Genome Editing of the CYP1A1 Locus in iPSCs as a Platform to Map AHR Expression throughout Human Development

Figure 1

Construction and validation of CYP1A1 reporter iPSCs. (a) Two vectors were created to achieve CAS9 targeted digestion at the CYP1A1 transcription start site and homologous recombination of a reporter construct. The donor plasmid contains a cassette that includes eGFP and luciferase separated by an internal ribosome entry sequence (IRES). Directly downstream of these reporter elements is a puromycin resistance gene (Puro) driven by a constitutive promoter (PGK) and flanked by loxP sites (denoted by black arrowheads). This cassette is flanked by regions that are homologous to the CYP1A1 endogenous locus (Left Homology, LH; Right Homology, RH) to facilitate homologous recombination. The guide RNA (gRNA) and Cas9 were encoded on the same plasmid, each driven by a separate constitutive promoter (U6 and CMV, resp.). (b) An idealized schematic of Cas9 digestion at the transcription start site (denoted by black arrow) of the CYP1A1 locus. (c) The integrated reporter construct is expected to specifically target the transcription start site of CYP1A1, and a PCR strategy was employed that creates amplicons in the 5′ and 3′ flanking regions of the cassette that include elements from the reporter construct as well as endogenous regions that are not encoded by the donor plasmid. (d) The 5′ amplicon (expected size = 962 bp) was exclusively detected in a properly targeted iPSC clone. (e) The 3′ amplicon (expected size = 704 bp) also could not be amplified in untargeted clones or the parental iPSC line but was detected in a properly targeted iPSC clone.

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