CRISPR/Cas9 Gene Editing of Human Histone H2A Variant H2AX and MacroH2A

Justin W.C. Leung, Lara E. Emery, Kyle M. Miller

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages255-269
Number of pages15
DOIs
StatePublished - 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1832
ISSN (Print)1064-3745

Keywords

  • CRISPR/Cas9
  • Chromatin
  • DNA damage
  • DNA repair
  • Gene editing
  • Genome stability
  • H2A
  • H2AX
  • Histone variant
  • macroH2A

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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