Continuous directed evolution of a compact Cj Cas9 variant with broad PAM compatibility

CRISPR–Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 ( Cj Cas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evo Cj Cas9, primarily recognizes N 4 AH and N 5 HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N 3 VRYAC PAM site. Moreover, evo Cj Cas9 exhibits higher nuclease activity than wild-type Cj Cas9 on canonical PAMs, with editing rates comparable to commonly used PAM-relaxed Sp Cas9 variants. Combined with deaminases or reverse transcriptases, evo Cj Cas9 enables robust base and prime editing, with the small size of evo Cj Cas9 base editors allowing for tissue-specific installation of A-to-G or C-to-T transition mutations from single adeno-associated virus vector systems.