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Abstract
Homologous recombination (HR) typically occurs during meiosis, between homologs, at a few unplanned locations along the chromosomes. There is less knowledge about HR between homologs in somatic cells. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via CRISPR-Cas9-induced DNA double-strand breaks (DSBs) repair in Solanum lycopersicum and Arabidopsis thaliana. Our experimental system includes DSB induction in “hot” and “cold” genomic regions, in somatic cells, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and Homologous Recombination repair. We present a series of evidence showing that targeted DSBs can be repaired via somatic HR using an homologous chromosome as the template leading to targeted crossover or to gene conversion. In both tomato and Arabidopsis we found similar rates of interhomolog, somatic HR in the 6-17% range. In addition, we demonstrated that some of these repair events can be transmitted to the next generations. HR events were classified into gene conversion events (nonreciprocal exchange of DNA patches - conversion tracks) and crossovers. Moreover, induction of somatic DSB at both “hot” and “cold” epigenetic contexts increased the crossover rate, suggesting that cold spots can be induced to recombine by a targeted DSB. Using long-range single molecule PacBio sequencing we were also able to characterize HR events in somatic tissue. Out of the repair events that have been transmitted to the next generation, gene conversion events were the most frequent outcome of HR. The length of the conversion tracks was quite variable ranging from 5 to 4,349 bp. In addition, a typical feature of these tracks was that they often were interrupted. We discuss whether this results from mismatch repair or multiple invasions. These results open new questions regarding the mechanism of somatic pairing and recombination in comparison to meiotic recombination and about the control of HR in heterochromatic regions. From a technological point of view, our findings pave the way for precise breeding methods based on somatic DNA DSB repair and may help overcoming limitations of classical breeding based on meiotic recombination.
