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

Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks 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 double-strand breaks in both alleles of F1 tomato seedlings to promote nonhomologous end joining and homology-directed repair; second, targeting a single allele in a heterozygous background to induce homology-directed repair in seedlings. CRISPR/Cas9 activity was confirmed in F1 seedlings by detecting nonhomologous end joining-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing 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 F1 progeny, no reliable targeted recombinations were found. 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 F1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs were insufficient to break the genetic linkage at the ToMV locus on chromosome 9 at a detectable level.