Lee Jae Young, Jang Yoo Jin, Bae Ji Hyun, Lee Yoon Hoo, Bae Hee Sook, Kim Seokjoong, Park Sin-Gi, Koo Ok Jae, Yeom Su Cheong
Toolgen Inc, Gasan Digital-Ro, Geumcheon, 08594, Seoul, Republic of Korea.
Graduate School of International Agricultural Technology, Designed Animal and Transplantation Research Institute, Seoul National University, 1447 Pyeongchang-Ro, Daewha, Pyeongchang, Kangwon, 25354, Republic of Korea.
Biochem Biophys Rep. 2020 Apr 1;22:100752. doi: 10.1016/j.bbrep.2020.100752. eCollection 2020 Jul.
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.
CRISPR/Cas9（SpCas9）系统目前被广泛用于生成基因工程小鼠；然而，一些研究提出了与该系统脱靶突变相关的问题。在此，我们利用Cas9（CjCas9）系统来生成基因敲除小鼠。我们设计了靶向小鼠或的sgRNA，并将其与CjCas9 mRNA一起显微注射到受精卵中。我们从显微注射的胚胎中获得了新生小鼠，并证实50%（Tyr）和38.5%（Foxn1）的新生小鼠在预期靶序列上存在双等位基因突变，这表明CjCas9能有效地进行基因组靶向。此外，我们通过靶向深度测序和全基因组测序分析了奠基突变小鼠中的脱靶突变。两种分析均显示，在预测的潜在脱靶位点未发现脱靶突变，且在分析的奠基动物中未发现意外的随机突变。总之，CjCas9系统可用于精确地生成基因编辑小鼠。
