CRISPR/Cas9-mediated Editing Of Tyr And Nanog Genes In Medaka (Oryzias Latipes)

CRISPR/Cas9 system is from adaptive immunity system of bacteria,and it can be used to modify the target genes.The system has the advantages of simple design,high efficiency,low cost,wide range of application and so on.After artificial modification by research,this system has been successfully applied to the precise genome modification of bacteria and a variety of animals and plants.CRISPR/Cas9 system has become a flourishing genome editing technology in clinical application.Nanog gene is one of the key factors to induce pluripotent stem cells,which plays an important role in the early development of embryo and the maintenance of the total energy of embryonic stem cells.In the study,we used the zebrafish codon-optimized Cas9 coding sequence to mutate the tyr and nanog locus in medaka.The experimental results show that the zebrafish codon-optimized CRISPR/Cas9 system can efficiently knock out the tyr and nanog genes.Simultaneously,the homologous recombination mechanism can also mediate the knockin of the nanog gene.In conclusion,the establishment of CRISPR/Cas9 gene editing system in medaka lays an important foundation for the research on the nanog gene.The main results are as follows:(1)We used the CRISPR/Cas9 system to knock out the tyr gene by using a single target method.In the F0 generation,obvious pigment loss phenotypes can be observed in the eye area.Applying the method of continuous selfing and phenotypic screening,we quickly obtained the new albino medaka which can stably inherit.(2)Using the CRISPR/Cas9 system,the double-target knockout method was used to knock out the nanog gene,resulting in a shortage of about 900 bp fragment after seqencing.Through PCR screening,we obtained three different mutant types of F2 homozygous.The F2 homozygous mutants were self-crossed and embryonic development was observed to show that the F3 embryos developed stagnantly after the late blastocyst.(3)According to the homologous recombination mechanism,the nanog gene knockin was mediated using the CRISPR/Cas9 system in medaka.Through fluorescence observation and sequencing analysis,the experimental results showed that the Hyg-EGFP fragment was precisely integrated into the target site of nanog gene.