Reconstruction of PAM-interacting domains to broad PAM compatibility

The protospacer adjacent motif (PAM) requirement limits CRISPR-Cas9 targetability. Here, we develop REPAM, an ancestral sequence reconstruction-based strategy that re-engineers PAM-interacting domains to expand PAM recognition. REPAM broadens the PAM specificities of Nme1Cas9 and Nme2Cas9 from N4GATT/N4CC to N4CNH, while additional mutations further extend Nme2Cas9 recognition to N4VHH. Applied to SpaCas9, REPAM expands PAM compatibility from NNGYRA to NNHH, increasing theoretical target coverage by approximately 36-fold. The engineered SpaCas9 achieves up to 76.6% editing efficiency in HEK293T cells, outperforming SpRY. Corresponding base editors efficiently generate protective variants and precise splice-site mutations, highlighting REPAM as a versatile platform for CRISPR engineering.