Directed evolution of CRISPR-Cas9 to increase its specificity

• DOI: 10.1038/s41467-018-05477-x
• Publication Date: 2018-07-30T09:47:00Z
• Authors: Jungjoon K. Lee; Euihwan Jeong; Joonsun Lee; Minhee Jung; Eunji Shin; Young-hoon Kim; Kangin Lee; Inyoung Jung; Daesik Kim; Seokjoong Kim; Jin-Soo Kim
• Keywords: Guide RNA

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 often poorly efficient in human cells. Here, we develop directed evolution approach E. coli to obtain Sniper-Cas9, which shows high without killing on-target activities Unlike other engineered variants, Sniper-Cas9 WT-level with extended or truncated sgRNAs further reduced and works well preassembled ribonucleoprotein (RNP) format allow DNA-free genome editing.