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bioRxiv · 10.1101/2022.04.12.488094

An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling

Namipashaki, A.·Liu, X.·Pugsley, K.·Lim, S. M.·Sun, G.·Herold, M. J.·Polo, J. M.·Bellgrove, M. A.·Hawi, Z.
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Abstract

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularised CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labour-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10 days. This was accomplished through improved viability of the transfected cells, paralleled with provision of enriched environment for the robust establishment and proliferation of singularised iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50-65% for NHEJ and 10% for HDR, indicative of the methods utility in stem cell disease modelling.

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Namipashaki, A., Liu, X., Pugsley, K., Lim, S. M., Sun, G., Herold, M. J., Polo, J. M., Bellgrove, M. A., Hawi, Z.. 2022-04-13. An Efficient CRISPR-Cas9 DNA Editing Methodology Applicable for iPSC Disease Modelling. https://doi.org/10.1101/2022.04.12.488094

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