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

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Abstract 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 F₁ 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. Article Summary This research targets plant biologists and geneticists interested in enhancing plant breeding techniques. The study used CRISPR/Cas9 technology to induce DNA breaks in tomato plants. It specifically targeted the Tomato Mosaic Virus (ToMV) resistance gene, which resists natural recombination. The aim was to induce genetic recombination via CRISPR/Cas9. The highly active CRISPR/Cas9 system did not increase the expected genetic changes, indicating challenges in achieving targeted recombination. These findings highlight the challenges in breaking genetic linkages in specific genome regions using current CRISPR methods. These findings are relevant for developing techniques for targeted recombination in plant breeding.

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