Directed evolution of CRISPR-Cas9 to increase its specificity

Jungjoon K. Lee (), Euihwan Jeong, Joonsun Lee, Minhee Jung, Eunji Shin, Young-hoon Kim, Kangin Lee, Inyoung Jung, Daesik Kim, Seokjoong Kim and Jin-Soo Kim ()
Additional contact information
Jungjoon K. Lee: Toolgen
Euihwan Jeong: Institute for Basic Science (IBS)
Joonsun Lee: Toolgen
Minhee Jung: Toolgen
Eunji Shin: Toolgen
Young-hoon Kim: Toolgen
Kangin Lee: Toolgen
Inyoung Jung: Toolgen
Daesik Kim: Seoul National University
Seokjoong Kim: Toolgen
Jin-Soo Kim: Institute for Basic Science (IBS)

Nature Communications, 2018, vol. 9, issue 1, 1-10

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-05477-x

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