title: Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system.
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.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Jin-Qing
authors.initials: JQ
authors.lastname: Wu
authors.affiliation: Institute of Animal Science, Guangdong Academy of Agricultural Sciences.
authors.forename: Gui
authors.initials: G
authors.lastname: Mei
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Zhi-Guo
authors.initials: ZG
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Yao-Sheng
authors.initials: YS
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Pei-Qing
authors.initials: PQ
authors.lastname: Cong
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Zu-Yong
authors.initials: ZY
authors.lastname: He
mesh_terms: D000818:Animals; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005818:Genetic Engineering; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013552:Swine / Q000235:genetics* / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D007335:Insulin-Like Growth Factor II; D003851:Deoxyribonucleases