Mutating Prmt5 In Medaka And Zebrafish By Genome Editing

Genome editing techniques have been widely used in various organisms to study gene function.Protein arginine methyltransferase 5(Prmt5)is a type ? enzyme to catalyze symmetric dimethylation of arginine in protein.Prmt5 plays important roles in embryonic development,cell-cycle progression,and immunity in mouse.Knock down of zebrafish prmt5 caused abnormal development of muscle.In this study,the TALEN and CRISPR/Cas9 technology were applied in medaka and zebrafish to mutate prmt5 for further studies of prmt5 function in fish.The TALEN technology was used at first.The TALEN constructs of prmt5 medaka and zebrafish were made via Unit Assembly.The TALEN mRNA synthesized in vitro was microinjected into the zygotes of zebrafish and medaka.prmt5 TALEN activity in F0 generation were 40%and 45%in medaka and zebrafish respectively.Seven of twenty F0 fish(35%)were identified as mutants in medaka.Four mutants were chosen to mate with wild type,and the mutation frequencies of F1 embryos were 15%,22%,20%,and 44.4%.The mutants were further screened in the adult by tail fin DNA testing.Four F1 mutants of the founder B were identified with same mutation,and 5%of F2 embryos were mutants by selfcrossing of these F1 fish.Five F1 mutants of the founder I were identified with same mutation,and 47.3%of F2 embryos were mutants by selfcrossing of these F1 fish.41.7%of the F2 fish from founder B were mutants.16.7%of the F2 fish from founder I were mutants.Two of 30(6.7%)FO fish were mutants in zebrafish.Next,CRISPR/Cas9 technology was used.The 116 bp of prmt5 gRNA for medaka or zebrafishand capped Cas9 mRNA were synthesized in vitro.Prmt5 gRNA and Cas9 mRNA were co-injected into the zygotes of medaka and zebrafish.30%of medaka embryos were mutants while no mutant was found in zebrafish.Eighteen of forty(41.9%)FO fish of medaka were identified as mutants.Ten mutants mating with wild type got mutate F1 with efficiency of 0,36.8%,30%,30%,12.5%,10%,12.5%,22.2%,16.7%,and 12.5%.T7EI still needs to use a combination of sequencing and further verification in order to obtain homozygous,providing guarantee for later use of technologies such as high-throughput transcriptome to analysis the function of prmt5.