Mechanism of expanded DNA recognition in xCas9

Abstract xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we establish the molecular determinants of xCas9’s expanded PAM recognition. We show that while Cas9 enforces strict guanine selection through the rigidity of its interacting arginine dyad, xCas9 modulates the flexibility of R1335 to recognize specific PAM sequences selectively. This modulation confers a pronounced entropic preference for the canonical TGG PAM over SpCas9. Moreover, xCas9 expands PAM recognition by enhancing DNA binding in the early evolution cycles and improving binding to the canonical PAM in the final evolution cycle. This dual capability explains how xCas9 expands PAM recognition while also enhancing recognition of the canonical TGG PAM. These findings will facilitate the engineering of Cas9 variants more effective and specific across a broader spectrum of genetic sequences.