Histone H2A variants play important roles in maintaining the integrity of the genome. For example, the histone variant H2AX is phosphorylated on Ser139 (called γH2AX) at DNA double-strand breaks (DSB) and serves as a signal for the initiation of downstream DNA damage response (DDR) factor recruitment and DNA repair activities within damaged chromatin. For decades, genetic studies in human cells involving DNA damage signaling and repair factors have relied mostly on either knockdown by RNA interference (i.e., shRNA and siRNA) or the use of mouse embryonic fibroblasts derived from knockout (KO) mice. Recent advances in gene editing using ZNF nuclease, TALEN, and CRISPR/Cas9 have allowed the generation of human KO cell lines, allowing genetic models for studying the DDR, including histone H2A variants in human cells. Here, we describe a detailed protocol for generating and verifying KO of H2AX and macroH2A histone H2A variants using CRISPR/Cas9 gene editing in human cancer cell lines. This protocol allows the use and development of genetic systems in human cells to study histone variants and their functions, including within the DDR.
Histone variant H2A H2AX macroH2A Chromatin CRISPR/Cas9 Gene editing DNA damage DNA repair Genome stability
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We thank Fade Gong from the Miller lab for insightful comments on the manuscript. The K.M.M. laboratory is supported by the NIH National Cancer Institute (R01CA198279 and RO1CA201268) and the American Cancer Society (RSG-16-042-01-DMC).
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