A paper published in the journal “Nature Communications” by the Cejka laboratory shows that the HLTF, a protein known to remodel replication forks, can unexpectedly dislocate Cas9 from the DNA after cleavage to initiate DNA double-stranded break repair.
Bellinzona – July 10 2024 – During gene editing, the repair of DNA double-stranded breaks generated with the CRISPR system is initially prevented by the presence of Cas9 on the DNA ends. Using single molecule and bulk biochemical assays, the authors directly show that the machineries responsible for DNA break repair are initially unable to process Cas9-mediated DNA breaks. The authors demonstrate that the DNA translocase HLTF can dislocate Cas9 from the DNA to initiate repair of the breaks. In this process, HLTF is recruited to the site of the break thanks to its HIRAN domain that binds the 3’-ssDNA that is released by Cas9 upon cleavage of the non-target strand of the DNA. Once HLTF is recruited to the site of the break, the translocase motor of the protein is engaged and Cas9 is evicted from DNA, allowing the repair of the break. This unexpected observation is relevant for some gene editing, because HLTF regulates the residency time of Cas9 at the DNA break site, which might affect the outcome or efficacy of the gene editing applications. The work was primarily carried out by Giordano Reginato and Maria Rosaria Dello Stritto from the Cejka laboratory at IRB, in collaboration with the teams of Martin Jinek (University of Zurich), Andre Nussenzweig (NIH, USA) and Taekjip Ha (Harvard University).
