High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.
CRISPR-Cas9
genome editing
human iPSCs
induced pluripotent stem cells
Journal
Cytotherapy
ISSN: 1477-2566
Titre abrégé: Cytotherapy
Pays: England
ID NLM: 100895309
Informations de publication
Date de publication:
10 2020
10 2020
Historique:
received:
14
01
2020
revised:
31
05
2020
accepted:
01
06
2020
pubmed:
10
8
2020
medline:
13
2
2021
entrez:
10
8
2020
Statut:
ppublish
Résumé
Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach. Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays. Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.
Sections du résumé
BACKGROUND AND AIMS
Genome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach.
METHODS
Here we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays.
RESULTS AND CONCLUSIONS
Our data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.
Identifiants
pubmed: 32768274
pii: S1465-3249(20)30754-4
doi: 10.1016/j.jcyt.2020.06.008
pii:
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
536-542Subventions
Organisme : CIHR
Pays : