Division of Hematology and Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.
O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.
Methods Mol Biol. 2022;2444:15-27. doi: 10.1007/978-1-0716-2063-2_2.
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.
DNA 双链断裂（DSBs）主要通过同源重组（HR）和非同源末端连接（NHEJ）来修复。HR 或 NHEJ 的选择部分取决于断裂的 DNA 末端是否被切除，以产生延伸的单链 DNA（ssDNA）突出端，这些突出端很快被三链 ssDNA 结合复合物 RPA 结合，这是 HR 的第一步。在这里，我们描述了一系列使用稳定整合的诱导型 Cas9 生成带有 Abelson 鼠白血病病毒转化的前 B 细胞（abl 前 B 细胞）的方案，可用于鉴定和研究调节 DNA 末端加工的新途径。这些方法涉及 CRISPR/Cas9 基因失活、全基因组向导 RNA（gRNA）文库介导的筛选以及 DNA 损伤后染色质结合的 RPA 的流式细胞术检测。
