A next-generation genome editing tool for plants has been unveiled

Genome editing represents one of the most transformative scientific discoveries of our time. It allows us to dive into the very 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. That’s the power of genome editing.

We use genome editing tools to edit the genetic sequences of microbes, animals and plants. Our goal? Develop desirable properties and eliminate undesirable ones. The impact of this technology has been felt across biotechnology, human therapy and agriculture, bringing rapid progress and solutions.

The most 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 such as core editing and primary editing require additional proteins to join Cas9 and Cas12a, making them even more bulky. This bulkiness presents a challenge to efficiently deliver these proteins to the 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 Plant Biotechnology Journalwe introduced TnpB, a smaller but highly efficient next-generation genome editing tool in plants.

TnpBs are the small precursors of the Cas12 nuclease

TnpB proteins are RNA-directed transposon-associated nucleases. They are considered the evolutionary ancestors of Cas12 nucleases. Although functionally similar to Cas12a, TnpB is much more compact, with a total amino acid number in the range of 350–500. To give an idea, TnpB is a third the size of Cas9 and Cas12a. If Cas9 and Cas12a are like soccer balls, TnpB are like baseballs.

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

The short RNA serves as a guide for TnpB, directing it to its target DNA sequence. TnpB specified by this RNA binds to the target and cleaves both DNA strands. When the broken ends are resealed by the cell, DNA letters can be inadvertently inserted or deleted. These insertions or deletions lead to modification of genetic sequences.

There is another 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. The specific TAM for TnpB from D. radiodurans is TTGAT, which must be present before the target sequence. In this sense, TnpB can access genomic loci that Cas9 cannot reach.

Reengineering TnpB for plant genome editing

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

Rice is a monocot, and systems that work well for monocots may not work as well for dicots. Therefore, we generated dicot-specific TnpB vectors and demonstrated successful editing in Arabidopsis. Interestingly, we observed that deletions mostly occurred at target loci in both rice and Arabidopsis. This makes TnpB suitable for efficient disruption of gene functions. TnpB could now be used to introduce genetic mutations to disrupt unwanted genes to remove anti-nutritional factors, increase nutrient content, resistance to biotic and abiotic stress, and more.

Dead TnpB to activate a gene and replace a single DNA letter

While TnpB functions as a programmable scissors in its native form, 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 the guide RNA. We then fused dTnpB with other cargo proteins to direct them to target genes, making those genes more active. This activation tool can enhance gene function and pave the way for creating better crops in the future.

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

We are using 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 plant biologists, biotechnologists and breeders to adopt TnpB for use in a variety of crops.

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

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

Research interests of Dr. Molly focuses on precision genome editing, using CRISPR-Cas and other advanced techniques for crop improvement. His lab at NRRI is dedicated to developing new genome editing tools and applying them to enhance crop performance.