RNA-guided transcriptional repression by TIGR-Tas systems

Abstract

Tandem interspaced guide RNA (TIGR)–TIGR-associated protein (Tas) systems are a widespread family of RNA-guided DNA-targeting proteins whose diversity has remained uncharacterized because their arrays, unlike CRISPR arrays, lack the sequence conservation required by existing annotation tools. We developed TIGRFinder, a motif-based pipeline that identified 6,685 TIGR arrays from genomic and metagenomic data. Phylogenetic and structural analysis of Tas proteins revealed clade-specific insertions in stem-loop binding Tas proteins that co-vary with features of their cognate tigRNAs. Cryo-electron microscopy structures of two stem-loop binding TasR ribonucleoprotein complexes demonstrate how these protein insertions directly accommodate extended tigRNA stems while maintaining DNA binding through catalytically inactive RuvC domains. We also show that these catalytically dead TasR proteins, along with the nuclease-lacking TasA, function as RNA-guided transcriptional repressors.These findings establish TIGR-Tas as a functionally diverse family of RNA-guided effectors, with comprehensive annotations available through TIGRSafari, which will be made publicly available upon publication, to support further exploration and engineering.