CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

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Here, we integrate fine-tuned protein language models and protein-protein interaction databases to develop a Structure-agnostic Language Transformer & Peptide Prioritization module that efficiently selects peptides from interaction interfaces, without the need for structural information. We experimentally fuse SaLT&PepPr-derived “guide” peptides to E3 ubiquitin ligase domains and reliably identify candidates that induce robust intracellular degradation of clinically-relevant targets, and exhibit high binding affinities, low off-targeting rates, and functional transcriptional effects.