Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 161 Public understanding and acceptance of genome-edited crops are equally important. Due to concerns over safety and ethical issues, the public is skeptical of genetically modified crops. It is of great significance for regulatory agencies to explain to the public the safety and benefits of genome editing technology and gain public trust (Arora and Narula, 2017; Kumar et al., 2024). Involving farmers, consumers and policymakers in discussions, the public can understand the potential of genome editing, which can increase sugarcane yields and thus make these genome-edited crops more socially acceptable (Arora and Narula, 2017; Kumar et al., 2024). Figure 2 Identification of gene targeting in sugarcane by restriction endonuclease assays and herbicide application (Adopted from Oz et al., 2021) Image caption: (A) Repair template compared to sugarcane acetolactate synthase (ALS) gene. The 1 833 bp template carries four nucleotide substitutions, one at the codon positions W574L (introducing the MmeI recognition site) and S653I (eliminating BfaI recognition site), as well as two modified PAM sites (PAM1 and PAM2) (preventing template cleavage by Cas9 complexed with sgRNA1 or sgRNA2). Amino acid residue numbering in the sugarcane ALS gene follows the Arabidopsis nomenclature. (B) Schematic representation of primer annealing positions. Primers DO1 and UP6 were used to amplify a 1,913 bp fragment. Using primer UP6 prevented amplification of randomly inserted template by annealing outside the repair template. Primers F1 and R1 were used to generate a 455 bp nested PCR amplicon for analyses by restriction enzyme digestion. (C) Restriction-digestion pattern of nested PCR amplicons from wild-type (WT) and edited lines after digestion with MmeI or BfaI. + indicates positive control that includes the MmeI site and lacks the BfaI site. (D) Vegetatively propagated edited line L1 with multi allele conversion of W574L and S653I was tolerant to application of the herbicide nicosulfuron (Accent® DuPont) at 95 g ha-1, in contrast to non-edited WT plants (Adopted from Oz et al., 2021) 6.3 Emerging tools and multi-omics integration Genome editing technology is developing rapidly. New tools such as base editing and start editing are integrated with CRISPR technology. These new technologies can directly modify genes without cutting the DNA strand, thereby enhancing the safety and accuracy of crop gene editing. Because the genome of sugarcane is very complex, the advancements of these technologies are of great significance to sugarcane. Using these new tools, researchers can make smaller and more precise alterations to the DNA of plants (Arora and Narula, 2017; Haque et al., 2018).
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