Construction Of Autism Risk Gene Katnal2 Zebrafish Mutation Model By Using CRISPR/Cas9 Technology

Objective: Knocking out the autism candidate gene katnal2 by CRISPR/Cas9 gene editing technology,to construct a zebrafish model with katnal2 gene mutation,and study the effects of katnal2 gene on development through zebrafish mutants.Methods: The expression of katnal2 gene in zebrafish at early development stage was observed by whole embryo in situ hybridization assay and qPCR assay.Through the katnal2 gene information of zebrafish,the appropriate target site was designed,then prepared corresponding gRNA and effective Cas9 mRNA.The microinjection technique of zebrafish embryo was used to inject the mixed system of gRNA and Cas9 mRNA into the single-cell embryo yolk,and the effectiveness of the target site was detected by T7E1 digestion and Sanger sequencing.F1 generation zebrafish mutants were screened by sequencing technique,F2 generation homozygous mutants were screened by genotyping-PCR reaction,and the expression of katnal2 in homozygous mutants was detected by qPCR.At the same time,the early developmental status of wild-type and F3 zebrafish homozygous mutant embryos were observed under microscope and the body length of the larva at 5 hpf was measured for comparative analysis,then the light and shade stimulation study was conducted on 6 dpf larva.Results: The qPCR experiment and in situ hybridization results showed that katnal2 was expressed at a low level in early development,with widely expression before 24 hpf,and limit expression in the brain after 24 hpf.T7E1 digestion and Sanger sequencing showed that gRNA1 located in exon 6 can mutate the katnal2 gene ofzebrafish.Among the various mutation types of the F1 generation,mutation types Mu-1 and Mu-2 which predicted the early termination of translation of the katnal2 protein were screened by reading frame analysis.The decrease of the expression level of katnal2 gene in two homozygous mutant katnal2 genes were confirmed by qPCR assay.Homozygous mutant embryos showed a phenotype of epibody and convergence and extension movements delay as well as the decrease of the body lenth.Homozygous mutants also showed phenotypes of neurobehavioral abnormalities.Conclusion: Compared with previous studies on katnal2 based on cellular level,this study used CRISPR/Cas9 gene editing technology to knock out katnal2 gene and constructed a stable animal model with katnal2 gene mutation.The zebrafish with homozygous mutation of katnal2 gene showed a phenotype of epibody delay and pre and post-body axis extension defects in the early morphogenesis stage,indicating that the katnal2 gene plays an important role in the early developmental stage of zebrafish.