Cas9 variants expand the targeting scope of base editing systems in bacteria

Abstract

Base editors (BEs) consist of partially active Cas9 homologs fused to nucleobase-converting DNA deaminases along with certain accessory proteins. The effectiveness of BE tools depends on the enzymatic efficiency of deaminases and the availability of protospacer adjacent motif (PAM) necessary for functioning of Cas enzyme. Furthermore, the target nucleotides must be situated within the editing window (EW) at the targeted genetic loci. In addition to SpCas9, which recognizes NGG PAM, there are various SpCas9 variants and orthologs recently characterized to recognize non-NGG PAMs or exhibit broader EW lengths when used in BEs. These Cas9 homologs have shown promise for BE tool development in plant and mammalian genome editing (GE). In this study, we engineered C-to-T modifying cytosine (CBE) and A-to-G modifying adenine (ABE) modules using SpCas9 variants for bacterial GE. These variants utilize circularly permuted SpCas9 (CP-Cas9) with extended EW lengths. Furthermore, BEs constructed by replacing SpCas9 nickase (nSpCas9) with the SaCas9 variant (nSaKKHCas9), which is compatible with NNNRRT PAM, expanded the targetable sites beyond the NGG PAM requirement. The SaCas9-based BE tool was successfully employed to evolve the rpoB gene, imparting antibiotic (rifampicin) resistance, highlighting the expanded potential of engineered BEs for bacterial GE applications.