| Background: ARID1 B is an important subunit of BAF complex,a chromatin remodeling complex.It is closely related to neurodevelopmental disorders such as Coffin-Siris syndrome(CSS),intellectual disability(ID),autism spectrum disorder(ASD)and Nicolaides-Baraitser syndrome(NCBRS).Patients with ARID1 B mutations have phenotypes such as developmental and intellectual disability,speech delay,hypertrichosis,and coarse facial features.Studies have shown that the pathogenic mechanism of ARID1 B haploinsufficiency may be related to impaired progenitor cell proliferation and differentiation,abnormal Wnt/β-catenin signaling pathway,excitatory-inhibitory(E/I)imbalance.Zebrafish is a favorable model for the study of neurodevelopmental diseases,and the construction of zebrafish arid1 b mutation model lays a foundation for further research on the pathogenesis of this gene.Objective: The mutation model of zebrafish arid1 b gene was constructed based on CRISPR/Cas9.By observing the differences between the mutants and wild types,the effects of arid1 b on the development of the nervous system can be preliminary discussed.Methods: The expression of arid1 b gene in zebrafish at early development stage was investigated by in situ hybridization assay and q PCR assay.The g RNA was designed and synthesized,and its mixture with Cas9 m RNA was injected into the single-cell embryo yolk.T7E1 digestion and Sanger sequencing were used to select the successful target sites.After crossing with the wild type,Sanger sequencing was used to screen for heritable mutants.The heterozygous mutants of the same mutation type were self-crossed to obtain experimental homozygous mutants and wild type.The body length of larval zebrafish of different genotypes was measured and compared to study the effect of arid1 b on the early growth of zebrafish.The ability of zebrafish to respond to stimuli was observed by light and dark stimulation experiments.Results: q PCR and in situ hybridization showed that arid1 b gene had a maternal effect and a low overall expression.It is widely expressed in the early embryo and mainly concentrated in the brain after 24 h.The constructed zebrafish mutant had a deletion of 74 bases in exon 8,and protein translation was terminated prematurely upstream of the main domain ARID.Larval homozygous mutant have slower development and shorter body length than the wild type.In the light-dark stimulation experiment,the mutant’s ability to respond to the stimulation decreased significantly.Conclusion: In this study,a zebrafish arid1 b gene mutation model was constructed based on CRISPR/Cas9.Larval homozygous mutant have shorter body lengths and a reduced ability to respond to stimuli.This shows that arid1 b gene plays an important role in the early development of the nervous system. |
