CRISPR/Cas9-mediated targeted recombination in somatic cells of Solanum lycopersicum
Project Description:This project investigates the feasibility and challenges associated with CRISPR/Cas9-induced targeted recombination in somatic cells of Solanum lycopersicum (tomato), specifically targeting the Tomato Mosaic Virus (ToMV) resistance locus, a known recombination cold spot. Despite the success of CRISPR/Cas9 technology in inducing DNA double-strand breaks (DSBs), achieving targeted recombination in this context remains difficult.The research focuses on two primary experimental strategies: (1) inducing DSBs in both alleles to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR), and (2) targeting a single allele in a heterozygous background to induce HDR. A robust CRISPR/Cas9 system was employed in the F1 progeny of tomato plants, with PacBio sequencing and a two-dimensional sample pooling system used to enhance the detection of true recombination events.However, despite the active CRISPR/Cas9 system, the study found no significant increase in recombination frequency compared to wild-type plants. These results suggest that CRISPR/Cas9-induced DSBs alone are insufficient to break the genetic linkage at the ToMV locus within the studied recombination cold spots. The findings provide valuable insights into the limitations of current genome editing techniques and underscore the need for further development of strategies to overcome genetic linkage in plant breeding.Relevance:The outcomes of this project are highly relevant to the field of plant breeding and genetic engineering. The ability to induce targeted recombination in somatic cells could revolutionize crop improvement by allowing breeders to break undesirable genetic linkages and introduce beneficial traits with greater precision. The specific focus on the ToMV resistance locus in tomato addresses a longstanding challenge in conventional breeding, where linkage drag has hindered the separation of desirable traits from unwanted genetic material. The insights gained from this study can be important for advancing tomato breeding but also have potential applications across a broad range of crops, contributing to the development of more efficient and precise genome editing tools in agriculture.Data Type:Raw sequencing data: PacBio Sequel II SMRT Cell sequencingProcessed data: SNP patterns indicative of recombination events, mutation analysis at CRISPR/Cas9 target sites.Organism:Species: Solanum lycopersicumVarieties: 'Moneymaker' and 'MoneyBerg'Keywords:CRISPR/Cas9, targeted recombination, genome editing, Tomato Mosaic Virus resistance, somatic cells, Solanum lycopersicum, plant breeding