Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling
Files
(Published version)
Date
2023
Authors
Namipashaki, A.
Pugsley, K.
Liu, X.
Abrehart, K.
Lim, S.M.
Sun, G.
Herold, M.J.
Polo, J.M.
Bellgrove, M.A.
Hawi, Z.
Editors
Advisors
Journal Title
Journal ISSN
Volume Title
Type:
Journal article
Citation
Stem Cell Reports, 2023; 18(12):2515-2527
Statement of Responsibility
Atefeh Namipashaki, Kealan Pugsley, Xiaodong Liu, Kirra Abrehart, Sue Mei Lim, Guizhi Sun, Marco J. Herold, Jose M. Polo, Mark A. Bellgrove, and Ziarih Hawi
Conference Name
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.
School/Discipline
Dissertation Note
Provenance
Description
Access Status
Rights
© 2023 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativeco mmons.org/licenses/by-nc-nd/4.0/).