Effects Of Knockout Of Faf1 Gene By CRISPR/Cas9 On Zebrafish Cartilage And Sarcomere Development

ObjectiveTo study the effect of faf1 gene on zebrafish by CRISPR/Cas9 gene editing technique.MethodsgRNA was designed and prepared for the faf1 gene of zebrafish,then mixed with Cas9 mRNA,were microinjected into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 were genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,then the same type of mutation F1 were genetically incrossed to get F2 homozygous zebrafish and the genotype of each generation was detected by microscopic observation.ResultsThe faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA6 located in the exon 6 and the gRNA7 located in the exon 4 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type 1(MU1)was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 dpf(days post fertilization,dpf),there were sarcomeric dysplasia and head shrinkage,Angle increased craniofacial cartilage deformity changes,and after fertilization 8~9dpf death.ConclusionThe knockdown of faf1 gene by Morpholino(MO)resulted in a phenotype of craniofacial cartilage changes in zebrafish.CRISPR/Cas9 knocked out the gene to produce a new phenotype,that is,pigment deposition delay and tail muscle section "Nodular" changes.Which provides a new clue for the study of the pathogenesis of cleft lip and/or cleft palate.