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Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling

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dc.contributor.authorNamipashaki, A.
dc.contributor.authorPugsley, K.
dc.contributor.authorLiu, X.
dc.contributor.authorAbrehart, K.
dc.contributor.authorLim, S.M.
dc.contributor.authorSun, G.
dc.contributor.authorHerold, M.J.
dc.contributor.authorPolo, J.M.
dc.contributor.authorBellgrove, M.A.
dc.contributor.authorHawi, Z.
dc.date.issued2023
dc.description.abstractThe 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.
dc.description.statementofresponsibilityAtefeh Namipashaki, Kealan Pugsley, Xiaodong Liu, Kirra Abrehart, Sue Mei Lim, Guizhi Sun, Marco J. Herold, Jose M. Polo, Mark A. Bellgrove, and Ziarih Hawi
dc.identifier.citationStem Cell Reports, 2023; 18(12):2515-2527
dc.identifier.doi10.1016/j.stemcr.2023.10.013
dc.identifier.issn2213-6711
dc.identifier.issn2213-6711
dc.identifier.orcidPolo, J.M. [0000-0002-2531-778X]
dc.identifier.urihttps://hdl.handle.net/2440/142050
dc.language.isoen
dc.publisherElsevier
dc.relation.granthttp://purl.org/au-research/grants/nhmrc/1146644
dc.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/).
dc.source.urihttp://dx.doi.org/10.1016/j.stemcr.2023.10.013
dc.subjectCRISPR-Cas9 editing
dc.subject.meshCell Line
dc.subject.meshHumans
dc.subject.meshDNA
dc.subject.meshCloning, Molecular
dc.subject.meshInduced Pluripotent Stem Cells
dc.subject.meshCRISPR-Cas Systems
dc.subject.meshGene Editing
dc.titleIntegration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling
dc.typeJournal article
pubs.publication-statusPublished

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