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

ABSTRACT: 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.

SUBMITTER: Ludwik KA 

PROVIDER: S-EPMC10382939 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature