CRISPR Exposes Genomic Evolutionary Mechanisms

      The continued evolution of any organism is based upon the plasticity of its genome and the dissemination of genetic modifications—mutations—onto subsequent generations. Now scientists at the Karlsruhe Institute of Technology (KIT) in Germany have been able to detect an important mechanism in the evolution of plant genomes using the model organism Arabidopsis thaliana. Employing the CRISPR-Cas system, the researchers studied the formation of tandem repeat DNA sequences and found out that these sequences form if both DNA strands are broken at a significant distance from each other.

    A guiding factor in genome evolution is the duplication of existing sequences, and such duplications may be caused by various mechanisms. Plant genomes often contain shorter DNA sequences that are duplicated in a tandem fashion. The KIT investigators believe they have found out how these sequences arise.

    “As we know, the DNA is a double-stranded helix. Our results show that the repair of single-strand breaks occurring at a significant distance from each other in the two opposite strands plays an important role in the generation of duplicates in plant genomes,” explained senior study author Holger Puchta, Ph.D., professor, and director of the Botanical Institute at KIT.

    The KIT team learned from their work with A. thaliana that the synchronized repair of two such single-strand breaks consistently led not only to deletions but also to tandem duplications of shorter sequences near the break locations. In a targeted manner, the scientists introduced differently spaced single-strand breaks into different regions of the genome and then analyzed the repair results by DNA sequencing.

    “In a recent study, the abundant presence of short direct repeats was documented by comparative bioinformatics analysis of different rice genomes, and the hypothesis was put forward that such duplications might arise due to the concerted repair of adjacent single-strand breaks (SSBs),” the authors wrote. “Applying the CRISPR/Cas9 technology, we were able to test this hypothesis experimentally in the model plant Arabidopsis thaliana. Using a Cas9 nickase to induce adjacent genomic SSBs in different regions of the genome (genic, intergenic, and heterochromatic) and at different distances (∼20, 50, and 100 bps), we analyzed the repair outcomes by deep sequencing. In addition to deletions, we regularly detected the formation of direct repeats close to the break sites, independent of the genomic context.”

    The findings from this study were published recently in PNAs through an article entitled “Repair of adjacent single-strand breaks is often accompanied by the formation of tandem sequence duplications in plant genomes.”

    In order to create the single-strand breaks exactly at the desired locations, the KIT team used a novel form of the CRISPR/Cas system.

    “Formerly, we could only work with molecular scissors that cut both strands at the same time, thus creating a double-strand break in the DNA,” Dr. Puchta noted. “With the modified CRISPR/Cas system, it is now possible for the first time to use scissors that only cut a single strand. This enables us to investigate in detail how such damages to the DNA are repaired.”

    Dr. Puchta and his team show that the presence of multiple single-strand breaks in the DNA can introduce genome modifications. Such single-strand breaks quite often occur naturally in plants, above all if they are exposed to UV light. “The newly discovered mechanism is therefore of enormous importance for understanding the evolution of plant genomes,” Dr. Puchta remarked.

    “Most interestingly, we found that even the induction of two SSBs on the same DNA strand can cause genome alterations, albeit at a much lower level,” the authors concluded. “Because such a scenario reflects a natural step during nucleotide excision repair, and given that the germline is set aside only late during development in plants, the repair of adjacent SSBs indeed seems to have an important influence on the shaping of plant genomes during evolution.”