Construction Of Autism Risk Gene Ppp2r5d Zebrafish Mutation Model And Research On Its Phenotype

Objective: Knocking out the autism risk gene ppp2r5 d by CRISPR/Cas9 gene editing technology,to establish a zebrafish mutant model,and study the effects of ppp2r5 d gene on the early development,behavior and transcriptome of zebrafish mutants.Methods: In situ hybridization experiment and RT-q PCR experiment were performed to obtain the expression of ppp2r5 d gene in the early developmental stage of zebrafish.Using CRISPR/Cas9 technology on zebrafish to obtain F0 generation,after passage and screening,obtain F3 generation homozygous mutants.Afterwards,the homozygous mutants and the control group of the same age under the same breeding environment were subjected to early development observation,morphological measurement and immunofluorescence staining.Conduct light and dark stimulation behavioral experiments on 5dpf fish and adult behavior experiments(new tank experiment,mirror experiment,triple tank experiment and cluster experiment)on 4 mpf adult zebrafish.The expression level of emotion-related genes in the brain of 4.5 mpf adult zebrafish was detected by RT-q PCR experiment.Transcriptomics analysis was performed by extracting total RNA and RNA sequencing(RNA-seq)from the head tissues of 5 dpf mutant and control larvae.Results: The zebrafish ppp2r5 d gene is a maternally expressed gene.We successfully obtained homozygous mutant model of ppp2r5 d by using CRISPR/Cas9 technology.Compared with the control group,the mutant zebrafishs have delayed development,shortened body length,increased interocular distance between optic apex and the expansion of the top cover.The length of the mutant adult fish is also shortened.According to the results of behavioral experiments,the ppp2r5 d homozygous mutant juvenile fish showed a decrease in startle avoidance response ability,learning and memory ability and the adult mutant fish showed increased anxiety and fear levels,increased aggression and social deficits.Compared with the control group,the expression level of HPA axis and other genes increased in the brain tissue of the mutant adult fish.The transcriptome sequencing results of the mutant also showed that the differentially expressed genes were enriched in the biological processes of nervous system development,glutathione metabolism,retinol metabolism and response to biological stimulus.In addition,the differential genes contain multiple ASD risk genes.Conclusion: The zebrafish ppp2r5 d mutant model was successfully obtained.Compared with the wild type,the mutant has early developmental abnormalities,abnormal morphological development,and obvious autism-like behavioral abnormalities.The brain tissues of adult fish with knockout gene have changes in the expression level of emotion-related genes.The transcriptomics results of the mutants showed that the mutation of this gene at the molecular level would cause abnormal functions and then affect the various biological processes involved.