Abstract
The realization of the full potential of human pluripotent stem cells (hPSCs), including human induced PSCs (iPSC), relies on the ability to precisely edit their genome in a locus-specific and multiplex manner. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) serve as a guide for the endonuclease Cas9 (CRISPR-associated protein 9) to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. CRISPR/Cas9–mediated editing has become the gold standard for precise genome manipulation as it offers a unique, versatile, and limitless tool for fast, robust, and efficient genome editing. Here, we provide a protocol to successfully generate gene knockout and/or knockin iPSCs. We include detailed information on the design of guide RNAs (gRNAs), T7 endonuclease assay to detect on-target CRISPR/Cas9 editing events, DNA electroporation of the iPSCs with a ribonucleoprotein complex, and single-cell cloning steps for the selection of the genome-edited iPSC clones.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Castano J et al (2017) Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the "safe harbor" AAVS1 locus. Stem Cell Res 21:137–140
Petazzi P et al (2020) Robustness of catalytically dead Cas9 activators in human pluripotent and mesenchymal stem cells. Mol Ther Nucleic Acids 20:196–204
Jinek M et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355
Zuris JA et al (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33(1):73–80
Ramakrishna S et al (2014) Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res 24(6):1020–1027
Kim S et al (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24(6):1012–1019
Acknowledgments
We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social La Caixa for their institutional support. Financial support for the study in PM’s laboratory was obtained from the Catalunya Goverment (SGR-AGAUR and PERIS 2017-2019) and the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2019-108160-R, RTC-2017-6367-1). Financial support in AS’s laboratory was obtained from MINECO, the European Development Regional Fund (FEDER, PGC2018-098626-B100) and the Merck Salud Foundation. AS is a recipient of a Ramón y Cajal contract (RYC-2016-19962). PM is a member of the Spanish Cell Therapy Network (TerCEL, ISCIII).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media New York
About this protocol
Cite this protocol
Petazzi, P., Menéndez, P., Sevilla, A. (2020). CRISPR/Cas9–Mediated Gene Knockout and Knockin Human iPSCs. In: Nagy, A., Turksen, K. (eds) Induced Pluripotent Stem (iPS) Cells. Methods in Molecular Biology, vol 2454. Humana, New York, NY. https://doi.org/10.1007/7651_2020_337
Download citation
DOI: https://doi.org/10.1007/7651_2020_337
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2118-9
Online ISBN: 978-1-0716-2119-6
eBook Packages: Springer Protocols