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Nat Commun. 2018 Aug 6;9(1):3048. doi: 10.1038/s41467-018-05477-x.

Directed evolution of CRISPR-Cas9 to increase its specificity.

Author information

1
Toolgen, Seoul, 08501, Republic of Korea. jj.lee@toolgen.com.
2
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, 34121, Republic of Korea.
3
Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea.
4
Toolgen, Seoul, 08501, Republic of Korea.
5
Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, 34121, Republic of Korea. jskim01@snu.ac.kr.
6
Department of Chemistry, Seoul National University, Seoul, 08826, Republic of Korea. jskim01@snu.ac.kr.

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.

PMID:
30082838
PMCID:
PMC6078992
DOI:
10.1038/s41467-018-05477-x
[Indexed for MEDLINE]
Free PMC Article

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