CRISPR-Cas9 Mediated Genome Editing of hPSCs

Advances in genome editing technologies have made possible the introduction of precise genetic modifications to hPSCs, facilitating a range of applications in basic and translational research.

In conjunction with the Transgenic Animal and Genome Editing Facility, we offer an hPSC gene editing service. We have successfully used CRISPR/Cas9 to introduce the following classes of edits into the genome of various hPSC lines:

1. Introduction of large DNA fragment removal to a generate gene knockout hPSC lines
2. Correction of disease-associated point mutations in patient-specific iPSCs
3. Introduction of disease-associated point mutations to control hPSCs
4. Introduction of reporter cassettes to monitor transcriptional activity of endogenous genes
5. Introduction of genetic elements to the AAVS1 safe harbor in hPSCs
• The hPSC gene editing service is available to both Cincinnati Children's/UC and external investigators.
• Gene editing projects are usually complete within 3-6 months. However, each project is different and occasionally delays are experienced. See an overview of a typical workflow below.

Availability of Transgenic Human hPSC Lines

We have generated several transgenic human iPSC lines that are available for distribution to the research community. These include lines for:

• Regulation of endogenous gene expression (CRISPRi/CRISPRa)
• Disruption of transcription factor binding to DNA binding motifs (CRISPRd)
• Expression of BirA ligase
• Expression of eGFP or mCherry
• Fucci2a-mediated cell cycle analysis

Lines are distributed from a well-validated cryobank. An executed MTA is required for transfer to an external institution. Please contact us to request lines.