Enhancing hypercompact CasPhi2 activity through EPICA.2, an optimized eukaryotic directed evolution platform
Compact Cas nucleases offer advantages over the widely used SpCas9 due to their smaller size, which enables more efficient delivery for in vivo applications. Among these, the phage-encoded CasPhi2 (Cas12j2) is highly promising because of its relaxed PAM requirement (5'-TTN-3') and compact size (757 aa). However, its translational potential is limited by low editing activity.To enhance the editing efficacy of CasPhi2, having nearly undetectable activity, we developed EPICA.2, a eukaryotic directed evolution platform from a previously reported system for optimizing Cas variants. EPICA.2 integrates additional yeast evolution rounds to enrich for active variants coupled with a low background mammalian reporter system that improves the detection and selection of enhanced variants. Finally, we set up a nanopore long read sequencing protocol which uses unique molecular identifiers (UMIs) to reduce sequencing errors, enabling the accurate identification of the combination of mutations present in each evolved variant. Among the most frequent variants, we obtained evoCasPhi2, which contains six activity-boosting mutations and outperformed both the wild-type nuclease and previously engineered variants nCasPhi2 and vCasPhi2.We report evoCasPhi2, an evolved CasPhi2 variant generated using a modified eukaryotic directed evolution platform EPICA.2, optimized to enhance Cas editors with very low activity. Compared to wild-type, evoCasPhi2 showed up to 70-fold increased activity in human cells and outperformed variants generated through a rational approach.