Gene-edited plants are typically generated throughAgrobacterium- or biolistic-mediated delivery of transgenesencoding gene editing reagents into plant cells, followed by re-generation of whole plants via tissue culture. However, tissueculture and regeneration are time-consuming, labour-intensive,require complex protocols, and are effective only in a limitednumber of plant species. Moreover, the transgenes encodinggene editing reagents, such as CRISPR/Cas, need to be removedthrough selfing or crossing to alleviate regulatory concerns overtransgenic plants. Therefore, tissue culture/regeneration andtransgene removal remain two major bottlenecks in fully realis-ing the potential of genome editing for plant functional genom-ics and crop improvement.

Here, we developed tissue culture-independent and transgene-free genome editing methods in Nicotiana benthamiana andtomato, utilising viral delivery of CRISPR/AsCas12f, a com-pact genome- editing tool (422 amino acids) derived fromAcidibacillus sulfuroxidans. AsCas12f cleaves DNA targetsbearing a TTR protospacer adjacent motif (PAM), where R rep-resents A or G, thereby broadening the range of editable targetsites compared to other compact genome- editing tools, such asthe TnpB enzyme ISYmu1, which recognises the more restric-tive TTGAT PAM (Ishibashi et al. 2024; Weiss et al. 2025). Ourmethod offers a scalable solution for plant genome editing, ad-dresses regulatory concerns associated with transgenic plants,and holds great promise for advancing both basic and appliedplant research.

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