Power of Small for Plant Genome Editing

Delivering genome editing machinery into plant cells remains a challenge despite the dire need for high yield crops with enhanced nutritional value that are resistant to disease and resilient in the face of climate change. “Our biggest problem is climate change. And the biggest solution is CRISPR,” said Rodolphe Barrangou, PhD, professor at the department of food, bioprocessing, and nutrition sciences at North Carolina State University (NCSU) in an interview with GEN.

Driven by the challenge to develop an efficient and versatile genome editor that works optimally in plant cells, a team of researchers at the ICAR-National Rice Research Institute in Cuttack, India, led by Kutubuddin Molla, PhD, and Mirza Baig, PhD, sought to exploit a transposase enzyme, ISDra2TnpB, in a bacterium (Deinococcus radiodurans) that can survive in extreme environments (an extremophile).

Molla and Baig’s group recently published their findings in an article in the Plant Biotechnology Journal, “A miniature alternative to Cas9 and Cas12: Transposon-associated TnpB mediates targeted genome editing in plants,” in collaboration with Yinong Yang, PhD, and Justin Shih, PhD, from the department of plant pathology and environmental microbiology and the Huck Institutes of the Life Sciences at the Pennsylvania State University.

Editing efficiency

The study claims the new TnpB-based system achieves an editing efficiency of 33.58% in an average plant genome and can target regions of the genome that are inaccessible to Cas9 or Cas12. The study also shows the new genome editor works in both types of flowering plant species—those with one or two “seed leaves” or cotyledons (monocots and dicots), establishing ISDra2TnpB as a versatile and promising tool for plant genome engineering.

 Earlier studies demonstrated the efficacy of TnpB-based genome editors in bacterial and mammalian cells but did not explore their application and effectiveness in plants. “It was not known whether TnpB can also be used to develop tools to activate gene function (activator) and swap DNA letters (base editor) [in the plant genome],” added Molla.

TnpB transposases are only 400–500 amino acids (aa) in length—much smaller than Cas9 (1000–1500 aa) or Cas12a (1100–1300 aa) that are believed to have evolved independently from a TnpB-like nuclease. Compactness makes TnpB-based transposase systems an asset in instances where protein or nucleic acid size is a limiting factor.

“[This compactness] provides convenience in delivery either via Agrobacterium-mediated T-DNA transformation or via viral vector-based delivery,” said Yiping Qi, PhD, professor at the department of plant science and landscape architecture at the University of Maryland. “In addition, it may be advantageous to use these small proteins to develop transcriptional regulation and epigenome editing tools via protein fusion.” (Yiping Qi was not involved in this study).

While Cas9 and Cas12 require the presence of a 2–6 base pair PAM (protospacer adjacent motif) to cut through double stranded DNA, cleavage by TnpB depends on the presence of a TAM (transposon-associated motif) upstream of the target sequence. Genome-wide analysis highlights TnpB’s enhanced targetability compared to the Cas nucleases. The TAM motif also lends the TnpB system a high degree of specificity.

“TnpB is highly specific to its motif (TTGAT). We have examined altering a single letter in this motif, and found none to negligible cleavage,” said Molla.

In this study, the researchers demonstrated the TnpB-based system’s efficacy in both gene activation and base editing in the plant genome. The catalytically dead TnpB utilized in the system introduces specific single nucleotide changes without double-strand breaks.

Qi added, “Improvements can be made to further enhance the editing efficiency of these TnpB nucleases as well as relax their TAM requirements to broaden the targeting range in the plant genomes,” added Qi.

ISDra2TnpB could potentially find a variety of applications in improving crops, precision plant breeding, and in developing environmentally friendly biological pesticides. To improve crops, the TnpB system can be used to introduce or enhance traits such as nutritional content, yield, and drought and pest resistance.

The TnpB system can also be used to modify microbes to enhance their ability to target and kill specific pests more effectively. On the other hand, the system can also be exploited to improve natural plant defense mechanisms or diminish the plant’s susceptibility to pests, reducing the need for external measures of pest control.

In upcoming projects, Molla and Baig’s team will explore and refine TnpB variants from various organisms to expand their applicability in different plant species.