The Effects Of CRISPR/Cas9 Knockout Stat1b Gene On Early Skeletal Development Of Zebrafish

Signal transducer and activator of transcription(STAT1)plays an important role in many biological processes including cell proliferation,apoptosis,cell differentiation and immune function regulation.Our early study from differential gene expression profiling analyses finds STAT1 gene is associated with osteoporosis in human.The gene homology between zebrafish and human is up to 87%,and was used in this study to explore the role of the stat1b gene in regulating bone development·In this study,we have established the mutant models of zebrafish stat1b(stat1b-/-)using CRISPR/Cas9 gene targeting technique to knock out the stat1b gene with high stability of the heredity.The gRNAs of the two target sites and Cas9-mRNA were mixed in a certain proportion and injected into a cell-level embryo of wild-type zebrafish.We have screened a total of four mutations of F0 zebrafish.with PCR amplification,DNA purification,T7E1 digestion and Sanger sequencing,and four different stable genetic mutations in F1 generation were named as,stat1b-S1,stat1b-S2,stat1b-S39 stat1b-S4 respectively.The delete a small fragment bases were deleted in the exon in stat1b-S1 and stat1b-S2 mutants to lead to a shift in the translation of the amino acid sequence bocause the number of missing bases was not a multiple of three.A large number of fragment bases were lost in the stat1b-S3 and stat1b-S4 including a part of exons and a complete fragment of intron without amino acid sequence shift.The spatial prediction results showed stat1b-S1 and stat1b-S2 were significant different from the wild type,while stat1b-S3 and stat1b-S4 had a high degree of similarity with the wild type.The phenotypic observation of stat1b-S2 mutant zebrafish showed that spinal deformity and caudal deformity.The osteoclast TRAP staining and osteoblast ALP staining showed statlb-S2 mutant zebrafish osteoclasts increased and osteoblasts reduced.The micro-CT scan found that there were some differences in skeletal form and development between the mutant and wild-type zebrafish.The BMD of mutant zebrafish significantly reduced compared with the wild type.All results indicated that statlb gene was associated with zebrafish bone development and it was involved in the regulation of the balance of osteoclasts and osteoblasts.