应用SSA报告载体提高ZFN和CRISPR/Cas9对猪IGF2基因的打靶效率

doi:10.16288/j.yczz.2015.01.008

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HEREDITAS(Beijing) ›› 2015, Vol. 37 ›› Issue (1): 55-62.doi: 10.16288/j.yczz.2015.01.008

• Research Articles • Previous Articles Next Articles

Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system

Jinqing Wu¹, Gui Mei², Zhiguo Liu¹, Yaosheng Chen¹, Peiqing Cong¹, Zuyong He¹

1. State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China; 2. Institute of Animal Science, Guangdong Academy of Agricultural Sciences; State Key Laboratory of Livestock and Poultry Breeding, Guangzhou 510640, China

Received:2014-06-19 Revised:2014-08-29 Online:2015-01-20 Published:2015-01-20

Abstract

Abstract: IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal development. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed——Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected population was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) reporter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay revealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.

Key words: IGF2 gene, ZFN, CRISPR/Cas9, SSA reporter system

Jinqing Wu, Gui Mei, Zhiguo Liu, Yaosheng Chen, Peiqing Cong, Zuyong He. Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system[J]. HEREDITAS(Beijing), 2015, 37(1): 55-62.

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Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system

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