TIGR-Tas

TIGR-Tas (Tandem Interspaced Guide RNA-associated proteins) is a family of RNA-guided DNA-targeting systems discovered in prokaryotes and their viruses. These systems utilize a dual-spacer targeting mechanism, compared to the single spacer required by CRISPR-Cas9-mediated gene targeting.

TIGR-Tas systems were reported in February 2025 by researchers at the Broad Institute of MIT and Harvard and MIT's McGovern Institute for Brain Research.^[1]

TIGR-Tas systems were discovered through computational mining approaches that began with structural analysis of the RNA-binding domain of SpCas9. Through iterative structural and sequence homology-based searches, protein were discovered that contain Nop domains—hallmarks of eukaryotic box C/D snoRNA ribonucleoproteins (RNPs)—associated with distinctive tandem interspaced guide RNA arrays.^[1]

The discovery process employed advanced computational methods, including protein large language models, to cluster related proteins based on their likely evolutionary relationships. This approach identified more than 20,000 different Tas proteins, predominantly from bacteriophages and parasitic bacteria.^[2]

TIGR arrays consist of repetitive sequences organized into dual-repeat units or stem-loop structures. Each unit contains:^[1][2]

• Edge repeats and loop repeats (8-12 nucleotides each)
• Spacer A and Spacer B (typically 9 nucleotides each)
• Conserved box C and box D motifs similar to those found in snoRNAs

TIGR-associated (Tas) proteins are classified into three main types:^[1][3]

• TasA (from Flavonifractor plautii): Contains only the Nop domain
• TasH (from Salicola phage): Nop domain fused with an HNH nuclease domain
• TasR (from Thermoproteota archaeon): Nop domain fused with a RuvC nuclease domain

TIGR arrays are transcribed and processed into 36-nucleotide guide RNAs called tigRNAs. Processing occurs at precise sites within edge repeats and requires the presence of Tas proteins, though the proteins themselves do not directly catalyze the cleavage.^[1][3]

Unlike CRISPR systems that use a single guide RNA to target one DNA strand, TIGR systems employ a tandem-spacer targeting mechanism:^[1][3][4]

• Spacer A pairs with one DNA strand
• Spacer B pairs with the complementary DNA strand
• Both spacers must be correctly paired for efficient cleavage
• No protospacer-adjacent motif (PAM) is required

TasR nucleases create double-strand breaks with 8-nucleotide 3' overhangs, cleaving 3' to the nucleotide complementary to the 5th base of each spacer (following a "C - 5 rule").^[1]

Cryo-electron microscopy studies revealed that TasR forms a C2-symmetric dimer that binds target DNA and tigRNA. The structure shows:^[1]

• Dramatic 180° DNA bending upon complex formation
• Nop domains that recognize box C/D motifs in tigRNAs
• RuvC domains positioned for coordinated cleavage of both DNA strands

TIGR systems are found primarily in:^[1][5][2]

• Bacteriophages and archaeal viruses
• Parasitic bacteria of the Candidate Phyla Radiation
• Various prokaryotic genomes

Two main architectural variants exist:

1. Dual-repeat arrays: Traditional TIGR organization
2. Stem-loop arrays: Alternative organization lacking separating repeats

TIGR systems show evolutionary connections to:^[1][2]

• IS110 transposases: Share structural domains and RNA-binding mechanisms
• Box C/D snoRNPs: Common Nop domain architecture and box C/D motifs

These relationships suggest TIGR systems may represent an ancestral form of RNA-guided systems.

TIGR-TasR systems can be successfully adapted for:^[1][2][4]

• Programmable DNA cleavage in human cells
• Genome editing with unique targeting properties

TIGR-Tas systems offer several potential advantages over CRISPR technology:^[5][2][4]

• No PAM requirement: Can theoretically target any genomic site
• Compact size: Tas proteins are approximately one-quarter the size of Cas9, potentially facilitating cellular delivery for therapeutic applications
• Dual-guide system: May enhance specificity by requiring correct recognition of both DNA strands
• Modularity: Distinct functional domains that could be engineered for various applications

The small size and modularity of TIGR-Tas systems make them promising candidates for therapeutic gene editing applications, potentially overcoming delivery challenges associated with larger CRISPR proteins.^[2][4]

While the exact biological roles remain unclear, proposed functions include:^[1][3]

• Mobile genetic element (MGE) interference
• Gene regulation
• Plasmid maintenance and inheritance
• Inter-MGE competition

• CRISPR-Cas systems
• RNA-guided systems
• Box C/D snoRNPs
• IS110 transposases

• The New TIGR-Tas Gene Editing System by Steven Novella
• Move Over, CRISPR. Smaller, Smarter Gene-Editing System Found
• New RNA-guided system TIGR-Tas could challenge CRISPR’s stronghold