A CRISPR-Cas9 based shuffle system for endogenous histone H3 and H4 combinatorial mutagenesis

Sci Rep. 2021 Feb 8;11(1):3298. doi: 10.1038/s41598-021-82774-4.

Abstract

Post-translational modifications of histone proteins greatly impact gene expression and cell fate decisions in eukaryotes. To study these, it is important to develop a convenient, multiplex, and efficient method to precisely introduce mutations to histones. Because eukaryotic cells usually contain multiple copies of histone genes, it is a challenge to mutate all histones at the same time by the traditional homologous recombination method. Here, we developed a CRISPR-Cas9 based shuffle system in Saccharomyces cerevisiae, to generate point mutations on both endogenous histone H3 and H4 genes in a rapid, seamless and multiplex fashion. Using this method, we generated yeast strains containing histone triple H3-K4R-K36R-K79R mutants and histone combinatorial H3-K56Q-H4-K59A double mutants with high efficiencies (70-80%). This CRISPR-Cas9 based mutagenesis system could be an invaluable tool to the epigenetics field.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • CRISPR-Cas Systems*
  • Histones / genetics*
  • Mutagenesis*
  • Mutation, Missense*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics*

Substances

  • Histones
  • Saccharomyces cerevisiae Proteins