University Of Tasmania

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

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.


Batten Disease Support and Research Association


Publication title

Methods in Molecular Biology

Article number

online ahead of print


online ahead of print




Wicking Dementia Research Education Centre


Humana Press

Place of publication

United States

Rights statement

Copyright 2021 Springer Science+Business Media, LLC

Repository Status

  • Restricted

Socio-economic Objectives

Clinical health not elsewhere classified