Figure 2

Detection of CRISPR/Cas9-induced indel mutations in the murine Ramp2 gene at the single blastocyst level. A: Schema of the Ramp2 guide RNA (R2gRNA) targeting exon 1 in the murine Ramp2 gene. The R2gRNA-coding sequence is shown in blue. The protospacer-adjacent motif (PAM) sequence is shown in red. The arrows indicate the locations of the PCR primers (see Table 3). B: Agarose gel electrophoresis of T7 endonuclease I-treated PCR products and surveyor-treated PCR products derived from 8 individual blastocysts (see Table 1, experiment 3). Lanes 1–8: PCR products amplified from the crude DNA solution of each blastocyst. Lane NIC (no injection control): PCR product amplified from the crude DNA of a single uninjected control blastocyst. M: lambda HindIII + 100-bp ladder markers. C: Sequencing results for the PCR products shown in lanes 1–8 and NIC of (B). The result of each sample was summarized as the two Ramp2 alleles. “wt” indicates the wild-type sequence. “-” indicates base deletion. D: Gel electrophoretic pattern of DNA products after whole genome amplification (WGA) of the crude DNA shown in lanes 1–8 and NIC of (B) (top panel) and after subsequent PCR amplification (middle panel) and T7 endonuclease I digestion (bottom panel). Note that the band pattern is similar to that shown in (B). NTC (no template control) indicates the WGA product obtained using water in place of template as a negative control (top panel); no PCR products were amplified by PCR (middle panel).