Heritable, Tissue Culture‐Independent and Transgene‐Free Genome Editing in Plants via Viral Delivery of CRISPR / AsCas12f

Gene-edited plants are typically generated through Agrobacterium- or biolistic-mediated delivery of transgenes encoding gene editing reagents into plant cells, followed by regeneration of whole plants via tissue culture. However, tissue culture and regeneration are time-consuming, labour-intensive, require complex protocols, and are effective only in a limited number of plant species. Moreover, the transgenes encoding gene editing reagents, such as CRISPR/Cas, need to be removed through selfing or crossing to alleviate regulatory concerns over transgenic plants. Therefore, tissue culture/regeneration and transgene removal remain two major bottlenecks in fully realising the potential of genome editing for plant functional genomics and crop improvement. Here, we developed tissue culture-independent and transgene-free genome editing methods in Nicotiana benthamiana and tomato, utilising viral delivery of CRISPR/AsCas12f, a compact genome-editing tool (422 amino acids) derived from Acidibacillus sulfuroxidans. AsCas12f cleaves DNA targets bearing a TTR protospacer adjacent motif (PAM), where R represents A or G, thereby broadening the range of editable target sites compared to other compact genome-editing tools, such as the TnpB enzyme ISYmu1, which recognises the more restrictive TTGAT PAM (Ishibashi et al. 2024; Weiss et al. 2025). Our method offers a scalable solution for plant genome editing, addresses regulatory concerns associated with transgenic plants, and holds great promise for advancing both basic and applied plant research. For our experiments, we cloned the AsCas12f variant (I123Y/D195K/D208R/V232A) with nuclear localization signal sequences (SV40 NLS) and sgRNA into tobacco rattle virus (TRV) vectors (Figure 1A). Hepatitis delta virus (HDV) ribozyme sequences and mobility motif sequences from tRNA isoleucine (tRNAIleu) were cloned downstream of the sgRNA. A single sgRNA targeting both PHYTOENE DESATURASE (NbPDS) homologues in N. benthamiana was inserted into the TRV2 vector, generating the TRV::AsCas12f-NbPDS sgRNA construct. Simultaneous disruption of both NbPDS-1 and NbPDS-2 genes impairs carotenoid biosynthesis, resulting in a characteristic photobleaching phenotype. To evaluate this system, TRV::AsCas12f-NbPDS sgRNA was introduced into Agrobacterium tumefaciens and delivered into N. benthamiana plants via leaf infiltration (Figure 1B). Photobleached spots appeared on systemic leaves approximately 2 weeks post-infection. As plants matured, photobleaching intensified and spread to stems and floral sepals (Figure 1C). Genomic DNA was extracted from systemic leaves, the target region was PCR-amplified, and the amplicons were analysed via next-generation sequencing (NGS). The total indel frequency was 83.08%, with multinucleotide deletions dominating the mutation profile (Figure 1D, Figure S1, and Table S1). We next evaluated whether the targeted mutagenesis observed in synthetically infected leaves is heritable. Seeds from the 1st to 16th seed pods (counting from the base of the inflorescence) were collected and germinated. Fully bleached seedlings, indicative of complete inactivation of both NbPDS homologues, were recovered from upper seed pods, particularly from the 10th to 16th pods at a frequency of 5.67% (Figure 1E,F). Albino and green seedlings from three seed pods were genotyped. All eight albino seedlings exhibited biallelic or homozygous mutations at NbPDS-1 (Figure S2 and Table S2). Among the green seedlings from the three seed pods, the total gene editing frequency at NbPDS-1 (including monoallelic, biallelic, and homozygous mutations) was 60%, 66.67%, and 100%, respectively (Figure 1G and Table S2). To confirm the absence of RNA virus vector integration, we performed PCR analysis on NbPDS-1-edited seedlings using primers specific to TRV1 and TRV2, and found no evidence of viral vector integration (Figure S3). These results demonstrate