Knockout Of Zebrafish Vap、C1H7orf55、mri1、BX284640.3 And Dscr6 Genes By CRISPR/Cas9 And TALENs System

In recent years methods of whole-genome sequencing and large-scale genome annotation projects which generate large amounts of information have been developing rapidly,and the major challenge for investigators is how to transform these massive data to functionally and clinically relevant knowledge.The core of the problem is the need for efficient and reliable methods allowing researchers to determine how the genotype affect the phenotype.Gene knockout technology is an effective method to study gene function and CRISPR/Cas9 and TALENs have been proven to be efficient and reliable tools for gene knockout.Zebrafish is a good animal model that can be used for functional genomics analysis and the study of human disease pathogenesis.Thus, zebrafish has become the first choice to use the most advanced genome editing techniques such as CRISPR/Cas9 and TALENs.CRISPR/Cas system is an immune system defending the exogenous genetic material,and it’s unique to prokaryotes.This system can be mediated by sequence-specific RNA to cut off the exogenous DNA including exogenous plasmid or phage.CRISPR/Cas system has three types, and the composition of type Ⅱ CRISPR/Cas system is the easiest, which can play a role in need of RNase Ⅲ, crRNA, tracrRNA and Cas9 protein.CRISPR/Cas9 mediated gene knockout includes the following steps:the selection and confirmation of Cas9 targeted point、the designation of detection primer; the building of gRNA in vitro transcript vector; the preparation of Cas9 mRNA and gRNA; the preparation of FO zebrafish and the detection of mutation efficiency of targeted point; the detection of FO reproductive system; screening F1 adult zebrafish with targeted point mutations.TALENs is a new method for gene knockout which developed in recent years.It takes advantage of the constant correspondence between the amino acid sequence of nucleic acid binding domain of TALEs protein,and can assemble modular protein that can bind any DNA sequence specifically to realize DNA editing and modification operation such as knock-out、base substitution or gene modification.Because of its fastand efficient advantages,TALENs technology has a profound impact on the field of bio-genetic research, and provides a new strategy for the treatment of genetic diseases.By methods such as chemical mutagenesis and accumulation of many years,zebrafish has more than one thousand kinds of mutants, but there are still tens of thousands of genes without corresponding mutants.By knocking out these genes to create a larger zebrafish mutant library,we can further study the function of these genes, and this will promote the development of biology and the study of human diseases.In this study, we knockouted zebrafish Vap、ClH7orf55、mril and BX284640.3 gene on the lth chromosome, and obtained corresponding mutants.For Vap gene,we screened out one type of mutation:7bp deletion mutation; for ClH7orf55 gene,we screened out two types of mutations:lbp insertion mutation andl6bp deletion mutation; for mril gene, we screened out two types of mutations:7bp deletion mutation and 40bp deletion mutation;for BX284640.3 gene, we screened out two types of mutations:lbp deletion mutation and 2bp deletion mutation.In addition, we also knockouted zebrafish dscr6 gene on the 10th chromosome, and obtained F0 generation of fish with germline mutation.The knockout of above genes laid the foundation for the further study of the function of these genes.