FIGURE 6. pKHR plasmid vectors for genome editing.

pKHR4: pKHR4 is an amp^R plasmid built from Bluescript SK(+) with its multiple cloning site (mcs) flanked by inverted head-to-head oriented I-SceI sites. pKHR5: pKHR5 is derived from pKHR4 by inserting the CV reporter gene cassette, FRT-CV-FRT-loxP, between the EcoRI and EcoRV sites. The cassette consists of the α-crystallin promoter driving expression of the Venus version of the GFP protein in the lens (Hesselson, Anderson, Beinat, & Stainier, 2009) flanked by FRT sites and bordered on the 3′ end with a single loxP site. pKHR5 contains two mcs for independently introducing left and right homology arms to flank the reporter cassette. pKHR7: pKHR7 contains a simple modification of pKHR4 that allows detection of imprecise or random insertion events. pKHR7 contains a cmlc2::mCherry (red heart) reporter gene that resides within the donor fragment that is produced by I-SceI digestion, but outside any homology region. Incorporation of cmlc2::mCherry with donor sequences can only occur as a consequence of imprecise recombination or random integration but never as a result of precise HR. pKHR8: pKHR8 contains a simple modification of pKHR5 that allows detection of imprecise or random insertion events. pKHR8 carries the CV reporter gene cassette, as in pKHR5, but also has a cmlc2::mCherry (red heart) reporter gene, which resides within the donor fragment produced by I-SceI digestion, but outside any homology region. Successful homologous recombination events of interest should acquire the green lens reporter but not the red heart reporter. Sequences for the donor vectors pKHR4, 5, 7, 8 have been deposited in GenBank (KU144822-KU144825). The plasmids are available through Addgene. (See color plate)