Abstract
Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.
Highlights
Gene-edited induced pluripotent stem cells provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds
These advances are of particular interest in the field of human genetics for disease modeling, where gene targeting in human induced pluripotent stem cells with nucleases enables the original patient iPSC line to act as an isogenic control[3]
Recent advances in nuclease technology have respectably improved gene targeting efficiencies for human embryonic stem cells (ESCs) or iPSCs, the deposition of single nucleotide variations which mimic or correct patient mutations remains difficult without a robust means for enrichment and selection, such that positive selection for antibiotic resistance markers remains a staple in gene targeting[4]
Summary
Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Error-prone repair via non-homologous end joining (NHEJ) is typically sufficient for gene disruption, while homology directed repair (HDR) can be usurped with custom template DNA that acts as a donor in the repair of targeted double-strand breaks, allowing for more specific gene editing These advances are of particular interest in the field of human genetics for disease modeling, where gene targeting in human induced pluripotent stem cells (iPSCs) with nucleases enables the original patient iPSC line to act as an isogenic control[3]. We expect this technique to have broad applications, even beyond scarless iPSC genome editing
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