SuperFi-Cas9 exhibits remarkable fidelity but severely reduced activity yet works effectively with ABE8e
Péter István Kulcsár, András Tálas, Zoltán Ligeti, Sarah Krausz, Ervin Welker
Abstract
Several advancements 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 first-generation IFNs. In this study, we characterize the on-target activity and the off-target propensity of SuperFi-Cas9 in mammalian cells, comparing it to first-generation IFNs. SuperFi-Cas9 demonstrates strongly reduced activity but high fidelity features that are in many aspects similar to those of some first-generation variants, such as evo- and HeFSpCas9. SuperFi-cytosine (CBE3) and -adenine (ABE7.10) base editors, as well as SuperFi-prime editor show no meaningful activity. When combined with ABE8e, SuperFi-Cas9, similarly to HeFSpCas9, executes DNA editing with high activity as well as high specificity reducing both bystander and SpCas9-dependent off-target base editing.