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RNA-guided transcriptional repression by TIGR-Tas systems

Peiyu XuiD, Lü LongiD, Annie Zhu, Stephanie Kim, Shai Zilberzwige‐TaliD, Natalia Quinones‐OlveraiD, Lilia EvgeniouiD, Daniel Flam-ShepherdiD, Rhiannon K. MacraeiD, Guilhem FaureiD, Feng ZhangiD

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 (https://tigr.bio) to support further exploration and engineering.

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Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (11 Sept 2026) · CC BY 4.0

TIGRFinder annotated 6,685 TIGR arrays; cryo-EM structures show inactive RuvC domains, and E. coli reporter assays show CsTasR and MbTasA repress transcription in a guide-dependent manner.

TIGR arrays lack the sequence conservation that CRISPR annotation tools depend on, so the diversity of Tas proteins and their link to guide RNA architecture was uncharacterized. This work adds a dedicated array finder, structures of two stem-loop TasR complexes, and evidence of a non-cleaving repressor function.

Key results

  1. ShownA motif-anchored pipeline for dual-repeat and stem-loop architectures greatly expanded the annotated TIGR array catalog over the earlier manually curated set. Dual-repeat loci carry longer arrays than stem-loop loci.

    Scanning 20,096 tas loci gave 6,685 TIGR arrays. Dual repeat arrays numbered 4,126 (65,635 tigRNAs); stem-loop arrays numbered 2,559 (17,756 tigRNAs). · TIGRFinder enables systematic discovery of TIGR…

  2. ShownStem-loop Tas proteins differ from dual-repeat Tas proteins in NOP segment lengths and lack the C-terminal Y/H processing loop. Their stem-loop tigRNA features (loop, linker, stem) vary by clade, consistent with protein-RNA co-evolution.

    Stem-loop Tas have significantly longer coiled-coil tips and insertion 1 and shorter CTD5; insertion 2 is longer in dual-repeat Tas. The Y/H dyad (Y289, H296) is absent across all stem-loop clades. Test details are not given. · Extensive structural diversification of…; Co-evolution of tigRNA architecture and Tas…; Stem-loop binding Tas proteins lack the Y/H…; Phylogenetic tree construction

  3. ShownCryo-EM structures of two stem-loop TasR complexes show inactive RuvC domains that still scaffold DNA binding. A shortened C-terminal loop and lineage-specific insertions accommodate extended tigRNA stems.

    PateTasR map 2.9 Å (34,352 particles; figure legend states 2.8 Å); CsTasR 3.3 Å (125,754 particles). PateTasR C-terminal loop is 8 aa vs 27 aa in SpTasH; CsTasR has an ~80-residue RuvC insertion; catalytic positions filled by R8 and V126. · Cryo-EM structure of PateTasR reveals a…; The CsTasR structure reveals co-adaptation of…; Cryo-EM data collection and processing; Figure legends

  4. ShownCsTasR and MbTasA repressed an mTurquoise2 reporter in E. coli in a guide-dependent, reversible manner, and neither PateTasR nor CsTasR cleaved DNA in vitro. Retargeting the guide to chromosomal cysG also lowered that transcript.

    No cleavage by PateTasR or CsTasR in vitro. CsTasR and MbTasA strongly reduced mTurquoise signal with targeting tigRNAs but not with native non-targeting tigRNAs. MbTasA EMSA gave a complete shift at low RNP. At least three biological replicates. · Catalytically inactive TasR and TasA proteins…; TIGR gene repression assays; Electrophoretic mobility shift assay (EMSA)

  5. SuggestedSmall RNA-seq of purified RNPs shows that stem-loop arrays are processed into discrete tigRNAs in E. coli. Processing boundaries often fall at a conserved CUU motif at the stem base, and extra flanking sequence sharpens the boundaries.

    CsTasR and BlTasA loci both gave multiple processed tigRNAs; BlTasA had intragenic units. Extended context including 2,215 nt of downstream flank gave sharper boundaries. The cleaving enzyme is not identified. · Stem-loop tigRNAs are expressed from flexible…; Discussion

Methods, data and reproducibility

Motif seed-and-extend array detection (TIGRFinder) was combined with structure-guided phylogenetics of Tas proteins. Cryo-EM of two TasR RNPs, small RNA-seq, in vitro cleavage and EMSA assays, and an E. coli fluorescent reporter assay tested function.

  • Previously identified tas loci scanned for TIGR arrays by TIGRFinder (phages, archaeal viruses, CPR bacteria; metagenomic) · viruses and CPR bacteria · n = 20,096 tas loci
  • Flanking genomic context for source annotation, host and viral origin prediction · metagenomic and bacterial sources · n = 4,817 loci
  • Cryo-EM dataset of PateTasR RNP co-expressed with its native array in E. coli · Patescibacteria group bacterium protein, expressed in E. coli · n = 2,898 images; 34,352 final particles
  • Cryo-EM dataset of CsTasR RNP with a 20-bp dsDNA target · Candidatus Staskawiczbacteria bacterium protein, expressed in E. coli · n = 6,284 images; 125,754 final particles
  • Small RNA-seq of purified CsTasR and BlTasA RNPs with native loci · E. coli host
  • In vivo mTurquoise2 reporter repression assays with CsTasR and MbTasA · E. coli BW25113 · n = at least three biological replicates
Methods & tools
TIGRFinder, DALI, IQ-TREE 3.0.1 (ModelFinder, BLOSUM62+R10), MMseqs2 with GTDB R232, geNomad v1.12.0, CryoSPARC v4.2.0, RELION 4.0, MotionCor2, CTFFIND4, Cutadapt, Bowtie 2, GraphPad Prism 10
Reagents & environment
IQ-TREE 3.0.1; geNomad v1.12.0 (database v1.9); GTDB R232; CryoSPARC v4.2.0; RELION 4.0; Leginon 3.6; GraphPad Prism 10.
Reproducibility gaps
TIGRFinder code, TIGRSafari, annotation CSVs, PDB/EMDB depositions and supplementary table are all stated to be available only upon publication.
Limitations (5)
  • The data do not report tigRNA 5' phosphorylation state, so processing of a longer precursor cannot be distinguished from independent promoter-driven transcription, especially for internal segments. · stated by the authors
  • The enzyme that cleaves stem-loop tigRNAs at the CUU motif is unknown (Tas protein, host RNase or auxiliary factor). Natural spacer targets are mostly undeterminable, and spacer acquisition is unexplained. · stated by the authors
  • At least one divergent branch of compact TasR proteins had no detectable arrays. It is unresolved whether these are arrayless systems or escape current detection. · stated by the authors
  • Repression was shown for only two effectors (CsTasR, MbTasA) in a heterologous E. coli host. PateTasR was not tested in the reporter assay, and the text gives no numeric repression magnitudes. · noted in this summary
  • The text is internally inconsistent: PateTasR resolution is 2.9 Å in the methods but 2.8 Å in the figure legend. Reporter inducer assignments differ between the legend and the methods. · noted in this summary

Who should read:Read this if you work on TIGR-Tas or other compact RNA-guided effectors and want annotation methods, stem-loop TasR structures, or evidence for nuclease-free transcriptional repression.

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