Knockout Of Lgalsla And The Other 8 Genes Of Zebrafish By CRISPR/Cas9 Technology

Clustered regularly interspaced short palindromic repeats and associated 9 (CRISPR/Cas9) technology is II type CRISPR/Cas system. This system is a kind of immune system specific bacteria. After the reformation by the researchers, it has become a more extensive application of site-specific gene editing technology, which followed by the zinc finger endonuclease (ZFN) and the transcription activator-like effector nuclease (TALEN) technology. In recent years, the technology has been successfully applied in human cells, rat, mouse, zebrafish, Arabidopsis and other organisms. At present, CRISPR/Cas9 have developed into a research tool with more features by repeated transformation,The purpose of this study is to realize the 9 genes in zebrafish on chromosome 1 deletion, and the preliminary analysis of the phenotype of the homozygous mutants, further study of gene function. At the same time, we also found abnormal phenomenon and statistical records, in order to provide more materials in study of zebrafish.We use CRISPR/Cas9 technology has realized 9 genes designated knockout. The mutation efficiency is between 20% and 100%. In the experiment of cloned TA of F0 samples, sequence analysis of 52 mutants showed that less base substitution accounted for a small proportion. It is indicated that the CRISPR/Cas9 technology not only has the high mutation efficiency, but also is very effective in the function deletion mutation application.The target analysis of gene was found, the best length of the target sequence is 19-20bp, and when increased to 21-22bp, the shooting efficiency is significantly reduced or not successfully detected the gene mutation. sgRNA whether targeting antisense strand or template strand, targeting efficiency has no obvious difference. In addition, although we have no quantitative analysis on the off-target, but from the perspective of the shooting results of high efficiency, is not as serious as we expected the predicted effects of "Miss Site".In the process of selection in heterozygous mutants from Fl generation, there is widespread non Mendel genetic phenomenon, the emergence of the "homozygous", or non wild, non homozygous and non heterozygous mutants. And the offspring of these mutants are not fully in line with Mendel genetic.In addition, we also found a handful of the screened mutant heterozygous adult males become available for normal mating female, the sex transformation phenomenon.