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

Qi Wang; Sueanne Chear; Kristof Wing; David Stellon; Minh Thuan Nguyen Tran; Jana Talbot; Alice Pébay; Alex W Hewitt; Anthony L Cook

doi:10.1016/j.ymeth.2021.02.009

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

Methods. 2021 Oct:194:18-29. doi: 10.1016/j.ymeth.2021.02.009. Epub 2021 Feb 17.

Authors

Qi Wang¹, Sueanne Chear², Kristof Wing¹, David Stellon², Minh Thuan Nguyen Tran¹, Jana Talbot², Alice Pébay³, Alex W Hewitt⁴, Anthony L Cook⁵

Affiliations

¹ Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia.
² Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia.
³ Department of Anatomy and Neuroscience, the University of Melbourne, Australia; Department of Surgery, the University of Melbourne, Australia.
⁴ Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia; Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Australia.
⁵ Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia. Electronic address: anthony.cook@utas.edu.au.

PMID: 33607266
DOI: 10.1016/j.ymeth.2021.02.009

Abstract

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.

Keywords: CRISPR/Cas ribonucleoprotein; Induced pluripotent stem cells; Isogenic.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems / genetics
Cell Line
Gene Editing*
Humans
Induced Pluripotent Stem Cells* / metabolism
Ribonucleoproteins / genetics
Ribonucleoproteins / metabolism

Substances

Ribonucleoproteins