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CRISPR/Cas-Mediated Knock-in of genetically encoded fluorescent biosensors into the AAVS1 locus of human-induced pluripotent stem cells

Citation

Stellon, D and Nguyen Tran, MT and Talbot, J and Chear, S and Mohd Khalid, MKN and Pebay, A and Vickers, JC and King, AE and Hewitt, AW and Cook, AL, CRISPR/Cas-Mediated Knock-in of genetically encoded fluorescent biosensors into the AAVS1 locus of human-induced pluripotent stem cells, Methods in Molecular Biology Article online ahead of print. ISSN 1064-3745 (2021) [Refereed Article]


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DOI: 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.

Item Details

Item Type:Refereed Article
Keywords:CRISPR, gene editing, stem cell, biosensor, AVS1, CRISPR/Cas, fluorescent biosensor, induced pluripotent stem cell, live cell imaging
Research Division:Biological Sciences
Research Group:Biochemistry and cell biology
Research Field:Cell development, proliferation and death
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Stellon, D (Mr David Stellon)
UTAS Author:Nguyen Tran, MT (Mr Minh Nguyen Tran)
UTAS Author:Talbot, J (Dr Jana Talbot)
UTAS Author:Chear, S (Ms Sueanne Chear)
UTAS Author:Mohd Khalid, MKN (Mr Mohd Mohd Khalid)
UTAS Author:Vickers, JC (Professor James Vickers)
UTAS Author:King, AE (Professor Anna King)
UTAS Author:Hewitt, AW (Professor Alex Hewitt)
UTAS Author:Cook, AL (Associate Professor Tony Cook)
ID Code:146575
Year Published:2021
Deposited By:Wicking Dementia Research and Education Centre
Deposited On:2021-09-14
Last Modified:2021-09-14
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