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.

Figure 1.

Strategy for episome-based Cas9 genome editing. (A) Key features of the Cas9- and the sgRNA-episome. Black arrow: PARP (procyclic acidic repetitive protein) promoter; HA: HA-epitope; NLS: SV40 nuclear localization signal; hSpCas9: human codon-optimized Cas9; PUR^R: puromycin N-acetyl transferase CDS conferring resistance to puromycin; G418^R: neomycin phosphotransferase conferring resistance to G418; HH: hammerhead ribozyme; crRNA: CRISPR-RNA; tracrRNA: trans activating RNA; HDV: hepatitis delta virus ribozyme; repair: sequence providing the DNA repair template carrying the desired mutations. (B) Sequence of the genomic target on Chr 11, 140,068-140,155. SCD6 ORF: black, uppercase; SCD6 3′ UTR: black, lower case; protospacer: purple highlight; PAM: green. (C) Strategy for the marker-free tagging of the SCD6 ORF. The purple triangle indicates the protospacer location targeted by the crRNA (not drawn to scale). The repair template encompasses the eGFP ORF flanked by 220 bp of the 3′-end of the SCD6 ORF and 220 bp of its 3′-UTR.

Figure 2.

Outline of transfection strategies. (A) Co-transfection strategy. (B) Sequential transfection strategy.

Figure 3.

Cas9 allows marker-free genome editing at high efficiency. Unless indicated otherwise, cells were sequentially transfected with the Cas9-episome and the sgRNA-episome(SCD6-GFP.1) and diluted 10 days after transfection. Cells were only analyzed if <20 wells per 96-well plate contained living cells. (A) Fluorescence microscopy analysis of cells transfected with sgRNA-episome(SCD6-GFP.1, clone B4) or a sgRNA-episome lacking a sgRNA (-crRNA). (B) Flow cytometry analysis of edited cells. Left panel: wild type cells (gray) and cells in which SCD6 was GFP-tagged using the pMOT system (55) (green). Right panel: wild type cells (gray) and a representative clone of cells transfected with the sgRNA-episome(SCD6-GFP.1, clone B4). (C) PCR-based analysis of wild type cells, six clones of cells transfected with sgRNA-episome(SCD6-GFP.1) and cells transfected with an sgRNA-episome lacking a sgRNA (-crRNA). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing the presence of homozygous (one PCR product) or heterozygous (two PCR products) tagging events. (D) Sanger-sequencing results of the SCD6 gene from wild type cells, the non-edited SCD6 allele of three heterozygous clones and cells transfected with a sgRNA-episome lacking a sgRNA (-crRNA).

Figure 4.

Cas9 allows multiple genome edits. (A) Strategy used for the removal of the sgRNA-episome. (B) PCR-based analysis of cells sequentially transfected with the Cas9-episome and sgRNA-episome(ΔH3.V) or sgRNA-episome(ΔH4.V). Top panel: outline indicating primer binding sites (half arrows). Bottom panel: agarose gel revealing presence or absence of H3.V (left) or H4.V (right). Homozygous deletions of H4.V were only observed after the culture had been diluted a second time. (C) Sanger sequencing demonstrated that all editing events exactly matched the repair template. The deleted CDSs of H3.V and H4.V are represented on top and highlighted in orange. The repair templates are shown in the middle (5′-UTR in green, 5′-end of the H3.V CDS in orange and 3′-UTR in purple. The sequencing chromatograms are depicted at the bottom.

Figure 5.

Cas9 allows editing of multicopy histone arrays. (A) Structure of acetylated lysine and arginine. (B) Sequences of the crRNA(H4R4) and the relevant regions of the H4R4 repair template. (C) Sanger-sequencing chromatograms from gDNA of cells transfected with the sgRNA-episome(H4R4). gDNA was extracted from multiple clones at different time points and revealed an accumulation of edited cells coding for H4R4 over time. Illumina-based gDNA-seq of clone B3-3-1 to 29x coverage indicated that after 5 months ∼90% of histone H4 transcripts code for H4R4. (D) gDNA-seq-based quantification of histone H4 copy number (for details see methods section).