efficient, heritable, tissue culture-free and transgene-free genome editing in N. benthamiana. We next applied this strategy in tomato by targeting ANTHOCYANIN 2 (SlAN2). Loss of SlAN2 suppresses anthocyanin biosynthesis, resulting in fully green petioles and stems (Liu et al. 2024). sgRNA targeting SlAN2 was designed and cloned into the TRV2 vector. The TRV vectors were then injected into the latent axillary meristematic tissues in tomato, following our established protocol (Liu et al. 2024) (Figure 1H). The infected tomato plants were grown at 26°C/22°C (light/dark), a temperature that supports AsCas12f activity in N. benthamiana. However, no edited shoots were recovered (Figure 1I and Table S3), likely due to antiviral defences suppressing viral replication and accumulation in tomato at this temperature (Liu et al. 2024). Host RNA-DEPENDENT RNA POLYMERASES (RdRPs) are central to plant antiviral defence, and lower temperatures have been shown to enhance TRV accumulation and genome editing efficiency, as demonstrated in our previous work using TRV to deliver sgRNAs or in combination with isopentenyl transferase into Cas9-expressing plants (Liu et al. 2024). Building on these findings, we tested two strategies to boost editing efficiency: (1) silencing RdRP1, RdRP6a, and RdRP6b using cucumber mosaic virus (CMV)-induced gene silencing, achieved by co-injecting TRV::AsCas12f-sgRNA with CMV::VIGS RdRP1/6a/6b into latent axillary meristems; and (2) growing plants under reduced temperature conditions (22°C light / 18°C dark). Both strategies successfully yielded SlAN2-edited shoots (Figure 1I and Table S3). Biallelic mutants were recovered at 9.09% and 15.79%, and monoallelic mutants at 9.09% and 15.79%, under CMV co-injection and low-temperature conditions, respectively. Biallelic knock-out mutants exhibited fully green stems and petioles, while wild-type plants retained their purple pigmentation (Figure 1J and Figure S4). PCR analysis on SlAN2-edited mutants using primers specific to TRV1 and TRV2 found no evidence of viral vector integration (Figure S5). We then evaluated whether the targeted mutagenesis observed in the shoots was heritable. Seeds from the shoot carrying −12 bp (51.6%)/−13 bp (48.4%) mutations at SlAN2 were germinated. All seedlings exhibited fully green stems and petioles, in contrast to the purple pigmentation observed in wild-type plants. Among the six seedlings genotyped, three were homozygous knockouts carrying −12 bp/−12 bp or −13 bp/−13 bp mutations, and three were biallelic knockouts carrying −12 bp/−13 bp mutations (Figure 1K). Taken together, these results demonstrate that TRV-mediated delivery of CRISPR/AsCas12f, combined with host antiviral suppression through CMV::VIGS RdRP1/6a/6b co-infection or low-temperature treatment, enables efficient, heritable, tissue culture-free and transgene-free genome editing in tomato. In conclusion, we have developed two methods for achieving heritable, tissue culture-free and transgene-free genome editing through viral delivery of CRISPR/AsCas12f: one in N. benthamiana via leaf infiltration, and the other in tomato via axillary meristematic cell injection, with success achieved through either RdRPs knockdown or low-temperature growth conditions. Our AsCas12f/sgRNA viral delivery methods hold significant potential to accelerate both fundamental plant research and targeted crop improvement. D.L. conceived and designed experiments, and wrote the manuscript. M.H. performed experiments, analysed the data, and wrote the manuscript. L.Z. assembled the CMV vector cloning backbone. L.H.-E. edited manuscript and provided advice and equipment. This study was supported by the Governor University Research Initiative program from the State of Texas. D.L., M.H. and L.H.-E. are in the process of filing a patent application covering this work. The data that supports the findings of this study are available in the Supporting Information of this article. Data S1: pbi70315-sup-0001-Supinfo.pdf. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.