Single base editing system mediates site-directed mutagenesis of genes <i>GDF9</i> and <i>FecB</i> in Ouler Tibetan sheep.

Yifan Zhao; Yingbing Zhang; Ruiluan YU; Ying WU; Yongzhong Chen; Ruolin Zhao; Chengtu Zhang; Jianmin SU

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Single base editing system mediates site-directed mutagenesis of genes GDF9 and FecB in Ouler Tibetan sheep.

Author: Yifan ZHAO ¹ ; Yingbing ZHANG ¹ ; Ruiluan YU ¹ ; Ying WU ² ; Yongzhong CHEN ² ; Ruolin ZHAO ¹ ; Chengtu ZHANG ² ; Jianmin SU ¹
Author Information

1. Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China.
2. Xining Center for Animal Disease Prevention and Control, Xining 810016, Qinghai, China.
Publication Type:Journal Article
Keywords: FecB gene; GDF9 gene; Ouler Tibetan sheep; single base editing system
MeSH: Animals; Sheep/genetics*; Gene Editing; Tibet; Mutation; Phenotype; Mutagenesis, Site-Directed
From: Chinese Journal of Biotechnology 2023;39(1):204-216
CountryChina
Language:Chinese
Abstract: In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.