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, efficien...

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Detalles Bibliográficos
Autores principales: Ludwik, Katarzyna A., Telugu, Narasimha, Schommer, Sandra, Stachelscheid, Harald, Diecke, Sebastian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10382939/
https://www.ncbi.nlm.nih.gov/pubmed/37481731
http://dx.doi.org/10.1016/j.xpro.2023.102406
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author Ludwik, Katarzyna A.
Telugu, Narasimha
Schommer, Sandra
Stachelscheid, Harald
Diecke, Sebastian
author_facet Ludwik, Katarzyna A.
Telugu, Narasimha
Schommer, Sandra
Stachelscheid, Harald
Diecke, Sebastian
author_sort Ludwik, Katarzyna A.
collection PubMed
description 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.
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spelling pubmed-103829392023-07-30 ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs Ludwik, Katarzyna A. Telugu, Narasimha Schommer, Sandra Stachelscheid, Harald Diecke, Sebastian STAR Protoc Protocol 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. Elsevier 2023-07-22 /pmc/articles/PMC10382939/ /pubmed/37481731 http://dx.doi.org/10.1016/j.xpro.2023.102406 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Ludwik, Katarzyna A.
Telugu, Narasimha
Schommer, Sandra
Stachelscheid, Harald
Diecke, Sebastian
ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title_full ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title_fullStr ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title_full_unstemmed ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title_short ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs
title_sort assured-optimized crispr protocol for knockout/snp knockin in hipscs
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10382939/
https://www.ncbi.nlm.nih.gov/pubmed/37481731
http://dx.doi.org/10.1016/j.xpro.2023.102406
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