Berkeley, California, USA
March 25, 2026

Transposons, the so-called “jumping genes,” are genetic elements that can excise themselves from DNA and insert themselves elsewhere in the genome. Some transposon families include a programmable endonuclease called TnpB, thought to be an ancestor of Cas enzymes used in CRISPR-Cas genome editing. Research teams at the Innovative Genomics Institute have recently been exploring the possibility of harnessing the jumping ability of TnpBs as a novel tool for genome editing, both in humans and plants. Because the use of TnPBs as genome-editing tools is so new, we wanted to provide an overview of TnpBs, how they work, and how they differ from CRISPR genome editors.

The advantages of being small

CRISPR genome editing has one significant limitation: the enzymes are large. Some TnpBs are less than half the size of CRISPR-Cas enzymes (~400 amino acids compared to >1000), meaning they aren’t restricted by many of the same physical barriers. 

In both plants and animals, viral delivery is commonly used to transport gene-editing proteins into specific cells. One problem is that viruses often have a finite carrying capacity, and cannot reliably transport large systems like CRISPR-Cas, but TnpBs are easier to carry. 

How TnpBs jump around the genome

Despite their shared evolutionary origins, TnpBs operate very differently from Cas enzymes. In order for a transposon to move across the genome, it must “jump” to a new site, a process that involves excision from its original site. This excision is performed by TnpA, a transposase enzyme that recognizes the beginning and end of the transposon and cuts at each sequence. The excised transposon can then insert itself into a new locus in the genome, but the original copy of the transposon must be restored.

To restore the transposon in the edited strand of DNA, TnpB initiates homologous recombination, a cellular mechanism that copies genetic information from one strand to another. This requires TnpB to cut the DNA at the excision site, which is made possible with guidance from the sequence encoded within the TnpB’s “right end element RNA” (reRNA), enabling TnpB to target a transposon-associated motif (TAM), a programmable site with a unique sequence requirement.

By locating the TAM and target site, the reRNA can position TnpB to make a double-stranded DNA (dsDNA) break at its target site, where the TnpB was originally excised. Recognizing that DNA damage has occurred, the cell performs homologous recombination, using DNA from the complementary strand as a reference to repair any potential mistakes. With a transposon in both strands of DNA, the transposon can excise itself once again, and the cycle continues.

Optimizing TnpB editors for practical use

The ability of TnpBs and their reRNA guides to recognize specific motifs and make precise, programmable cuts is what powers their potential as a novel tool in genome editing. However, with the RNA-guided endonuclease activity of TnpB only being experimentally characterized in 2021, this field remains new. As such, TnpBs have not been optimized for high editing efficiency enough to compete with other systems like CRISPR-Cas. Brittney Thornton and Rachel Weissman of the Savage and Doudna labs at the IGI are two of the researchers working to improve TnpB systems for implementation in modern biotechnology. In a new paper in Nature Biotechnology, the research team evaluated the effects of changing every individual amino acid of TnpB through deep mutational scanning (DMS). 

Favorable mutations were identified by using DMS libraries of TnpB, encompassing nearly all single–amino acid substitutions in the protein and single-nucleotide mutations in the reRNA. These pooled variant libraries were transformed into a yeast reporter strain, and they measured performance of each variant by programming TnpB to repair an essential gene, based on an assay previously used to improve CRISPR-Cas9. Edited yeast cells can survive and grow into colonies, while unedited cells do not proliferate. By sequencing DNA from the colonies that grew, they counted how many times each variant appeared in edited yeast cells. By scoring TnpB mutants against the unmodified (WT) TnpB, mutations can be associated with a positive or negative effect on activity. 

The DMS analysis revealed the effects, both positive and negative, of changing every individual amino acid, allowing researchers to identify patterns in performance. 

 “We were about to do this big screening assay and we wanted to be very rigorous about being able to interpret mutational effects,” explains Thornton, a co-author of the study. 

Having a good understanding of the biochemical structure enabled them to ensure that their results made sense, and the enriched mutations were rational. Studying the physical shape of the TnpB reRNA uncovered a region they called the “hinge,” hypothesized to regulate when the TnpB is active. This provides a possible explanation for why mutations in the “hinge” were enriched in the DMS: changes in this region may leave TnpB in a state free to make more edits. Along with increasing confidence in their results, this strong foundation enabled them to make hypotheses about other gain- or loss-of-function mutations. 

Remarkably, 20% of single amino acid mutations resulted in an increase in activity compared to WT TnpB. Considering TnpB’s role as a transposon-associated endonuclease, it makes sense that evolution might be selecting against highly active variants: excessive double-stranded breaks is not advantageous if it compromises host fitness. A hyperactive transposon risks disrupting essential genes, which would harm its host, reducing its evolutionary success — but these variants could be valuable as genome-editing tools. 

From their library of mutations, researchers chose 33 of the best reRNA mutations and combined them to create about 5,000 TnpB mutants. After more assays in yeast, five of the best mutants were selected for further testing in plants, using model organism Nicotiana benthamiana. All variants exhibited editing efficiencies 4–40 times that of WT TnpB.

Paired with WT reRNA, the top two TnpB mutants outperformed other novel engineered small RNA-guided endonucleases at editing in N. benthamiana, and were subsequently used in a second study in collaboration with the Dinesh-Kumar lab at UC Davis, recently published in Nature Plants. Using these enhanced mutants for genome editing in N. benthamiana, researchers found the eTnpBc variant was particularly successful, achieving up to 90% editing efficiency at targeted sites in the genome. Also, this method of genome editing is entirely transgene free, meaning no foreign DNA is inserted into the genome. This advancement demonstrates the potential of TnpB research, both in its current capabilities and scientists’ ability to spur it forward. By adapting nature’s tools to humanity’s needs, this exciting field promises to advance human knowledge while inventing new possibilities.

Read more

Thornton, B.W., Weissman, R.F., Rodriguez, J.E. et al. (2026) Engineered TnpB genome editors for plants and human cells identified by ribonucleoprotein mutational scanning. Nature Biotechnology: https://doi.org/10.1038/s41587-026-03059-7

Nagalakshmi, U., Rodriguez, J.E., Nguyen, T. et al. (2026) High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB. Nature Plants: https://doi.org/10.1038/s41477-026-02237-4

Website: http://innovativegenomics.org/

Published: March 25, 2026