Abstract
Repair of DNA Double-Strand Breaks (DSBs) can be error-free or highly mutagenic, depending on which of multiple mechanistically distinct pathways repairs the break. Hence, DSB-repair pathway choice directly affects genome integrity, and it is therefore of interest to understand the parameters that direct repair towards a specific pathway. This has been intensively studied using genomic reporter constructs, in which repair of a site-specific DSB by the pathway of interest generates a quantifiable phenotype, generally the expression of a fluorescent protein. The current developments in genome editing with targetable nucleases like Cas9 have increased reporter usage and accelerated the generation of novel reporter constructs. Considering these recent advances, this review will discuss and compare the available DSB-repair pathway reporters, provide essential considerations to guide reporter choice, and give an outlook on potential future developments.
Highlights
The integrity of our genome is constantly challenged by DNA damaging lesions that arise during normal cell growth and division, and are caused by exposure to environmental mutagens and irradiation (Hoeijmakers, 2009)
Double-Strand Breaks (DSBs) can be repaired by Homologous Recombination (HR), which is initiated by extensive nuclease-mediated resection of the DSBends to generate 3’ single strand overhangs (Figure 1A; Jasin and Rothstein, 2013)
ASeveral variants of EJ7-GFP were constructed that contain 1–4 nucleotides microhomology. bEither HR or HITI can be studied, depending on the provided repair template. cLoss of BFP expression can result from mutagenic repair by either end-joining or Single-Strand Annealing (SSA). dThe I-SceI target site is located 39 bp behind the GFP sequence, so only repair resulting in deletions >39 bp will disrupt GFP expression. eWhich repair pathway is responsible for the large deletions that are detected by the CAT-R system has not been determined
Summary
DSB-repair pathway choice directly affects genome integrity, and it is of interest to understand the parameters that direct repair towards a specific pathway. This has been intensively studied using genomic reporter constructs, in which repair of a sitespecific DSB by the pathway of interest generates a quantifiable phenotype, generally the expression of a fluorescent protein. The current developments in genome editing with targetable nucleases like Cas have increased reporter usage and accelerated the generation of novel reporter constructs. Considering these recent advances, this review will discuss and compare the available DSB-repair pathway reporters, provide essential considerations to guide reporter choice, and give an outlook on potential future developments
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