NanoPAMDA enables rapid PAM characterization of metagenome-derived Cas9 effectors
CRISPR-Cas9 orthologs remain functionally underexplored despite the rapidly growing scale of publicly available metagenomic sequencing data. Here, we applied a multi-stage computational discovery pipeline to two independent sources of metagenomic sequence data, a multi-biome collection of 660,185 metagenome-assembled genomes spanning 17 public catalogs, and raw sequencing reads from human…
CRISPR-Cas9 orthologs have yet to be fully explored, despite the increasing availability of metagenomic sequencing data. Researchers utilized a multi-stage computational discovery pipeline on two sets of metagenomic sequences - a collection of 660,185 metagenome-assembled genomes from 17 public databases, and raw sequencing reads from human microbiome studies. This pipeline identified 14 potential Cas9 orthologs for further testing.
To assess the preference for PAM sequences at a large scale, researchers created a cost-effective, long-read sequencing-based adaptation of the high-throughput PAM determination assay, named NanoPAMDA. This allowed them to rapidly and simultaneously profile the PAM preferences of multiple Cas9 candidates.
One of these Cas9 effectors, designated E10, exhibited PAM-specific activity in vitro. Furthermore, researchers confirmed that E10 was also catalytically active when introduced into E. coli bacteria. These results showcase a scalable, long-read sequencing-based approach for the discovery and biochemical characterization of novel Cas9 orthologs derived from metagenomic data.
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