ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs


CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening. Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Authors Ludwik KA, Telugu N, Schommer S, Stachelscheid H, Diecke S
Journal STAR protocols
Publication Date 2023 Sep 15;4(3):102406
PubMed 37481731
PubMed Central PMC10382939
DOI 10.1016/j.xpro.2023.102406

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