Exploiting CRISPR–Cas9 technology to investigate individual histone modifications

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.

Universal highly efficient conditional knockout system in Leishmania , with a focus on untranscribed region preservation

“…In our experience, tagging the C‐terminus is generally safer than tagging the N‐terminus in Leishmania . This is contrary to what is known in T. brucei where the tag is best positioned at the N‐terminal end (Kelly et al, ; Vasquez, Wedel, Cosentino, & Siegel, ). However, this has to be tempered and likely depends on the targeted gene either due to the presence of targeting motifs that have a pivotal role in the protein localisation and addressing to specific cellular compartment and/or to the role of the endogenous UTR in post‐transcriptional regulation.…”

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…; Vasquez et al. ; Table ). Several approaches have been successfully used to adapt the CRISPR/Cas9 system and modify these parasites’ genomes.…”

“…; Vasquez et al. ; Zhang and Matlashewski ). Other factors could affect the efficiency of the system, such as the DNA delivery system (nucleofection is a more efficient electroporation method than transfection); the possibility of performing cell sorting during the selection period, to enrich the population of genetically modified parasites or even get mutant clones; and the presence of a resistance marker in the DNA donor template to ensure the selection of parasites with properly edited genomes.…”

“…; Vasquez et al. ; Zhang and Matlashewski ). In addition, the transient expression of in vitro‐transcribed sgRNAs could reduce the efficiency of the method.…”

“…; Vasquez et al. ) and the honey bee trypanosomatid Lotmaria passim (Liu et al. ) and the system has been successfully used to investigate the role of several proteins in trypanosomatids, but mainly in T. cruzi (Beneke et al.…”