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Nucleic Acids Res. 2018 Oct 12;46(18):e106. doi: 10.1093/nar/gky517.

Exploiting CRISPR-Cas9 technology to investigate individual histone modifications.

Author information

1
Research Center for Infectious Diseases, University of Würzburg, 97080 Würzburg, Germany.
2
Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, 80752 Munich, Germany.
3
Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152 Planegg-Martinsried, Germany.

Abstract

Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.

PMID:
29912461
PMCID:
PMC6182134
DOI:
10.1093/nar/gky517
[Indexed for MEDLINE]
Free PMC Article

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