Construction Of Zebrafish Mutants Of Keratin92 Gene Using CRISPR/Cas9 Gene Editing Technology And Its Phenotype Analysis

Background Craniofacial anomalies are the most common developmental malformations,representing a group of malformations associated with the differentiation and growth of the head and facial bones,an umbrella term for a multifactorial disorder that accounts for one-third of all congenital birth defects one.Craniofacial deformities may present as syndromic forms involving other organs or tissues,or nonsyndromic forms affecting only craniofacial tissues and organs.Common craniofacial deformities include cleft lip and palate,orofacial cleft,craniosynostosis,and hemifacial infantile deformity.Craniofacial deformities can cause chewing,language,aesthetic,and psychological problems,placing a heavy burden on affected individuals and societies.At present,the comprehensive use of a series of means such as surgical intervention,prosthetic appendage repair,and orthodontic treatment can significantly improve the treatment effect of craniofacial deformity,the facial appearance and oral function of patients,but the overall effect is still very limited,and the craniofacial deformity can be significantly improved.Facial deformity patients are often accompanied by a family history of genetic disease.The etiology of craniofacial deformities can be divided into environmental factors and genetic factors.Environmental factors include physical,chemical,radiation,trauma,etc.Although many potential and established environmental factors can affect craniofacial development,existing studies have demonstrated that genetic factors are more important causative factors for craniofacial deformities.and treatment is more valuable.This research project will use zebrafish as an animal model,use CRISPR/Cas9 gene editing technology to knock out Keratin92 gene in zebrafish,screen out the homozygote of Keratin92 gene zebrafish mutant,and study the effect of Keratin92 gene on zebrafish maxillofacial development.From the perspective of developmental biology,the genetic factors and pathogenesis of craniofacial malformations were studied,and whether Keratin92 could regulate the development of zebrafish maxillofacial bones through the BMP signaling pathway.ObjectiveIn this study,the CRISPR/Cas9 gene editing technology was used to successfully construct a Keratin92 gene-deficient zebrafish mutant model,and the gene function of Keratin92 was preliminarily discussed.The molecular mechanism of development,to clarify the mechanism of Keratin92 gene mutation leading to congenital craniofacial deformities,and to provide theoretical basis and genetic basis for the molecular regulation of jaw development and the diagnosis and treatment of craniofacial deformities.Materials & Method The target site and detection primers were designed on the protein coding region(CDS)of the Keratin92 gene through the website,and the gRNA plasmid was used as a template to carry out Prime STAR PCR by in vitro amplification,and the obtained product was purified and transcribed in vitro to obtain the gRNA of the Keratin92 gene.The gRNA was formulated and mixed with Cas9 enzyme,and injected into the 1-cell stage of Wild Type(WT)zebrafish embryos by microinjection.The mutant sample embryos were retained by sequencing,cultured to sexual maturity,and then crossbred with WT zebrafish to produce F1 mutant zebrafish heterozygotes,and then sequenced to retain the mutant embryos with stable inheritance and continue to cultivate to adulthood,the mutation mode of each F1 mutant zebrafish was determined by fish scale sequencing,and the F1 generation zebrafish with the same mutation mode and frameshift mutation were selfed to obtain the F2 generation Keratin92 mutant zebrafish homozygote.The expression pattern of Keratin92 gene in the development of WT zebrafish was explored by whole embryo in situ hybridization,and the region and time of the gene function were explored;the embryonic development process of Keratin92 mutant was photographed and recorded;Alcine-Alizarin was used The 120 Hour PostFertilization(hpf)mutant zebrafish was stained for the jawbone by double-staining experiment without acid,and its skeletal structure was analyzed.Morphological structure and tissue arrangement;real-time quantitative PCR experiments were used to detect whether the expression of genes related to bone development in the mutants was affected.Results We successfully constructed a Keratin92 homozygous mutant zebrafish with five base deletions(-AGAGG)in the first exon of the Keratin92 gene and stable inheritance.The results of whole embryo in situ hybridization showed that the Keratin92 gene was widely expressed in the early embryonic development period,and concentrated in the jaw,pharyngeal arch and intestine of zebrafish after 24 hpf.The embryonic development of Keratin92 mutant zebrafish is almost arrested during early embryonic development(4.70hpf-8.00hpf),which is also an important period of early neural crest development.The results of jawbone staining showed that the 120 hpf mutant zebrafish had abnormal jawbone development,the width of Merkel cartilage increased,and the angle between the hyoid bones on both sides increased.The results of HE staining of the jaw sections showed that the morphological structure and tissue arrangement of the jaw chondrocytes of the mutant zebrafish were not significantly different from those of the WT type.The results of real-time quantitative PCR showed that the expression of the mutants’ craniofacial structure development-related genes Dlx2,Zic2 a,Fzd4,Dkk1 b,osteogenic marker genes Fgf8 a and cartilage development-related genes Col2a1 a,Nkx3.2,PAX9 were compared.Down-regulated with WT type.Conclusion This study successfully knocked out the Keratin92 gene in zebrafish using the CRISPR/Cas9 system and obtained stable inheritance,and successfully constructed a zebrafish Keratin92 mutant.The study found that this gene is widely expressed in the early developmental stage of zebrafish embryos,and is concentrated in the zebrafish jaw and pharyngeal arch after 72 hpf.The deletion of Keratin92 gene will cause abnormal development of the zebrafish jaw.Provides a certain theoretical and experimental basis.It also demonstrated the feasibility of constructing a craniofacial deformity model in zebrafish by CRISPR/Cas9 gene editing technology.Moreover,this method can also realize the screening of clinical craniofacial disease-related genes on a large scale,and provide a new treatment plan for the treatment of this genetically related craniofacial deformity.