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

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 F₁ 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 F₁ 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 F₁ 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.