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Reconstruction of PAM-interacting domains to broad PAM compatibility
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DOI：10.1186/s13059-026-04241-5.png)
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Abstract
En 中文
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.
Keywords:
CRISPR-Cas9
Genome editing
PAM specificity
Ancestral sequence reconstruction
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