A recent study has explored the potential of TnpB proteins as an innovative genome editing tool, revealing promising results for their application in plant systems. Traditionally, Cas9 and Cas12a nucleases have been the go-to genome editors, but their large size presents challenges for cell delivery, particularly through viral vectors. TnpB proteins, being significantly smaller, offer an attractive alternative. Although TnpBs have been shown to function as RNA-guided nucleases in human cells, their efficacy in plants was previously untested.
Researchers focused on developing a TnpB genome editing system using TnpB protein from Deinococcus radiodurans ISDra2. This study achieved an average editing efficiency of 33.58% in the plant genome. The research team codon-optimized the ISDra2TnpB and cloned it under the OsUbi10 promoter. They used a protoplast system workflow to evaluate TnpB-mediated editing, constructing the pK-TnpB1 vector, which also included the reRNA component essential for target DNA recognition and cleavage.
A notable aspect of TnpB is its dependence on the transposon-associated motif (TAM) sequence 5′-TTGAT-3′. Genome-wide analysis in rice revealed a 0.35% TTGAT TAM coverage, highlighting TnpB’s unique ability to target regions inaccessible to Cas9 or Cas12a. The study designed guide RNAs for six rice genomic loci, assessing the effectiveness by transfecting rice protoplasts and using restriction enzyme analysis. Results showed that TnpB-induced mutations were confirmed by Sanger sequencing, with deletions ranging from 7 to 53 base pairs.
Further analysis confirmed TnpB’s high specificity for the canonical TTGAT TAM sequence, with minimal activity on noncanonical sequences. The study also demonstrated TnpB’s suitability for multiplex genome editing by targeting multiple genes simultaneously in rice, achieving notable indel efficiencies.
The researchers extended their experiments to a dicot model, Arabidopsis thaliana, using the TnpB-D1 vector. Initial editing efficiencies were modest, but by replacing the promoter with the eCaMV35S promoter, the enhanced TnpB-D2 vector showed improved results.