas (Beijing), 2016, 38(5)：391–401. [25] Wang H, Al E. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.[J]. Cell, 2013, 153 (4)：910. [26] 马元武, 马婧, 路迎冬,等. 利用 CRISPR/Cas9敲除大鼠胰岛素受体底物1(Irs1)基因[J]. 中国比较医学杂志, 2014(3)：55-60.[27] Gupta A, Hall VL, Kok FO, Shin M, McNulty JC, Lawson ND, Wolfe S A. Targeted chromosomal deletions and inversions in zebrafish. Genome Res, 2013, 23(6)：1008–1017. [28] Xiao A, Wang ZX, Hu YY, Wu YD, Luo Z, Yang ZP, Zu Y, Li WY, Huang P, Tong XJ, Zhu ZY, Lin S, Zhang B. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res, 2013, 41(14)：e141.[29] Carroll D. Genome engineering with targetable nucleases. Annu Rev Biochem, 2014, 83：409–439. [30] Mojica FJ, Díez-Villase?or C, García-Martínez J, Almendros C. Short mot if sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology, 2009, 155(Pt 3)：733–740. [31] Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S. The CRISPR/Cas bacterial immunesystem cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320)：67–71. [32] Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-en coded small RNA and host factor RNase III. Nature, 2011, 471(7340)：602–607. [33] Fu YF, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013, 31(9)：822–826. [34] Pattanayak V, Lin S, Guilinger JP, Ma EB, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol, 2013, 31(9)：839–843. [35] Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).[36] Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).[37] Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell 154, 1380–1389 (2013). [38] Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).[39] Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014). [40] Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically in active Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).[41] Kleinstiver, B. P. et al. High-fidelity CRISPR Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016). [42] Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).[43] Osborn, M. J. et al. Evaluation of TCR gene editing achieved by TALEN s, CRISPR/Cas9 and megaTAL nucleases. Mol. Ther. 24, 570–581 (2016).[44] Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015). [45] Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015). [46] Chiarle, R. et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–1 19 (2011). [47] Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015). [48] Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013). [49] Kim, D. et al. Digenome-seq： genome-wide profiling of CRISPR Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015). [50] Tsai S Q, Joung J K. Defining and improving the genome-wide specificityes of CRISPR-Cas9 nucleases[J]. Nature Reviews Genetics, 2016, 17(5)：300.
