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SuperFi-Cas9 exhibits extremely high fidelity but reduced activity in mammalian cells

P. Kulcsár,András Tálas,E. Welker

Published 2022 in bioRxiv

ABSTRACT

Several advances have been made to SpCas9, the most widely used CRISPR/Cas genome editing tool, to reduce its unwanted off-target effects. The most promising approach is the development of increased fidelity nuclease (IFN) variants of SpCas9, however, their fidelity has increased at the cost of reduced activity. SuperFi-Cas9 has been developed recently, and it has been described as a next-generation high fidelity SpCas9 variant, free from the drawbacks of the first-generation IFNs. In this study, we characterized the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells comparing it to first-generation IFNs. SuperFi-Cas9 demonstrated strongly reduced activity but exceptionally high fidelity exhibiting features that are in many aspects similar to those of the first-generation variants, such as evo- and HeFSpCas9. When combined with ABE8e, SuperFi-Cas9 produced DNA editing with high activity rate as well as high specificity by reducing both bystander and SpCas9-dependent off-target base editing.

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