Authors and Affiliations

Authors and Affiliations

Jungjoon K. Lee^1,*, Euihwan Jeong^2,3, Joonsun Lee¹, Minhee Jung¹, Eunji Shin¹, Young-hoon Kim¹, Kangin Lee¹, Inyoung Jung¹, Daesik Kim³, Seokjoong Kim¹ & Jin-Soo Kim<sup>2,3,*

1</sup> Toolgen, Seoul 08501, Republic of Korea. ²Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 34121, Republic of Korea. ³Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. These authors contributed equally: Jungjoon K. Lee, Euihwan Jeong. C

 

^*Correspondence to Jungjoon K. Lee or Jin-Soo Kim.

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.