File(s) under permanent embargo
Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells
journal contributionposted on 2023-05-20, 21:50 authored by Wang, Q, Sueanne ChearSueanne Chear, Kristof WingKristof Wing, David Stellon, Tran, MTN, Jana TalbotJana Talbot, Pebay, A, Alexander HewittAlexander Hewitt, Anthony CookAnthony Cook
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.
Batten Disease Support and Research Association
Department/SchoolMenzies Institute for Medical Research
PublisherAcademic Press Inc Elsevier Science
Place of publication525 B St, Ste 1900, San Diego, USA, Ca, 92101-4495
Rights statementcopyright 2021 Elsevier Inc.