Structure-Led Exploration of the Metagenome Yields Novel RNA-Guided Nucleases with Broad PAM Diversity

Compact RNA-guided nucleases with favorable targeting properties are challenging to discover due to their low natural abundance. Here, we develop a structure-led search strategy -leveraging predicted protein folds and sequence-independent similarity metrics -to systematically identify extremely low-homology compact RNA-guided nucleases across vast metagenomic datasets with high computational efficiency. Homology clustering resolved these proteins into distinct groups, for which we performed comprehensive PAM profiling and evaluated editing efficiency in eukaryotic cells. This structure-guided discovery revealed a previously undiscovered landscape of compact nuclease subtypes that exhibit extensive protospacer-adjacent motif (PAM) diversity, expanding the targeting potential of compact editors. Comparative analysis across the novel RNA-guided nuclease families demonstrates that compact systems are not intrinsically limited to highly constrained PAMs but instead have a broad and previously unknown breadth of genome targeting capabilities, comparable to that of Cas9 and far exceeding common transposon-derived systems. Additionally, this search revealed that a compact transposon-associated motif (TAM) is a prerequisite for the emergence of a CRISPR-Cas system from ancestral transposons, before protein domain expansions increase the target length and specificity constraints. These results enrich the catalog of RNA-guided nuclease architectures and contribute validated compact genome editing tools with broad and diverse PAM recognition, which may have therapeutic applications.