The team focused on a gene called lemon white1, or lw1, a key regulator of chlorophyll and carotenoid biosynthesis. The choice was strategic. When lw1 function is knocked out across enough gene copies, the resulting plants display a pale, yellowish foliage that can be scored visually, giving the researchers an immediate, unambiguous readout of whether their editing constructs were actually working in a genome where sequencing every allele is a formidable chore. Visible markers of this kind have become a favorite calibration tool in polyploid crop editing, and the sugarcane community had previously used a magnesium chelatase gene for the same purpose with Cas9. Extending the approach to Cas12a, however, required solving several technical problems at once.

Cas12a, originally characterized as Cpf1, differs from the more familiar Cas9 nuclease in ways that matter for crops like sugarcane. It recognizes a T-rich DNA motif called a PAM, which makes it particularly well suited for targeting AT-rich genomic regions, including many cis-regulatory elements that control when and where genes are switched on. It also produces staggered DNA cuts and processes its own guide RNAs, simplifying multiplexed editing. Until now, despite these advantages, Cas12a had never been demonstrated in sugarcane. The Florida group compared two engineered versions of the Lachnospiraceae bacterium-derived enzyme: an intron-containing LbCas12a-R variant, in which added intron sequences boost expression in plant cells, and LbCas12a-RRV, a more recently developed high-activity variant.

The results were striking. Editing efficiencies across the experimental lines ranged from 31 to 85 percent, and the LbCas12a-RRV variant consistently outperformed the intron-containing LbCas12a-R in both overall editing frequency and the proportion of alleles edited within individual plants. In a genome where every gene may be present in a dozen or more copies, allelic editing efficiency, the fraction of homologous copies that carry a mutation, is the metric that ultimately determines whether an edited phenotype appears at all. The demonstration that a modern Cas12a variant can push multiallelic disruption to visually scorable levels in sugarcane opens a substantially larger fraction of the genome to precision editing, particularly the AT-rich promoter and enhancer regions that Cas9 systems reach less comfortably.

The second half of the study addresses a problem that goes beyond efficiency: the size of the edits themselves. Standard CRISPR cutting typically produces small insertions and deletions as the cell repairs the break, and in a polyploid genome these tiny changes can be difficult to detect and may not fully abolish gene function. The researchers therefore co-expressed mTREX2, the mouse version of Three prime repair exonuclease 2, alongside the Cas12a machinery. TREX2 is a processive exonuclease that chews back DNA ends after the nuclease cut, enlarging the gap that the repair machinery must fill. When the cell reseals the break, the result is a deletion that is often far larger than what standard repair would produce. The strategy has precedent in other systems, where TREX enzymes fused to or co-expressed with Cas nucleases have been shown to enlarge deletion sizes in both plants and human cells, but its application to Cas12a editing in a highly polyploid crop is new.

The effect on the sugarcane mutation spectrum was dramatic. Co-expression of mTREX2 shifted the profile of recovered mutations toward moderate and large deletions, with the class of deletions measuring at least 51 base pairs increasing approximately seven-fold in both Cas12a variants. Larger deletions matter for two reasons. First, they are far more likely to completely disrupt gene function rather than leaving a partially active protein. Second, and arguably more important in polyploids, they are much easier to detect. A deletion of 51 or more base pairs creates a size difference that standard fragment analysis can resolve, even when dozens of unedited homologous copies are present in the same sample. In effect, mTREX2 co-expression converts the hardest part of polyploid genotyping, distinguishing edited from unedited alleles, into a comparatively simple measurement.

That measurement itself was a focus of the study. Rather than relying on next-generation sequencing, which is expensive and computationally demanding when applied to the tangled allele sets of sugarcane, the team used capillary electrophoresis-based genotyping, a fragment analysis approach that separates DNA molecules by size. The method proved to be a cost-effective, high-throughput alternative that accurately predicted both allelic editing efficiency and indel size. For breeding programs and laboratories in sugarcane-producing countries that may not have routine access to deep sequencing infrastructure, this is a practical advance as significant as the editing system itself. It lowers the barrier to screening the hundreds of independent transgenic events that polyploid editing typically requires before a useful line emerges.

The broader significance lies in what Cas12a unlocks for sugarcane improvement. Much of the regulatory information that governs yield, stress tolerance, and sugar accumulation resides in AT-rich cis-regulatory DNA, and fine-tuning those elements, rather than knocking out coding sequences outright, is increasingly recognized as the most sophisticated use of genome editing in crops. Previous work from the same laboratory and others has shown that multiallelic editing of genes such as LIGULELESS1 can tune leaf architecture and biomass yield in sugarcane, and that promoter editing can adjust gene expression levels in rice and maize. A reliable Cas12a platform, especially one enhanced with mTREX2 to generate large deletions, gives breeders a tool for reshaping regulatory landscapes that were previously out of reach, whether the goal is reduced lignin for easier ethanol extraction, altered flowering, or enhanced disease resistance.

The work, funded by the US Department of Energy’s Center for Advanced Bioenergy and Bioproducts Innovation and by the USDA National Institute of Food and Agriculture, arrives at a moment when genome editing is accelerating across global crop improvement. Sugarcane occupies a unique position at the intersection of food security and renewable energy, and its genomic complexity has kept it a generation behind rice, wheat, and maize in editing-based improvement. With this study, the crop gains not only a new nuclease platform but also a validated workflow, from construct design through visual phenotyping to affordable genotyping, that other teams working on polyploid crops such as switchgrass, Miscanthus, and potato can adapt. The seven-fold boost in large deletions from a single co-expressed exonuclease suggests that the remaining technical gaps in polyploid editing may be closed not by entirely new technologies, but by clever combinations of components that already exist.

For now, the lemon white1 seedlings glowing pale yellow in the Florida greenhouse stand as proof of concept that Cas12a can function in one of nature’s most demanding genomes. The next steps will be to apply the LbCas12a-RRV and mTREX2 system to agronomically meaningful targets, to test whether large deletions can be directed toward promoter regions for fine-tuned expression changes, and to determine how edited lines perform under field conditions. If those efforts succeed, the study may be remembered as the moment sugarcane, long the most genomically intimidating of the major crops, finally became a tractable target for the full toolkit of modern plant synthetic biology.

Subject of Research: CRISPR/Cas12a genome editing with mTREX2 co-expression in the highly polyploid sugarcane genome

Article Title: mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome

Article References: mTREX2 co-expression promotes large deletions in CRISPR/Cas12a editing of the highly polyploid sugarcane genome. (n.d.). https://doi.org/10.1007/s00299-026-03935-1

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03935-1

Keywords: sugarcane, CRISPR/Cas12a, mTREX2, LbCas12a-RRV, polyploid genome, genome editing, lw1 gene, large deletions, capillary electrophoresis, plant biotechnology, biofuel feedstock, cis-regulatory elements