CRISPR-Cas systems provide microbes with adaptive immunity by employing short sequences, termed spacers, that guide Cas proteins to cleave foreign DNA 1,2 . Class 2 CRISPR-Cas systems are streamlined versions in which a single Cas protein bound to RNA recognizes and cleaves targeted sequences 3,4 . The programmable nature of these minimal systems has enabled their repurposing as a versatile technology that is broadly revolutionizing biological and clinical research 5 . However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving untapped the vast majority of enzymes from organisms that have not been cultured. Metagenomics, the sequencing of DNA extracted from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms 6,7 . Here, using genome-resolved metagenomics, we identified novel CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet identified. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in E. coli. Interrogation of environmental microbial communities combined with in vivo experiments allows access to an unprecedented diversity of genomes whose content will expand the repertoire of microbe-based biotechnologies.
