Efficient and specific generation of knockout mice using CRISPR/Cas9 system.

The CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse or and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner.