MEK抑制剂PD0325901显著提高猪胎儿成纤维细胞ssODN介导的HDR效率

doi:10.16288/j.yczz.18-294

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Hereditas(Beijing) ›› 2019, Vol. 41 ›› Issue (4): 327-336.doi: 10.16288/j.yczz.18-294

• Research Article • Previous Articles Next Articles

MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts

Hao Ou¹,Guoling Li¹,Haoqiang Wang¹,Guangyan Huang¹,Gengyuan Cai^1,²,Zicong Li¹,Zhenfang Wu^1,²,Xianwei Zhang^1,²()

^1. National Engineering Research Center for Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China
^2. Wens Foodstuff Group Co., Ltd, Xinxing 527400, China

Received:2018-10-29 Revised:2019-03-02 Online:2019-04-20 Published:2019-03-25
Contact: Zhang Xianwei E-mail:zxianw@163.com
Supported by:
the National Natural Science Foundation of China(31772555);the National Transgenic Major Projects(2016ZX08006002)

Abstract

Abstract:

There are two major pathways, homology-directed repair (HDR) and nonhomologous end joining (NHEJ), involved in double-strand break (DSB) repair. Single-stranded oligodeoxyribonucleotide (ssODN)-mediated homologous recombination repair is commonly used for animal site-directed genome editing, with great scientific and practical value. To improve ssODN-mediated HDR efficiency in the pig genome, we investigated the effect and molecular mechanism of mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor PD0325901 on the HDR efficiency in porcine fetal fibroblasts (PFFs). The results showed that PD0325901 obviously increased the percentage of G₂ and S phase cell populations and reduced the cell population ratio in the G₁ phase of PFFs, and promoted the expression of HDR repair factor. At the optimal concentration of 250 nmol/L, PD0325901 increased the repair efficiency of ssODN-mediated GFP reporter vector by 58.8% and the directed editing efficiency of PFF DMD and ROSA26 locus by 48.16% and 17.64%, respectively. The results show that MEK inhibitor PD0325901 significantly promotes the efficiency of ssODN-mediated homologous-directed repair in the porcine genome, thus offering a new idea to generate genetically modified pigs more effectively.

Key words: MEK inhibitor, homologous-directed repair (HDR), PD0325901, gene editing

Hao Ou,Guoling Li,Haoqiang Wang,Guangyan Huang,Gengyuan Cai,Zicong Li,Zhenfang Wu,Xianwei Zhang. MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts[J]. Hereditas(Beijing), 2019, 41(4): 327-336.

References

[1]	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.
[2]	Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U . Breaking the code of DNA binding specificity of TAL-typeⅢeffectors. Science, 2009,326(5959):1509-1512.
[3]	Hsu PD, Lander ES, Zhang F . Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014,157(6):1262-1278.
[4]	Xu S, Cao S, Zou B, Yue Y, Gu C, Chen X, Wang P, Dong X, Xiang Z, Li K, Zhu M, Zhao Q, Zhou G . An alternative novel tool for DNA editing without target sequence limitation: the structure-guided nuclease. Genome Biol, 2016,17(1):186.
[5]	Huang YQ, Li GL, Yang HQ, Wu ZF . Progress and application of genome-edited pigs in biomedical research. Hereditas(Beijing), 2018,40(8):632-646.
[5]	黄耀强, 李国玲, 杨化强, 吴珍芳 . 基因编辑猪在生物医学研究中的应用. 遗传, 2018,40(8):632-646.
[6]	Difilippantonio MJ, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE, Ried T, Nussenzweig A . DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature, 2000,404(6777):510-514.
[7]	Capecchi MR . Altering the genome by homologous recombination. Science, 1989,244(4910):1288-1292.
[8]	Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis G . High- frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods, 2011,8(9):753-755.
[9]	Shen B, Zhang X, Du Y, Wang J, Gong J, Zhang X, Tate PH, Li H, Huang X, Zhang W . Efficient knockin mouse generation by ssDNA oligonucleotides and zinc-finger nuclease assisted homologous recombination in zygotes. PLoS One, 2013,8(10):e77696.
[10]	Hu Z, Shi Z, Guo X, Jiang B, Wang G, Luo D, Chen Y, Zhu YS . Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells. Cell Biosci, 2018,8(1):12.
[11]	Richardson CD, Kazane KR, Feng SJ, Zelin E, Bray NL, Schäfer AJ, Floor SN, Corn JE . CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat Genet, 2018,50(8):1132-1139.
[12]	Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL . Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2015,33(5):538-542.
[13]	Ma X, Chen X, Jin Y, Ge W, Wang W, Kong L, Ji J, Guo X, Huang J, Feng XH, Fu J, Zhu S . Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells. Nat Commun, 2018,9(1):1303.

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MEK inhibitor PD0325901 significantly boosts ssODN-mediated HDR efficiency in porcine fetal fibroblasts

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