Open Access Repository

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

Stellon, D, Nguyen Tran, MT, Talbot, J, Chear, S, Mohd Khalid, MKN, Pebay, A, Vickers, JC ORCID: 0000-0001-5671-4879, King, AE ORCID: 0000-0003-1792-0965, Hewitt, AW ORCID: 0000-0002-5123-5999 and Cook, AL ORCID: 0000-0003-1770-7910 2021 , 'CRISPR/Cas-Mediated Knock-in of genetically encoded fluorescent biosensors into the AAVS1 locus of human-induced pluripotent stem cells' , Methods in Molecular Biology , doi: 10.1007/7651_2021_422.

Full text not available from this repository.

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 Type: Article
Authors/Creators: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
Keywords: CRISPR, gene editing, stem cell, biosensor, AVS1, CRISPR/Cas, fluorescent biosensor, induced pluripotent stem cell, live cell imaging
Journal or Publication Title: Methods in Molecular Biology
Publisher: Humana Press
ISSN: 1064-3745
DOI / ID Number: 10.1007/7651_2021_422
Copyright Information:

Copyright 2021 Springer Science+Business Media, LLC

Related URLs:
Item Statistics: View statistics for this item

Actions (login required)

Item Control Page Item Control Page
TOP