This blog post was written by Dr. Kutubuddin Molla, investigator at ICAR-Central National Rice Research Institute.
When it comes to genome editing, CRISPR is a name that resonates with nearly every biologist, academic, and researcher. Among the most well-known CRISPR-associated nucleases are Cas9 and Cas12a, the heavyweights in the genome editing toolbox. However, both of these proteins are quite large — comprising approximately 1000–1400 amino acids — which poses a challenge for efficient delivery into cells, particularly via viral vectors. While viral delivery is a highly effective method for gene editing and allows targeting of specific cell types, it has a limited capacity to carry large DNA cargos. As a result, the search for smaller, compact genome editors has become a key focus in recent years.
TnpB nucleases represent an exciting class of compact genome editors, typically only around 400 amino acids in size. To give a visual analogy: if Cas9 or Cas12a is a soccer ball, TnpB is a baseball — much smaller, but still powerful. Our lab recently harnessed TnpB from Deinococcus radiodurans (Dra2TnpB) to create a genome editing system for plants (Karmakar et al., 2024). This version of TnpB is just 408 amino acids long — making it one of the most compact genome editors to date. We have developed multiple vector versions for TnpB-mediated genome editing, and two of our top-performing constructs are now available through Addgene (pKb-TnpB1 and pKb-TnpB2).
How TnpB Works: The Uniqueness of TnpB, TAMs, and ωRNA (reRNA)
TnpBs are thought to be evolutionary ancestors of Cas12 proteins. Functionally, while Cas9 contains two nuclease domains (HNH and RuvC-like), and Cas12a has one (RuvC-like), TnpB also operates with a single RuvC-like domain. In simple terms, Cas9 cuts DNA using two "scissors" (one for each strand), while TnpB, like Cas12a, uses just one to cleave both strands.
Similar to how Cas proteins require a PAM (protospacer adjacent motif) near the target DNA, TnpBs also need a short sequence motif called a TAM (transposon-associated motif), which is positioned like Cas12a PAM sites. For Cas9, the PAM is located just downstream (3′ end) of the target sequence, while for Cas12a and TnpB, the motif is found upstream (5′ end) of the target site. Additionally, the TAM changes based on the species of origin of the TnpB protein, much like how PAM sites change for different Cas species. For instance, the TnpB from Deinococcus radiodurans (Dra2TnpB) recognizes the TAM sequence 5′-TTGAT-3′, while TnpB from Deinococcus geothermalis (Dge10TnpB) targets a different TAM (5′-TTAT-3′). The unique TAM requirements expand the number of genomic sites that can be targeted with TnpB, offering access to regions previously unreachable with Cas-based editors.
TnpBs have a familiar targeting mechanism. Small RNA elements called right-end RNA (reRNA) or omega RNA (ωRNA) guide the TnpB to its DNA target, much like the single guide RNA (sgRNA) used in Cas systems. The structure is also reminiscent of sgRNAs, with reRNA containing two parts: a structural scaffold derived from the transposon's right-end element, and a 20-nucleotide (nt) guide sequence that is complementary to the DNA target.
The 20-nt guide region binds to the DNA sequence immediately downstream of the TAM. This RNA-DNA hybrid formation recruits TnpB to the site and activates its nuclease function. Once bound, TnpB cleaves the DNA at a position 15–21 base pairs downstream of the TAM, creating staggered double-strand breaks like those created by Cas12a.
Applications of TnpB
One of the major challenges in genome editing is the efficient delivery of editing reagents into cells. Viral vectors, because of their natural ability to infect cells, have emerged as promising tools for delivering these reagents in both plant and animal systems. Importantly, they allow transient delivery, enabling transgene-free genome editing (no transgene integration), which is especially valuable for regulatory approval and public acceptance. Recently, viral vectors have been successfully used to generate gene-edited plants without the need for tissue culture (Maher et al., 2020; Qiao et al., 2025).
However, a key limitation of viral vectors is their restricted cargo capacity, which makes it difficult to accommodate the large coding sequence of commonly used nucleases like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the size of Cas9 or Cas12a, TnpB is a much smaller genome editing tool that can be efficiently packaged into viral vectors. Even for advanced genome editing approaches like base editing and prime editing (Molla et al., 2021), which require fusing Cas proteins with additional enzymes or domains (increasing the overall size), using TnpB as the core nuclease could help keep the total construct size smaller than Cas9 alone.
