DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of HR or NHEJ is dictated in part by whether the broken DNA ends are resected to generate extended single-stranded DNA (ssDNA) overhangs, which are quickly bound by the trimeric ssDNA binding complex RPA, the first step of HR. Here we describe a series of protocols for generating Abelson murine leukemia virus-transformed pre-B cells (abl pre-B cells) with stably integrated inducible Cas9 that can be used to identify and study novel pathways regulating DNA end processing. These approaches involve gene inactivation by CRISPR/Cas9, whole genome guide RNA (gRNA) library-mediated screen, and flow cytometry-based detection of chromatin-bound RPA after DNA damage.
Keywords: CRISPR/Cas9; DNA end resection; Genome-wide screen; HR; NHEJ; RPA.
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