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. 2014 Aug 8;5:4516.
doi: 10.1038/ncomms5516.

Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs

Vinod Ranganathan  1 , Karl Wahlin  2 , Julien Maruotti  2 , Donald J Zack  3
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Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs

Vinod Ranganathan et al. Nat Commun. .
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Abstract

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ~15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species.

Figures

Figure 1
Evaluating the ability to direct CRISPR targeting via gRNA synthesis from the H1 promoter. (a) Schematic illustration depicting the gRNA expression constructs. Above, the U6 promoter only expresses gRNAs with a +1 guanosine nucleotide; below, the H1 promoter can drive expression of gRNAs initiating at either purine (adenosine or guanosine) nucleotide. On the right, a cartoon depiction of the Cas9 protein with gRNA targeting genomic sequence AN19NGG. The location of the +1 A is indicated. (b) Schematic overview of the eGFP targeted disruption assay. eGFP fluorescence is disrupted by CRISPR targeting followed by error-prone NHEJ-mediated repair resulting in frameshift mutations that disrupt the coding sequence, resulting in loss of fluorescence. (c) Microscope images demonstrating successful CRISPR targeting by U6 or H1 promoter expressed gRNAs. H7 ES cells were stained and colonies were visualized to show nuclei (left, magenta), eGFP fluorescence (middle, green), and merged images (right) indicating areas of GFP fluorescence mosaicism in the colony. To the right is shown the quantification of eGFP fluorescence loss by flow cytometry for the respective constructs. Below is a higher magnification of an H7 colony targeted by an H1 expressed gRNA showing expression mosaicism. Scale bar, 50 µM. (d) Surveyor assay-based quantitation of the frequency of NHEJ. Bioanalyzer gel image depicting control (first lane), U6 expressed gRNA (second lane), H1 expressed gRNA (third lane), and marker (fourth lane). The % indel (as calculated by the fraction of uncut (u) to cut (c) bands) is indicated below.
Figure 2
Bioinformatics analysis of GN19NGG and AN19NGG sites in the genome. (a) Circos plot depicting the frequency of CRISPR sites in the human genome. The outside circle depicts the human chromosome ideograms. Moving inwards, GN19NGG (orange), AN19NGG (blue), and RN19NGG (purple) CRISPR sites frequency is indicated along the chromosomes. Plotted inside the circle is the human exon density (black), and OMIM disease loci (blue). (b) Frequency and distance between of CRISPR sites in the genome. Barplot of the frequency and distance of adjacent GN19NGG (orange), AN19NGG (blue) sites in the genome. The mean and median values are inset within the plot including RN19NGG sites. (c) Barplot quantification of GN19NGG vs AN19NGG site frequency at human genes (left) or OMIM disease loci (right). (d) Barplot quantifying the GN19NGG vs. AN19NGG frequency in six genomes: human, cow, mouse, rat, chicken, and zebrafish.
Figure 3
CRISPR targeting of AN19NGG at an endogenous gene (MERTK) in H7 ES cells. (a) Schematic diagram of the MERTK locus and various protein domains. Target site in exon 2 is shown below in larger scale, indicating the CRISPR AN19NGG target site. (b) Quantification of CRISPR targeting at exon2 by the Surveyor assay. The CRISPR site in exon 2 is depicted above, with the various primers (arrows) used in the Surveyor assay; both F1:R1 and F2:R2 span the target site, while the control PCR product, F3:R3, is just outside the target site. The gel from the Surveyor assay is shown below with the three control products shown on the left, and targeting is shown on the right. Below the % indel frequency is indicated. (c) Sanger sequencing of mutant lines. Clonal lines were isolated and sequenced indicating that CRISPR targeting at the AN19NGG sites resulted in mutagenesis at this region. The aligned chromatograms show the 6 unique mutations that were cloned. (d) Western Blot analysis for Mertk expression in H7-derrived RPE cells. Lanes 1, 3, and 4 indicate knockout lines and lane 2 indicates expression from heterozygous line.
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