ena-STUDY-INSTITUTE FOR MOLECULAR INFECTION BIOLOGY-04-07-2017-14:14:01:344-86
Exploiting CRISPR-Cas9 technology to investigate individual histone modifications
Histone modifications can modulate DNA accessibility and are therefore a key regulator of all DNA-templated processes. However, barring few exceptions, mutational analyses of histones in vivo are lacking and for most histone modifications it is not known whether mutation of a modified histone residue reproduces the phenotype caused by mutation of the modifying enzyme. Reasons for the lack of such mutational analyses are that histones are encoded by multigene families and that tools to simultaneously edit multiple genomic loci at high efficiency are being only recently developed.To overcome these challenges and to study the function of individual histone modifications we took advantage of the recently uncovered potential Cas9 for precise genome editing and the fact that in the protozoan parasites Trypanosoma brucei most DNA repair occurs via homologous recombination. We established an episome-based CRISPR-Cas9 system for T. brucei that allowed us to edit wild type cells without the need to insert selectable markers, to insert a GFP tag between the ORF and its 3´UTR, to delete both alleles of a gene in a single transfection, and to perform precise editing of genes existing in multi-copy arrays (e.g. histone H4K4 to H4R4) with no detectable off-target effects.The newly established genome editing toolbox will be highly useful for the trypanosome community and beyond as it allows for the generation of precise mutants without the need 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.
Histone modifications can modulate DNA accessibility and are therefore a key regulator of all DNA-templated processes. However, barring few exceptions, mutational analyses of histones in vivo are lacking and for most histone modifications it is not known whether mutation of a modified histone residue reproduces the phenotype caused by mutation of the modifying enzyme. Reasons for the lack of such mutational analyses are that histones are encoded by multigene families and that tools to simultaneously edit multiple genomic loci at high efficiency are being only recently developed.To overcome these challenges and to study the function of individual histone modifications we took advantage of the recently uncovered potential Cas9 for precise genome editing and the fact that in the protozoan parasites Trypanosoma brucei most DNA repair occurs via homologous recombination. We established an episome-based CRISPR-Cas9 system for T. brucei that allowed us to edit wild type cells without the need to insert selectable markers, to insert a GFP tag between the ORF and its 3´UTR, to delete both alleles of a gene in a single transfection, and to perform precise editing of genes existing in multi-copy arrays (e.g. histone H4K4 to H4R4) with no detectable off-target effects.The newly established genome editing toolbox will be highly useful for the trypanosome community and beyond as it allows for the generation of precise mutants without the need 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.