pcDNA3.1(+)-eGFP-HR/NHEJ-reporter plasmid vector哺乳动物荧光报告质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心

pcDNA3.1(+)-eGFP-HR/NHEJ-reporter plasmid vector哺乳动物荧光报告质粒载体

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms
to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dualfluorescent DSB Repair), that enables the detection
and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based
on the introduction and subsequent resolution of
one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and
sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone nonhomologous end-joining (NHEJ), as well as between
proximal and distal NHEJ repair. Furthermore, CDDR
can detect homology-directed repair (HDR) with high
sensitivity. Using CDDR, we found HF-NHEJ to be
strictly dependent on DNA Ligase IV, XRCC4 and XLF,
members of the canonical branch of NHEJ pathway
(c-NHEJ). Loss of these genes also stimulated HDR,
and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand,
stimulated HF-NHEJ and suppressed HDR. These
findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

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