Jungjoon K. Lee1,*, Euihwan Jeong2,3, Joonsun Lee1, Minhee Jung1, Eunji Shin1, Young-hoon Kim1, Kangin Lee1, Inyoung Jung1, Daesik Kim3, Seokjoong Kim1 & Jin-Soo Kim2,3,*
1 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.
Abstract
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.