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 OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

NT-CRISPR: Combining natural transformation and CRISPR/Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

“…Anti-CRISPR proteins have been used in eukaryotes to achieve spatiotemporal control over CRISPR/Cas9 activity, e.g. through an optogenetic approach (Bubeck et al ., 2018), by selective expression in certain cell types (Hoffmann et al ., 2019) or to reduce off-target effects (Shin et al ., 2017; Aschenbrenner et al ., 2020) and also in the prokaryotic species Clostridium acetobutylicum to allow cells to carry cas9 and gRNA simultaneously without toxic effects (Wasels et al ., 2020). In a similar fashion, we used constitutive expression of acrIIA4 to compensate for the basal production of Cas9 and gRNA.…”

Challenges and Opportunities for the Veterinary Pathologist in Biomedical Research

“…However, in the rapid development and application of this technology, there have been concerns particularly for off-target effects, safety, and selection of appropriate controls in the use of these tools. 44,89,94 While gene editing tools are often described as "specific" for their targets, research studies continue to report new approaches to improve specificity, 3,45 which also raises questions as to how thoroughly off-target effects are being routinely evaluated. Since pathologists serve as key professional resources on multidisciplinary teams and provide critical expertise in phenotypic and safety studies, they continually update their expertise to provide effective guidance in defining and evaluating appropriate controls, safety assessments, evaluating target specificity, and screening for off-target effects.…”

Tuning up Transcription Factors for Therapy

“…Kinetic models suggest that the cleavage of off-target sequences can be reduced by accelerating the dissociation of the enzyme from the DNA or by reducing its catalytic activity [94]. Following these predictions, an enhancement of cleavage specificity has been realized by designing enzymes with attenuated cleavage rate or by fusing Cas9 to inhibitor proteins [95,96]. In the above model, the specificity is affected by both the binding and catalytic reactions because they are coupled, since both of them involve the same molecular species, the enzyme.…”