Efficient Editing of Mouse Galt Gene Based on CRISPR/cas9 System

doi:10.13560/j.cnki.biotech.bull.1985.2020-0237

Abstract

Abstract: GALT gene mutation is the main cause of human type I galactosemia. This paper intends to target the mouse Galt gene through the CRISPR/cas9 system for simulating human GALT gene mutation,so as to lay the foundation for the establishment of an accurate animal model of type I galactosemia. We first analyzed pathogenic mutation sites of the GALT gene of type I galactosemia in China,and designed the sgRNA-directed sequence of the specifically targeted mouse Galt gene that can mimic the pathogenic mutation. Then sgRNA expression plasmid was then constructed and co-transfected with the cas9 expression plasmid into mouse 3T3 cells. The positive transfected cells were selected by puromycin and blasticidin,and the genomic DNA of the positive cells was extracted and the DNA fragment of the target site was amplified by PCR. The gene editing was identified and the editing efficiency was analyzed by TA clone sequencing. The results showed that the three sgRNA-directed sequences designed in this efficiently edited the mouse Galt gene through the CRISPR/Cas9 system,and the editing efficiency was 100%.

Key words: sgRNA, CRISPR/Cas9, Galt, gene editing, galactosemia

YUE Peng-peng, GUO Jun-fan, YU Hong-hao, FU Can, WANG Xiao-yan, GAO Jin-tao. Efficient Editing of Mouse Galt Gene Based on CRISPR/cas9 System[J]. Biotechnology Bulletin, 2020, 36(8): 235-342.

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