Abstract: Objective To establish a mouse model of H435Y mutation of Gata4 gene using CRISPR/Cas9- mediated gene targeting. Methods The single-stranded guide RNA (sgRNA) specific to the H435Y loci of Gata4 gene was designed based on the sequence of Gata4 gene. After activity assessment, the active sgRNA and Cas9 were in vitro 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 Gata4 gene mutation confirmed by PCR and gene sequencing. Gata4 gene mutations in the offsprings of the F0 generation mice were analyzed. Results Gene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of Gata4 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 Gata4 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 Gata4 gene in F2 mice was found, indicating the mousemodel of Gata4 gene mutation in H435Y was established and propagated successfully. Conclusion We successfully established Gata4 gene H435Y mutant mouse models using CRISPR/Cas9 technique.