Targeted Mutagenesis Of Tyrp1b, Chmp7 And Kank3 In Zebrafish Mediated By CRISPR/Cas9 System

The experiment was designed to investigate the function of tyrp1b, chmp7 and kank3 and the effects on the functional traits in fishes, the zebrafish as the test object, we adopt the three gene site directed mutagenesis mediated by CRISPR/Cas9 system. After target site designed, 100 ng/?L of cas9/gRNA were microinjected into single-cell zebrafish embryos, then Nest-PCR, fragment sequencing and T7EI digestion were conducted to detect mutation effect of three genes and F1 mutation frequency. Test results were as follows:1. The single and efficient target site of three genes were determined by PCR sequencing and T7EI digestion, especially the b1 target loci of gene tyrp1b, the t1 target loci of gene chmp7, the c3 target loci of kank3 gene, can be applied to breeding work in future.2.100 ng/?L of cas9/gRNA were microinjected by CRISPR/Cas9 system in this study, the reasonable control of the Cas9/gRNA dose is very important, high dose of Cas9/gRNA can lead to fish deformity or death.3. F0 sequencing, F1 sequencing and TA cloning results showed that Cas9 mediated gene mutation could be inherited to the next generation with germ line cells, which showed that the gene mutation mediated by CRISPR/Cas9 system was hereditary.4. The statistical mutation rate of each gene showed that cas9 mediated tyrp1b mutation rate reached 45.8%-38.5%; chmp7 mutation rate reached 54.5%-51.6%; kank3 mutation rate reached 40%-44.7%. These mutations showed that individuals with high mutation rate were more likely to develop stagnation than those who acquired later.5. Most of the cas9 evoked mutation effect the upstream 5-10 kilobases pairs of PAM area, TA cloning of tyrp1b gene showed that four bases have occurred, deletion and insertion in the probability of basically the same; and TA cloning results of kank3 gene showed that, the deletion mutation probability is higher than that of the insertion mutation, and the mutation significantly genetic to the next generation.These results indicate that the mutation rate induced by CRISPR/Cas9 system is highly efficient and can be inherited.