Katarzyna A. Ludwik, Narasimha Telugu, Sandra Schommer, Harald Stachelscheid, and Sebastian Diecke
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 (afford- able, 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 auto- mated cell isolation followed by clone screening.
STAR Protocols 4, 102406, September 15, 2023
