Establishing a CRISPR-Cas9 system targeting at mouse Krt14 that simulates pathogenic site mutations of human

Objective To establish an effective CRISPR-Cas9 system targeting at mouse Krt14 that may simulate the pathogenic site mutation of human KRT14. Methods Databases such as OMIM, ExAC and ClinVar were retrieved to identify the strong pathogenic sites of human KRT14, the causative gene of human epidermolysis bullosa simplex. Then, the human and mouse KRT14 protein sequence alignment analysis was carried out to identify the strong pathogenic sites on the mouse genome. Based on the gene targeting principle of the CRISPR-Cas9 system, 4 sgRNAs targeting the mouse Krt14 and 4 expression plasmids were constructed. Then, the sgRNA expression plasmids and Cas9 expression plasmid were co-transfected into mouse NIH 3T3 cells simultaneously. PCR product sequencing, TA clone sequencing of the positive cells screened by drugs were performed to verify the target efficiency of the 4 sgRNAs. Results The p.Arg125 site of human KRT14 protein was identified to be a strong pathogenic site, which corresponded to the p.Arg131 site of mouse KRT14 protein. According to the DNA sequence of the mouse p.Arg131 site, 4 sgRNA expression plasmids targeting at this site was successfully constructed. Sequencing of PCR amplification products of the target site from drug screen positive cells showed that 4 sites were all mutated. The results of TA cloning sequencing showed that the mutation efficiency of the four sites was 70%, 90%, 65% and 100% respectively. Conclusions The above results indicates that efficient CRISPR-Cas9 system targeting at the mouse Krt14 based on the highly pathogenic sites of the human KRT14 has successfully constructed. This result lays a foundation for the in-depth study of Krt14 function and for the establishment of Krt14 gene editing mouse model in order to explore the mechanism and treatment strategy of disease.