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

被引：0

作者：

Namipashaki, Atefeh ^{[1
]}

Pugsley, Kealan ^{[1
]}

Liu, Xiaodong ^{[2
,3
,4
]}

Abrehart, Kirra ^{[1
]}

Lim, Sue Mei ^{[2
,3
,4
]}

Sun, Guizhi ^{[2
,3
,4
]}

Herold, Marco J. ^{[5
,6
]}

Polo, Jose M. ^{[2
,3
,4
,7
,8
]}

Bellgrove, Mark A. ^{[1
]}

Hawi, Ziarih ^{[1
]}

机构：

[1] Monash Univ, Turner Inst Brain & Mental Hlth, Sch Psychol Sci, Melbourne, VIC, Australia

[2] Monash Univ, Dept Anat & Dev Biol, Melbourne, VIC 3800, Australia

[3] Monash Biomed Discovery Inst, Dev & Stem Cells Program, Melbourne, VIC 3800, Australia

[4] Monash Univ, Australian Regenerat Med Inst, Melbourne, VIC 3800, Australia

[5] Walter & Eliza Hall Inst Med Res, Melbourne, VIC, Australia

[6] Univ Melbourne, Dept Med Biol, Melbourne, VIC, Australia

[7] Univ Adelaide, Adelaide Ctr Epigenet, Adelaide, SA, Australia

[8] Univ Adelaide, South Australian Immunogen Canc Inst, Adelaide, SA, Australia

来源：

STEM CELL REPORTS | 2023年 / 18卷 / 12期

基金：

英国医学研究理事会;

关键词：

PLURIPOTENT STEM-CELLS; TRANSFECTION; TRANSIENT; ELECTROPORATION; FIBROBLASTS; GENERATION; MUTATIONS; APOPTOSIS; SURVIVAL; RNA;

D O I：

10.1016/j.stemcr.2023.10.013

中图分类号：

Q813 [细胞工程];

学科分类号：

摘要：

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.

引用

页码：2515 / 2527

页数：13