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 six unique mutations that were cloned. (d) Western blot analysis for Mertk expression in H7-derrived retinal pigment epithelium cells. Lanes 1, 3 and 4 indicate knockout lines and lane 2 indicates expression from heterozygous line. Rabbit monoclonal anti-MERTK IgG: Abcam ab52968 (1:10,000).