Directed evolution of CRISPR-Cas9 to increase its specificity
- Lee, Jungjoon K.; Jeong, Euihwan; Lee, Joonsun; Jung, Minhee; Shin, Eunji; Kim, Young-hoon; Lee, Kangin; Jung, Inyoung; Kim, Daesik; Kim, Seokjoong; Kim, Jin-Soo
- Issue Date
- Nature Communications, Vol.9, p. 3048
- The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.