Allelic DNA synthesis followed by template switching underlies microhomology-mediated tandem duplication in BRCA1-deficient cells

Microhomology-mediated short tandem duplication (TD) is among specific mutational signatures associated with BRCA1-deficient tumors. Several mechanisms have been proposed for its generation; however, our understanding of these mechanisms is largely gained through DNA repeat-based reporters and may not be applicable in approximately 50% of the human genome due to lack of neighboring repeats. We thus developed a red fluorescent protein-based repeat-less TD reporter and a PCR-based endogenous site-specific TD assay to analyze short TDs induced by one-ended DNA double strand breaks (DSBs) converted from DNA nicks in Brca1-deficient mouse embryonic stem cells. We found that the level of short TDs induced by DNA nicks are detectable in normal cells but significantly elevated in Brca1-deficient cells. Because SpCas9 nickases (Cas9n) were used to induce DNA nicks, the on-target TD formation indicates a source of safety concerns in Cas9n-based genome editing such as base editing and prime editing. Analysis of TD products reveals that the TD formation is partly mediated by template switching of displaced nascent strand after allelic DNA synthesis. This suggests either allelic DNA synthesis or the strand annealing step of allelic break-induced replication, the primary homologous recombination pathway in repair of one-ended DSBs, might be more easily aborted in Brca1-deficient cells, thus promoting TD. Neither depletion of Rad51 or Brca2 nor inactivation of the Brca1 coiled-coil domain stimulates nick-induced TD, indicating that RAD51 loading by BRCA1 is dispensable for BRCA1-mediated TD suppression. These results together provide novel insights into the mechanisms underlying BRCA1-linked TD formation in cancer.