Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors

Base editing requires that the target sequence satisfy the PAM requirement of the Cas9 domain and that the target nucleotide is located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG PAMs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing.