Mutation Of Prmt5 And Mep50 In Medaka By Genomic Editing Techniques

PRMT5 is a kind of protein arginine methyltransferase,it plays an important role in the biosynthesis of ribosomes,golgi assembly,cell growth,cell proliferation,apoptosis,cell differentiation,cell totipotency and carcinogenesis.MEP50 is the chaperone of PRMT5.MEP50 also plays an important role in cell growth,cell cycle,cell differentia-tion and cancer,etc.Gene editing technology is to produce double-strand breaks at the DNA target sites,thereby inducing homologous recombination and/or non-homologous terminal linkages,and then editing the target gene in the genome.In this study,we used TALEN,CRISPR/Cas9 and NgAgo-gDNA to mutate the prmt5 and mep50 of medaka,so as to study the functions of prmt5 and mep50 in the development of medaka.?1?Mutation of mep50 using the CRISPR/Cas9 technique.In order to avoid off-target effect,we chose three gRNA target sites.One single target site was at the first exon and the double target sites were at the second exon.We found that injection of the samples were obviously toxic to the embryos.The embryonic abnormality rates was 1.4%,and the mortality rate was as high as 16.61%.Statistical analysis showed a signifi-cant difference between the experimental group and the control group?P<0.01?.Ge-nomic DNA of F0 generation embryos was extracted,and the target fragment was ampli-fied by PCR.T7EI digestion showed that only the second exon had been mutated.Se-quencing results showed obvious mutations at the upstream and downstream of the target sites of the second exon.There were three types of mutation,insert,deletion and substi-tution.The mutation efficiency was 53.3%.The results showed off-target effect of CRISPR/Cas9 technology.This experiment provided an experience for the subsequent studies,it is better to use multi-site mutation to reduce the off-target effect.?2?Application of the NgAgo-gDNA technique.We constructed pNgAgo-IRES2-eGFP-nos by insertion of the T7 promoter,SV40 NLS,nucleoplasmin NLS,NgAgo CDS and the 3' UTR of zebrafish nanos3 into pIRES2-eGFP vector.The target sites of medaka mep50 for NgAgo-gDNA technique was the same as that for CRISPR/Cas9 technology.The NgAgo mRNA was synthesized in vitro and mixed with gDNA for microinjection.FO embryos were collected and checked after microinjection.T7EI digestion showed the mutations at the first exon but not at the second exon.Se-quencing showed no mutation at the target site but at the upstream of the targets.The mutation efficiency was 10%.This results indicated that the NgAgo-gDNA technique possibly worked in medaka but needed further exploration in later.?3?Screening of the progeny of prmt5 mutants.In the early stage of this study,the mutation of prmt5 was carried out by TALEN technique.Based on the previous study,the progeny of prmt5 mutants were screened.A total of 20 fish were screened,and 6 of them were heterozygotic mutants.Sequencing showed the deletion of four bases.The heterozygotic mutants were mated to obtain F₂ generation.The deformities and albino were found in F₂ embryos.The mortality rate of F₂ embryos was as high as 46.1%,of which 17.1%died in 1 to 2 days of development.Statistical analysis showed a significant difference between the experimental group and the control group?P<0.01?.Possibly,it was lethal for the embryos with homozygous mutant.A total of 45 F₂ individuals were screened,and 13 were identified as homozygote by T7EI digestion.However,sequencing results showed all of the 13 individuals were actually heterozygote.It indicated that ho-mozygous mutation of prmt5 was lethal to the embryos so that no homozygous mutants were obtained.In the future,the embryos dying in 1 to 2 days of development can be tested for the mutant types of homozygte or not.Transcriptome analysis can also be per-formed in these embryos.In conclusion,the progeny of prmt5 and mep50 mutants were obtained for further studies of the functional relationship between the prmt5 and mep50 in medaka.This study involves of three different gene edit techniques.Especially,the NgAgo-gDNA technology was improved and applied in this study.The trial showed that this technology needed further exploration.