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 enhanced GFP (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.