T-GUIDE

TnpB is a hypercompact RNA-guided DNA endonuclease encoded within IS200/IS605 family transposons in bacteria and archaea, widely regarded as an evolutionary precursor to type V CRISPR-Cas12 systems. Typically around 400 amino acids in length — significantly smaller than Cas9 or Cas12 — TnpB uses a single guide RNA known as ωRNA (reRNA) and recognizes a 5′ target-adjacent motif (TAM) for DNA cleavage. Its compact size makes it particularly attractive for applications where delivery cargo capacity is limited.

T-GUIDE is a web tool developed for TnpB ωRNA guide design, accepting three types of target input: gene symbols (retrieved via Ensembl), NCBI accessions, and direct DNA sequences. It identifies candidate ωRNA guides based on selected TnpB orthologs and their specific TAM requirements, provides TEEP deep learning-based efficiency predictions (CNN and RNN scores), performs genome-wide off-target analysis using an adapted Cas-OFFinder engine configured for 5′ TAM orientation, and generates primer pairs using Primer3. Results include interactive visualization of targets and direct links to the UCSC Genome Browser for genomic context.