The LbCas12ai-mTREX2 system enhances large deletions, and the LbCas12ai-RRV enables efficient editing of lw1 in sugarcane. Sugarcane (Saccharum spp. hybrids) is a major feedstock for biofuel production and the primary source of global table sugar; however, its highly polyploid and complex genome constrains the effective deployment of functional genomics and precision breeding strategies. While CRISPR/Cas9-mediated genome editing has been reported in sugarcane, Cas12a-based editing remains unexplored despite its advantages in targeting AT-rich genomic regions. In this study, we report efficient CRISPR/Cas12a-mediated targeted mutagenesis of the lw1 gene in sugarcane. The lemon white1 (lw1) is a key regulator of chlorophyll and carotenoid biosynthesis, and targeted lw1 mutagenesis results in a visible phenotype. We compared the editing performance of intron-containing LbCas12a-R (LbCas12ai-R) and LbCas12a-RRV (LbCas12ai-RRV) variants and assessed the impact of Mus musculus Three prime repair exonuclease 2 (mTREX2) co-expression on editing efficiency and deletion profiles. Editing efficiencies ranged from 31 to 85%, where LbCas12ai-RRV consistently outperformed LbCas12ai-R in both overall editing efficiency and allelic editing efficiency. Co-expression of the mTREX2 shifted the mutation spectrum toward larger deletions, increasing the frequency of moderate-to-large deletions. The ≥ 51 bp deletion class increased ~7-fold in both variants. Capillary electrophoresis-based genotyping provided a cost-effective, high-throughput alternative to next-generation sequencing (NGS) and accurately predicted allelic editing efficiency and indel size. This first report of CRISPR/Cas12a-mediated genome editing in sugarcane provides a framework for future engineering of AT-rich cis-regulatory elements in the polyploid sugarcane genome.
