Figure 2.

Confirmation of sxc^H537A and sxc^K872M mutant lines derived by the CRISPR/Cas9 technique. A, representative gels demonstrating the loss of TaqI (above) or XhoI (below) restriction sites in potential sxc^H537A or sxc^K872M mutants, respectively. Genomic DNA from F1 males was extracted and subjected to PCR amplification followed by restriction digest with TaqI or XhoI. Shown are restriction digests of genomic DNA from five F1 males, each derived from two injected parents. The arrowheads mark the digested band, whereas the asterisk marks the band resistant to TaqI (above) or XhoI (below). B, sequencing chromatograms of WT (top), the putative sxc^H537A homozygote line 1.5 genomic DNA (second), WT (third), and the putative sxc^K872M heterozygote line 7.11 (bottom). These data confirm the incorporation of a desired mutation that would lead to the His-537 to Ala mutation in addition to the two silent mutations that were introduced into the wobble positions in the adjacent codons. For the Lys-872 to Met mutants, the presence of multiple peaks in the chromatogram demonstrates the heterozygosity of the locus.