Use CRISPR/Cas9 To Repair Pig Eye Development Defects Caused By MITF Mutation

In our group,a mutant pig with albinism,hearing loss,and iris hypopigmentation was screened from 1119 F1 offsprings created by ENU mutagenesis,consistent with the phenotype of human Waardenburg syndrome type II?WS2?.This mutant was then mated with wild type sow,the mutant phenotype was shown to be inherited in a dominant autosomal inheritance pattern,about 50%of the resulting F2 offsprings exhibited the WS2 phenotype similar as the F1 mutant.The F2 mutants were further mated with each other,and and new phenotype appeared in F3generation:about 1/4 of the F3 offsprings exhibited developed defect in the eyeballs besides albinism,iris hypopigmentation and hearing loss.Linkage analysis and family-based genome-wide association analysis?GWAS?were performed to identify the causative gene.Results showed that a point mutation?T>C?at the CDS740bp site on exon 8 of MITF gene is the causative mutation of the phenotype.Owning to the highly similarities with human anatomy,metabolism,and physiology,pigs are considered as excellent models for human diseases,especially for eye disease and neurodegenerative disease.Therefore,the F3 homozygous mutant pigs can be served as ideal large animal models for studying eye development defects.With the advantage of simple design and highly efficiency,CRISPR/Cas9 technology develops rapidly in the gene editing field in recent years.Since about 2/3 of human genetic disease is caused by point mutations,the introduction of precise point mutations in genome by gene editing technology is of great significance in the treatment of human monogenic genetic diseases.Here we performed gene repair study based on the model of eye development defect pigs.A sgRNA targeting the L247S mutation site was designed.The gRNA-Cas9 expressing plasmid,99 nt ssODN-donor and pCAG-GFP plasmid were co-transfected into the MITF^L247S/L247S porcine fetal fibroblast cells,and single cell colonies were screened by FACS.A total of 34 colonies were obtained,and genotyping result showed that one of them?#27?was mono-allelic repaired at the CDS740 site.This colony was then used as the donor cells for somatic cell nuclear transfer?SCNT?,a total of 2380 embryos were transferred to 9 surrogates and 3 pregnancies were established and developed to term.Nine live-born piglets and 3 stillborn piglets were obtained.One of these pigs lived for more than 6 months.The eyes of these piglets appear normal,which are significantly different from the eyes of MITF^L247S/L247S pigs.OCT,fundus imaging and ERG test on live pigs showed that the eyes of the repaired pigs were functional,and the morphology were normal.HE staining and immunofluroscence in eyes of the dead pigs further confirmed that the eyes of the repaired pigs were functional and structural normal.Above all,the eye development defect was corrected by CRISPR/Cas9 technology,which provides prospects for future gene therapy in pig models.However,the efficiency of point mutation correction in the current study was relatively low,and new mutations may be introduced.Therefore,in the future,we will study how to improve the efficiency and precision of point mutation correction.