Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling
- Author(s)
- Namipashaki, A; Pugsley, K; Liu, X; Abrehart, K; Lim, SM; Sun, G; Herold, MJ; Polo, JM; Bellgrove, MA; Hawi, Z;
- Journal Title
- Stem Cell Reports
- Publication Type
- epub ahead of print
- 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 singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-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 is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized 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 method's utility in stem cell disease modeling.
- Publisher
- Cell Press
- Keywords
- CRISPR-Cas9 editing; clonal homogeneity; electroporatio; human iPSC; lipofection; single-cell cloning; transfection
- Research Division(s)
- Blood Cells And Blood Cancer
- PubMed ID
- 37977144
- Publisher's Version
- https://doi.org/10.1016/j.stemcr.2023.10.013
- Open Access at Publisher's Site
https://doi.org/10.1016/j.stemcr.2023.10.013- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2023-11-20 03:32:55
Last Modified: 2023-11-20 03:37:30