CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells

David Stellon; Minh Thuan Nguyen Tran; Jana Talbot; Sueanne Chear; Mohd Khairul Nizam Mohd Khalid; Alice Pébay; James C Vickers; Anna E King; Alex W Hewitt; Anthony L Cook

doi:10.1007/7651_2021_422

CRISPR/Cas-Mediated Knock-in of Genetically Encoded Fluorescent Biosensors into the AAVS1 Locus of Human-Induced Pluripotent Stem Cells

Methods Mol Biol. 2022:2549:379-398. doi: 10.1007/7651_2021_422.

Authors

Affiliations

¹ Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia. david.stellon@utas.edu.au.
² Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia.
³ Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia.
⁴ Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC, Australia.
⁵ Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Parkville, VIC, Australia.
⁶ Wicking Dementia Research and Education Centre, University of Tasmania, Hobart, TAS, Australia. anthony.cook@utas.edu.au.

PMID: 34505269
DOI: 10.1007/7651_2021_422

Abstract

Genetically encoded fluorescent biosensors (GEFBs) enable researchers to visualize and quantify cellular processes in live cells. Induced pluripotent stem cells (iPSCs) can be genetically engineered to express GEFBs via integration into the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus. This can be achieved using CRISPR/Cas ribonucleoprotein targeting to cause a double-strand break at the AAVS1 locus, which subsequently undergoes homology-directed repair (HDR) in the presence of a donor plasmid containing the GEFB sequence. We describe an optimized protocol for CRISPR/Cas-mediated knock-in of GEFBs into the AAVS1 locus of human iPSCs that allows puromycin selection and which exhibits negligible off-target editing. The resulting iPSC lines can be differentiated into cells of different lineages while retaining expression of the GEFB, enabling live-cell interrogation of cell pathway activities across a diversity of disease models.

Keywords: AAVS1; CRISPR/Cas; Fluorescent biosensor; Induced pluripotent stem cell; Live cell imaging.

Publication types

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

MeSH terms

Biosensing Techniques*
CRISPR-Cas Systems / genetics
Cell Differentiation / genetics
Genetic Engineering
Humans
Induced Pluripotent Stem Cells* / metabolism