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.