Structure‐Guided Engineering of a Cas12i Nuclease Unlocks Near‐PAMless Genome Editing

ABSTRACT The therapeutic and research applications of CRISPR‐Cas nucleases are constrained by their reliance on specific Protospacer Adjacent Motifs (PAMs), which limit the accessible sites in the genome. To overcome this critical barrier, we performed structure‐guided engineering of SF01, a compact Cas12i nuclease. Using AlphaFold‐predicted structural models, we identified and systematically mutagenized 38 residues at the PAM‐interacting interface. This iterative engineering process yielded three superior variants—KR, IKRR, and STKRR—that exhibit dramatically relaxed PAM specificity, enabling efficient editing at a broad spectrum of 5'‐NNTN‐3' sites. Importantly, while the most broad‐spectrum variant (STKRR) shows a trade‐off at canonical sites, the IKRR variant retains high activity at canonical 5'‐NTTN‐3' PAMs while simultaneously enabling efficient editing at 5'‐NNTN‐3' sites. This near‐PAMless activity expands the targetable portion of the genome to over 25%, a four‐fold increase over the parental nuclease. Furthermore, adenine base editors (ABEs) constructed with these variants achieve high‐efficiency editing (∼80%) at endogenous loci with expanded targeting scope. Comprehensive off‐target analysis using GUIDE‐tag and Digenome‐seq revealed that the enhanced on‐target activity of the SF01 variants is not accompanied by a loss of specificity. These engineered nucleases represent a powerful and versatile expansion of the genome editing toolkit, enabling applications previously inaccessible due to PAM constraints.