Targeted recombination between homologous chromosomes for precise breeding in tomato

Journal Article / 2017

Filler Hayut, Shdema; Melamed Bessudo, Cathy; Levy, Avraham A.

Homologous recombination (HR) between parental chromosomes occurs stochastically. Here, we report on targeted recombination between homologous chromosomes upon somatic induction of DNA double-strand breaks (DSBs) via CRISPR-Cas9. We demonstrate this via a visual and molecular assay whereby DSB induction between two alleles carrying different mutations in the PHYTOENE SYNTHASE (PSY1) gene results in yellow fruits with wild type red sectors forming via HR-mediated DSB repair. We also show that in heterozygote plants containing one psy1 allele immune and one sensitive to CRISPR, repair of the broken allele using the unbroken allele sequence template is a common outcome. In another assay, we show evidence of a somatically induced DSB in a cross between a psy1 edible tomato mutant and wild type Solanum pimpinellifolium, targeting only the S. pimpinellifolium allele. This enables characterization of germinally transmitted targeted somatic HR events, demonstrating that somatically induced DSBs can be exploited for precise breeding of crops.