In our recent study, we demonstrated that TnpB can also function as an efficient gene activator when fused to a transcriptional activation domain (Karmakar et al., 2024). Additionally, we showed that TnpB can be used to simultaneously target multiple genes, enabling efficient multiplexed genome editing in a single experiment (Karmakar et al., 2024).
Practical Guide: Using pK-TnpB1 and pK-TnpB2 Vectors
The vectors pK-TnpB1 and pK-TnpB2 are designed for plant genome editing using TnpB. The main difference between them lies in the choice of promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene as the plant-selectable marker and a kanamycin resistance gene for bacterial selection.
The process for cloning the 20-nt guide RNA is identical for both vectors. Here, we provide a step-by-step guide for using a Dra2TnpB.
Step 1: Design the 20-nt Guide Sequence
- Identify a TAM site (5′-TTGAT-3′) in your target gene.
- Choose a 20-nt sequence immediately downstream of the TAM for targeting.
- While plant-specific tools for predicting efficiency of guides are currently unavailable, you can use TEEP — a tool based on animal data.
Step 2: Prepare Oligos
- Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1).
- Oligo 2: Add 'ggcc' to the 5′ end of your reverse complement guide (see Figure 1).
- Perform standard phosphorylation and annealing of the two oligos.
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| Figure 1: Components of TnpB, schematic of pKb-TnpB1 and pKb-TnpB2, and guide design rules. |
Step 3: Vector Digestion
- Digest either pK-TnpB1 or pK-TnpB2 with BsaI.
- Run on a gel and purify the digested vector.
Step 4: Ligation
- Ligate the annealed oligos into the digested vector using your method of choice.
Step 5: Colony Screening
- Use Primer 92F (5′-cattacgcaattggacgacaac-3′) and Oligo 2 (specific to your target) to screen colonies via PCR.
- A 354-bp product confirms successful insertion.
- Alternatively, use M13R and Oligo 2 to screen.
- A 667-bp product confirms successful insertion.
Step 6: Confirmation
- Pick 2–3 positive colonies, grow them, and extract plasmids.
- Confirm the correct guide insertion by Sanger sequencing using either 92F or M13R as the sequencing primer.
Step 7: Transformation
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Your confirmed plasmid is now ready for Agrobacterium-mediated, biolistic, or PEG-mediated transformation into plant cells.
Conclusion & Future Directions
TnpB represents a new generation of compact, efficient, and versatile genome editing tools. Its small size and unique features make it especially promising for delivery via viral vectors and for multiplexed gene editing applications. As research into TnpB continues to evolve, we anticipate its use will expand into diverse plant and animal systems, potentially enabling more precise and accessible genome engineering. More elaborate protocols are available in our earlier publications (Karmakar et al., 2024; Karmakar et al., 2025).
We invite researchers to explore and use our TnpB-based genome editing vectors. If you have specific questions, feel free to contact the Molla Lab (X account: @Kutub_joy).
Dr. Kutubuddin Molla is an investigator at the ICAR-Central National Rice Research Institute in India. He specializes in genome editing tools with a focus on plants.
References and Resources
References
Karmakar, S., Panda, D., Baig, M. J., & Molla, K. (2025). A unified protocol for genome editing in monocot and dicot plants using a Transposon-Associated TNPB system. In Springer protocols handbooks/Springer protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20
Karmakar, S., Panda, D., Panda, S., Dash, M., Saha, R., Das, P., Avinash, S., Shih, J., Yang, Y., Nayak, A. K., Baig, M. J., & Molla, K. A. (2024). A miniature alternative to Cas9 and Cas12: Transposon‐associated TnpB mediates targeted genome editing in plants. Plant Biotechnology Journal, 22(10), 2950–2953. https://doi.org/10.1111/pbi.14416
Maher, M. F., Nasti, R. A., Vollbrecht, M., Starker, C. G., Clark, M. D., & Voytas, D. F. (2019). Plant gene editing through de novo induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi.org/10.1038/s41587-019-0337-2
Molla, K. A., Sretenovic, S., Bansal, K. C., & Qi, Y. (2021). Precise plant genome editing using base editors and prime editors. Nature Plants, 7(9), 1166–1187. https://doi.org/10.1038/s41477-021-00991-1
Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X., Hu, W., Wang, Y., Han, C., Li, D., & Wang, X. (2025). Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat. Nature Plants. https://doi.org/10.1038/s41477-025-02023-8
Additional Resources on the Addgene Blog
- CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9
- RNA Interference in Plant Biology: New Tools for an Old Favorite
- A History of Genome Editing in Popular Culture
Additional Resources on addgene.org
Topics: Other Plasmid Tools
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