Efficient generation of knock out mice by the CRISPR/Cas9 system with two guide RNAs

The CRISPR/Cas9 system is a powerful genome editing tool for generating knockout mice. To generate mutant mice, single guide RNA （sgRNA） for tyrosinase was transcribed in vitro with template Oligo DNA and a mixture of either one or two sgRNAs and Cas9 mRNA were microinjected into the fertilized eggs of C57BL/6J（ black hairs and eyes）. To determine whether tyrosinase gene is successfully knocked out, their phenotype（ black hairs/eyes or albino） was observed. In this study, to investigate whether knockout efficiency can be increased by microinjection of two sgRNAs, either one or two sgRNAs were microinjected. Results indicated that when one sgRNA was microinjected, albino mice were obtained at relatively low efficiency （3/16, 19% or 13/23, 65%）; however, the efficiency was increased by microinjecting two sgRNAs （24/26 92.9%）. In conclusion, we demonstrate here that knockout mice at F0 can be obtained by microinjection of two sgRNA and Cas9 mRNA at high efficiency