Knockout And Identification Of The Trdmt1 Gene In Zebrafish

Epigenetic regulation refers to the regulation of gene expression without changes in the DNA sequences.RNA methylation,as an important mediating method,plays an important role in the development of early mammalian embryo,mouse sperm development and cell differentiation.TRDMT1(DNMT2)is one type of RNA m5C methyltransferases and it mainly catalyzes the following three types of tRNA methylation:tRNAsp,tRNAval,and tRNAGly,which can stabilize the genome and help protein synthesis.The sequencing results showed that the similarity between zebrafish genome and human genome was up to 87%,so researchers can study the function of the same human genes by studying the zebrafish genes.In this research,CRISPR/Cas9 gene editing technology was used to knock out Trdmt1 so as to study its function.By using CRISPR/Cas9 technology,the targets on the exon of Trdmt1 gene of zebrafish were designed in order to knockout the Trdmt1 gene.Random insertion and deletion of bases were introduced,and code shifting mutation was generated to edit the gene.The homozygous mutation of the Trdmt1 gene was screened out from the mutant individuals to study the effect of Trdmt1 gene knockout on the growth,development and phenotype of zebrafish,so as to provide an ideal model for further study in the future.The results are as follows:(1)Establishing Trdmt1 knockout zebrafish line.The online(website chopchop)was used to design target gRNAs,multiple gRNAs with high scores were selected,gRNA and Cas9 mRNA was coinjected into small batches of zebrafish embryos,the target for subsequent large batch injections were based on the digesting efficiencies with enzyme.Then FO zebrafish with the same mutant genotype were selected for mating to obtain the offsprings.After that,primers were designed according to the mutant genotype,and then Trdmt1-/-fishes were screened based on PCR-sequencing results.The results showed that gRNAl was the most efficient target among the designed targets.After a large number of injections,a variety of FO generation fishes with gene mutations were screened out.The mutation types were 8 bp deletion,22 bp insertion and 39 bp deletion,respectively.The male and female zebrafishes with the same mutation type were selected formating,then F1 generation was obtained.Trdmt1-/-fishes were identified by specific primers and the PCR-sequencing.Finally,the results showed that the Trdmt1 knockout zebrafishes were successfully obtained.(2)The effects of Trdmtl knockout on the growth and phenotypes of zebrafishes.The F1 generation of Trdmt1 knockout zebrafish was mated to obtain homozygous mutant F2 zebrafishes.The phenotypes of Trdmt1 knockout zebrafish embryos were observed,including hatching rate,survival rate and deformity rate.The results showed that Trdmt1 knockout was not conducive to the hatching of zebrafish.The hatching rate of Trdmt knockout zebrafish was 50.06%.It was significantly lower than 89.67%of wild type zebrafish(p<0.01).There was no significant effects on the survival and deformity rate of zebrafish.The survival and deformity rate of Trdmt1 knockout zebrafish respectively was 84.17%and 4.21%while those of wild type zebrafishes was 89.64%and 3.48%(P>0.05).The data showed that the ratio of female to male of Trdmt1 knockout zebrafish was 1:1.872,whereas that of wild type zebrafish was 1:0.836 with significant difference(p<0.01),suggesting that Trdmtl knockout affected the sex ratio of zebrafishes.In this study,CRISPR/Cas9 was used to knock out zebrafish’s Trdmt1 and Trdmt1-/-fishes were obtained by screening.It was found that Trdmt1 gene knockout was not conducive to the hatching of zebrafish,and had no significant effect on the survival and deformity rate of zebrafish.In addition,it also affects the sex differentiation of zebrafish.This will provide reference for the sex selection of aquatic animals in animal husbandry and a useful model for further research in the future.