User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9

Figure 1

Schematic of CRISPR/Cas9-mediated conditional targeting. (a) Plasmid map of LNL and FNFL vectors. Schematic diagrams of LNL plasmid and FNFL plasmid. LoxP sites are shown by purple arrowheads, and FRT sites are indicated by grey arrowheads. (b) Sequential targeting of LNL and FNFL cassettes in two steps. First, the LNL cassette was inserted to the 5 site of the region intended to delete. And then the neo^r cassette was removed upon 4-OHT treatment, with one LoxP site left. Subsequently, the FNFL cassette was inserted to the 3 site of the target region. Next, part of the FNFL cassette was depleted by FRT-FLP recombination upon Dox treatment, with one FRT site and one LoxP site left. Finally, the floxed exon was eliminated by Cre-LoxP recombination induced by 4-OHT treatment. LoxP sites and FRT sites are indicated by purple and grey arrowheads, respectively. The CRISPR targeting sites are labelled by red arrows. (c) Simultaneous targeting of LNL and FNFL cassettes in one step. The CRISPR/Cas9 technique was employed to introduce biallelic insertion of LNL and FNFL cassettes to the targeting locus simultaneously. The FNFL cassette was depleted by Dox-inducible FLP-FRT recombination, with one FRT site and one LoxP site being left. Lastly, the LoxP-flanked genomic region was deleted by 4-OHT treatment to induce knockout.

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