| dc.description.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. | en |
