Tiny TnpB: the next-generation genome editing tool for plants unveiled

Genome editing is one of the most transformative scientific breakthroughs of our time. It allows us to delve into the code of life and make precise adjustments. 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 desirable traits and eliminate undesirable traits. The impact of this technology has been felt in biotechnology, human therapy and agriculture, bringing rapid progress and solutions.

The most commonly 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 large, consisting of 1,000-1,350 amino acids. Advanced editing technologies such as base editing and prime editing require the fusion of additional proteins to Cas9 and Cas12a, making them even larger. This size poses a challenge for efficiently delivering these proteins 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 Journalwe introduced TnpB, a smaller but highly effective next-generation tool for plant genome editing.

TnpBs are small ancestors of Cas12 nuclease

TnpB proteins are transposon-associated nucleases that are controlled 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 number of amino acids 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 developed a hyper-compact 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, derived from D. radiodurans, is only 408 amino acids long.

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

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

Repurposing TnpB for plant genome editing

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 sufficient guide RNA for highly efficient 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 well in dicots. Therefore, we generated dicot-specific TnpB vectors and demonstrated successful editing in Arabidopsis. Interestingly, we observed that deletions occurred primarily at the target loci in both rice and Arabidopsis. This makes TnpB suitable for effectively disrupting gene functions. TnpB could now be used to introduce genetic mutations to disrupt unwanted genes for removing antinutrient factors, improving nutritional content, biotic and abiotic stress resistance, and more.

A dead TnpB for gene activation and the exchange of single DNA letters

While TnpB functions as a programmable scissors in its native form, it can also be modified to recruit factors that activate genes. By inactivating its cutting ability, we developed inactivated TnpB (dTnpB). dTnpB retains its ability to bind to target DNA specified by guide RNA. We then fused dTnpB to 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.

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

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

This story is part of Science X Dialog, where researchers can report findings from their published research articles. Visit this page for information about Science X Dialog and how to participate.

Dr. Kutubuddin Molla is a scientist specializing in agricultural biotechnology at ICAR-National Rice Research Institute (NRRI), Cuttack, India. He received his Ph.D. from the University of Calcutta, Kolkata. Dr. Molla did postdoctoral research at the Pennsylvania State University on a Fulbright scholarship.

Dr. Molla’s research interests focus on precision genome editing, using CRISPR-Cas and other cutting-edge techniques for crop improvement. His lab at NRRI is dedicated to developing novel genome editing tools and applying them to improve crop performance.