Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.

Item Type:	Refereed Article
Keywords:	CRISPR, gene editing, stem cell, induced pluripotent stem cells, isogenic
Research Division:	Biological Sciences
Research Group:	Biochemistry and cell biology
Research Field:	Biochemistry and cell biology not elsewhere classified
Objective Division:	Expanding Knowledge
Objective Group:	Expanding knowledge
Objective Field:	Expanding knowledge in the biomedical and clinical sciences
UTAS Author:	Wang, Q (Miss Qi Wang)
UTAS Author:	Chear, S (Ms Sueanne Chear)
UTAS Author:	Wing, K (Mr Kristof Wing)
UTAS Author:	Stellon, D (Mr David Stellon)
UTAS Author:	Talbot, J (Dr Jana Talbot)
UTAS Author:	Hewitt, AW (Professor Alex Hewitt)
UTAS Author:	Cook, AL (Associate Professor Tony Cook)
ID Code:	143340
Year Published:	2021
Web of Science® Times Cited:	3
Deposited By:	Wicking Dementia Research and Education Centre
Deposited On:	2021-03-12
Last Modified:	2022-08-22
Downloads:	0