Novel CRISPR/Cas9-based assay for studying DNA repair associated mutations

Abstract

DNA double-strand breaks (DSBs) are a lethal type of DNA damage and are primarily repaired by two main pathways: homologous recombination (HR) and classical non-homologous end joining (C-NHEJ). Impairment of HR or C-NHEJ leads to DSB repair through a less characterised pathway termed alternative non-homologous end joining (Alt-EJ). Recent studies suggest that Alt-EJ contributes to the formation of insertions/deletions and chromosomal translocations, which may cause cancer onset and progression. However, there is currently no existing Alt-EJ assay in the native chromatin context, and thus the underlying mechanism of the Alt-EJ pathway remains poorly understood.

In this Master project, I aimed to develop and implement a novel Alt-EJ assay termed Quantitative Multiplex Analysis of Translocations (QMAT-seq) that can quantify the frequency of translocation events and identify mutational signatures at the repair junctions of translocations. The assay exploits the RNA-guided CRISPR/Cas9 system to generate multiple DSBs in the genome, and the repair of these breaks by Alt-EJ leads to translocations. In this study, five guide-RNA (gRNA) sequences were selected, allowing for assessment of outcomes at 20 distinct translocation junctions. CRISPR/Cas9-mediated DSB formation was verified by the Surveyor nuclease assay and the translocation junctions were captured by nested PCR. Sanger sequencing of the translocation amplicons revealed that translocations form as expected, thus strongly supporting the feasibility of QMAT-seq.

However, QMAT-seq is still in its preliminary stage, and future perspectives include quantification of translocation frequencies, high-throughput sequencing, and an extensive bioinformatical analysis of mutational patterns at the translocation junctions. Moreover, the sensitivity of the assay needs to be quantified by perturbing known Alt-EJ factors and assessing if the resulting changes in translocation frequency and mutational patterns are consistent with what is known in the literature.

Upon completion, QMAT-seq will provide a means of identifying novel Alt-EJ factors, subtypes of Alt-EJ, and their underlying mechanism. Furthermore, characterisation of the Alt- EJ mutational signature can serve as a biomarker for deficiency in HR and C-NHEJ, which can aid in developing personalised cancer therapies. QMAT-seq thus promises to make a substantial contribution to the field of DNA repair and cancer biology.