Next-generation genome editing tool for plants discovered

Genome editing stands as one of the most transformative scientific discoveries of our time. It allows us to dive into the code of life itself 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 fix the genetic sequences of microbes, animals and plants. Our goal? To develop desired traits and eliminate unwanted ones. The impact of this technology has been felt across biotechnology, human therapy and agriculture, bringing rapid advances 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 large, consisting of 1000-1350 amino acids. Advanced editing technologies such as base editing and core editing require fusing additional proteins to Cas9 and Cas12a, making them even larger. This measure poses a challenge for delivering these proteins efficiently 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 last article on Journal of Plant Biotechnologywe introduced TnpB, a smaller but highly effective next-generation tool for genome editing in plants.

TnpBs are minor progenitors of the Cas12 nuclease

TnpB proteins are RNA-guided transposon-associated nucleases. 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 that 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 in extreme environments and its exceptional 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 its target DNA sequence. Specified by this RNA, TnpB binds to the target and cleaves both DNA strands. When broken ends are resealed by the cell, inadvertent 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 TnpB from D. radiodurans, the specific TAM is TTGAT, which must be present upstream of the target sequence. In this sense, TnpB can access genomic sites that Cas9 cannot reach.

Repurposing TnpB for plant genome modification

We first 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 sufficient guide RNAs 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 work so well in dicots. Therefore, we generated dicot-specific TnpB vectors and demonstrated successful editing in Arabidopsis. Interestingly, we observed that the deletions occurred predominantly at the target sites in both rice and Arabidopsis. This makes TnpB well suited to effectively disrupt gene functions. TnpB can now be used to introduce genetic mutations to disrupt unwanted genes for removal of anti-nutrient factors, increased nutrient content, resistance to biotic and abiotic stress, and more.

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

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

Similarly, we coded another cargo protein with dTnpB to develop a tool capable of exchanging one DNA letter for another. This precise tool will enable crop innovation by altering the genetic code with 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 very 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 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 the ICAR National Rice Research Institute (NRRI) in Cuttack, India. He earned his Ph.D. from Calcutta University, Calcutta. Dr. Molla conducted postdoctoral research at Pennsylvania State University on a Fulbright scholarship.

The research interests of Dr. Apples focus on precise genome editing, using CRISPR-Cas and other advanced techniques for crop improvement. His laboratory at NRRI is dedicated to the development of new genome editing tools and their application to improve crop performance.