Tiny TnpB: The next

Genome editing stands as one of the most transformative scientific breakthroughs of our time. It allows us to dive into the very code of life and make precise modifications. Imagine being able to rewrite the genetic instructions that determine almost everything about an organism—how it looks, behaves, interacts with its environment, and its unique characteristics. This is the power of genome editing.

We use genome editing tools to tweak the genetic sequences of microbes, animals, and plants. Our goal? To develop desired traits and eliminate unwanted ones. This technology's impact has been felt across biotechnology, human therapeutics, and agriculture, bringing rapid advancements and solutions.

The most widely used proteins in genome editing are Cas9 and Cas12a. These proteins are like the scissors of the genetic world, allowing us to cut and edit DNA. However, they are quite bulky, consisting of 1,000–1,350 amino acids. Advanced editing technologies like base editing and prime editing require the fusion of additional proteins with Cas9 and Cas12a, making them even bulkier. This bulkiness poses a challenge to delivering these proteins efficiently into cells, where the genetic material resides.

But now, we have an exciting development—a miniature alternative that promises to overcome this limitation. In our recent article in the Plant Biotechnology Journal, we introduced TnpB, a smaller, yet highly effective next-generation tool for genome editing in plants.

TnpB proteins are transposon-associated nucleases guided by RNA. They are considered the evolutionary ancestors of Cas12 nucleases. Although TnpB is functionally similar to Cas12a, it is much more compact, with a total amino acid count ranging from 350–500. To put it in perspective, TnpB is one-third the size of Cas9 and Cas12a. If Cas9 and Cas12a are like soccer balls, TnpBs are like baseballs.

We have developed a hypercompact genome editor using the TnpB nuclease from Deinococcus radiodurans. This bacterium is known for its ability to survive extreme environments and its remarkable resistance to radiation. Our TnpB, sourced from D. radiodurans, is only 408 amino acids long.

A short RNA serves as a guide for TnpB, directing it to the target DNA sequence. Specified by this RNA, TnpB binds to the target and cleaves both strands of DNA. When the broken ends are re-sealed by the cell, insertions or deletions of DNA letters can inadvertently occur. These insertions or deletions result in the modification of genetic sequences.

An additional level of specificity exists: The target sequence must be adjacent to a Transposon Associated Motif (TAM) sequence. This TAM is analogous to PAM sequence of Cas9 and Cas12. For the TnpB from D. radiodurans, the specific TAM is TTGAT, which must be present upstream of the target sequence. In that sense, TnpB can access genomic loci that Cas9 can't reach.

We first codon-optimized the sequence for the TnpB protein to develop a genome editor for plant systems. We also optimized the combinations of regulatory elements to produce enough guide RNA for high-efficiency plant genome editing. By testing four different versions of genome editing vector systems in rice protoplasts, we identified the most effective version.

Rice is a monocot, and systems that work well in monocots may not perform as well in dicots. Therefore, we generated dicot-specific TnpB vectors and demonstrated successful editing in Arabidopsis. Interestingly, we observed that most deletions occurred at the target loci in both rice and Arabidopsis. This makes TnpB suitable for effectively disrupting gene functions. TnpB could now be used for introducing genetic mutations to disrupt undesired genes for removing antinutrient factors, enhancing nutrient content, biotic and abiotic stress resistance, and more.

While TnpB in its native form acts as a programmable scissors, it can also be adapted to recruit factors that activate genes. By inactivating its cutting ability, we developed deactivated TnpB (dTnpB). dTnpB retains its ability to bind to target DNA specified by guide RNA. We then fused dTnpB with additional cargo proteins to channel them to target genes, making these genes more active. This activation tool can boost gene function, paving the way for creating better crops in the future.

Similarly, we fused another cargo protein with dTnpB to develop a tool capable of swapping one DNA letter for another. This precise tool will enable crop innovation by altering the genetic code with single-letter resolution.

We are leveraging this miniature genome editor to create rice plants with improved yields and increased climate resilience. Our research highlights TnpB as a highly versatile and promising tool for plant genome engineering. We expect that plant biologists, biotechnologists, and breeders will adopt TnpB for use in a variety of crops.

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More information: Subhasis Karmakar et al, A miniature alternative to Cas9 and Cas12: Transposon‐associated TnpB mediates targeted genome editing in plants, Plant Biotechnology Journal (2024). DOI: 10.1111/pbi.14416

Journal information: Plant Biotechnology Journal

Dr. Kutubuddin Molla is a scientist specializing in agricultural biotechnology at the ICAR-National Rice Research Institute (NRRI) in Cuttack, India. He earned his Ph.D. from the University of Calcutta, Kolkata. Dr. Molla has conducted post-doctoral research at Pennsylvania State University with the Fulbright scholarship.

Dr. Molla's research interests focus on precise genome editing, employing CRISPR-Cas and other advanced techniques for crop improvement. His laboratory at NRRI is dedicated to developing novel genome editing tools and applying them to enhance crop performance.