TnpB disrupted the gene responsible for green colour, which generated albino rice plants. Credit: Kutubuddin Molla and Subhasis Karmakar
Scientists are using genetic scissors one-third the size of CRISPR Cas9/Cas12a to develop a more resilient rice strain. Sourced from a hardy archaebacteria, the repurposed protein1 can easily slip into plant cells, has a wider targeting scope, while maintaining high editing efficiency, they say.
A team led by the Indian Council of Agricultural Research-National Rice Research Institute, in Cuttack, Odisha demonstrated that transposase enzyme TnpB, a programmable RNA-guided DNA endonuclease from radiation-resistant Deinococcus radiodurans, cleaves target DNA sequences with 34% efficiency in both monocot and dicot plants.
“Using the TnpB system, we are trying to reduce the height of an aromatic rice variety so that it doesn’t fall over (lodge) in extreme weather conditions,” says study co-author, Kutubuddin Molla. It can also be used to develop tools to activate genes and swap DNA letters (base editing) in plant genomes.
Considered the evolutionary ancestors of Cas12 nucleases, TnpB is functionally similar to Cas12a, but smaller — 350 to 500 amino acids compared with Cas12a’s 1300 and Cas9’s 1000–1400.
For gene editing, Cas proteins need a short DNA stretch — the protospacer adjacent motif (PAM) — immediately after the target sequence to bind, besides an RNA molecule to guide them to the targeted stretch. But TnpB recognises a broad range of Transposon Associated Motif (TAM) sequences for gene editing. The TnpB system from the archaebacteria prefers specific TAM (TTGAT motif) upstream of the target sequence, which means it can access genomic loci that Cas9 can't reach and expand the target area, says Molla.
Testing the hypercompact gene scissors
The team first improved the TnpB protein sequence to work better in plants and fine-tuned parts of the genetic instruction to ensure there was enough guide RNA for high-efficiency gene editing.
They then engineered four different TnpB vectors targeting six loci in the rice genome. Key target genes included OsHMBPP and OsSLA4, which are important for chloroplast development and function – the site of photosynthesis – and overall plant health. The TnpB constructs were introduced into rice plants using Agrobacterium-mediated transformation, a common method for plant genetic engineering.
After culturing and plant growth, they noted plants with white or pale leaves (albino phenotypes) because they lacked chlorophyll, indicating successful disruption of the targeted genes.
Sanger sequencing confirmed that the system worked by revealing insertions or deletions (indels) at the target sites. By measuring the frequency of these indels at the target loci, the researchers could conclude 33.58% efficiency on average.
The system was tested in dicots by using dicot-specific TnpB vectors in Arabidopsis. “Mostly deletions occurred at the target loci in both rice and Arabidopsis. This makes TnpB suitable for effectively disrupting gene functions,” says Molla. The group is currently working to enhance the efficiency by truncating the guide RNA to remove unnecessary portions.
Kailash Bansal, former director at the National Bureau of Plant Genetic Resources in Delhi, says as the next steps, the focus could be on exploring more TnpBs from diverse microbial species and making them versatile by tweaking the TAM specificity to expand the targeting scope.
“The potential applications of TnpB in crop improvement can include trait enhancement for climate resilience, disease and pest resistance, yield improvement, and nutritional enrichment,” Bansal adds.
