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Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells

Wang, Q, Chear, S, Wing, K, Stellon, D, Tran, MTN, Talbot, J, Pebay, A, Hewitt, AW ORCID: 0000-0002-5123-5999 and Cook, AL ORCID: 0000-0003-1770-7910 2021 , 'Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells' , Methods , pp. 1-12 , doi: 10.1016/j.ymeth.2021.02.009.

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Abstract

Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regardto predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cassystem have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Casgene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants providesa 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/Casribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs andhomology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery vianucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifyingiPSC clones for further characterization.

Item Type: Article
Authors/Creators:Wang, Q and Chear, S and Wing, K and Stellon, D and Tran, MTN and Talbot, J and Pebay, A and Hewitt, AW and Cook, AL
Keywords: CRISPR, gene editing, stem cell, induced pluripotent stem cells, isogenic
Journal or Publication Title: Methods
Publisher: Academic Press Inc Elsevier Science
ISSN: 1046-2023
DOI / ID Number: 10.1016/j.ymeth.2021.02.009
Copyright Information:

copyright 2021 Elsevier Inc.

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