CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus of Solanum lycopersicum

Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks (DSBs) for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the Tomato Mosaic Virus (ToMV) resistance locus in Solanum lycopersicum . We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing DSBs in both alleles of F 1 tomato seedlings to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR); second, targeting a single allele in a heterozygous background to induce HDR in seedlings. CRISPR/Cas9 activity was confirmed in F 1 seedlings by detecting NHEJ-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing single nucleotide polymorphisms (SNPs) between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F 1 progeny, no increase in recombination frequency was observed compared to wild-type plants. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F 1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs are insufficient to break the genetic linkage at the ToMV locus on chromosome 9 in recombination cold spots within somatic cells.