OBJECTIVETo establish a mouse model of H435Y mutation of gene using CRISPR/Cas9- mediated gene targeting.

METHODSThe single-stranded guide RNA (sgRNA) specific to the H435Y loci of gene was designed based on the sequence of gene. After activity assessment, the active sgRNA and Cas9 were transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target gene mutation confirmed by PCR and gene sequencing. gene mutations in the offsprings of the F0 generation mice were analyzed.

RESULTSGene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57BL/6J mice to obtain the F1 generation mice, and PCR confirmed the presence of H435Y mutations of gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435Y mutation of gene in F2 mice was found, indicating the mousemodel of gene mutation in H435Y was established and propagated successfully.

CONCLUSIONSWe successfully established gene H435Y mutant mouse models using CRISPR/Cas9 technique.