Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

ABSTRACT: The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OF

SUBMITTER: Aschenbrenner S 

PROVIDER: S-EPMC7002122 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature