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Directed evolution of CRISPR-Cas9 to increase its specificity.

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.

SUBMITTER: Lee JK

PROVIDER: S-EPMC6078992 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Directed evolution of CRISPR-Cas9 to increase its specificity.

Lee Jungjoon K JK Jeong Euihwan E Lee Joonsun J Jung Minhee M Shin Eunji E Kim Young-Hoon YH Lee Kangin K Jung Inyoung I Kim Daesik D Kim Seokjoong S Kim Jin-Soo JS

Nature communications 20180806 1

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 Cas ...[more]

PMID: 30082838

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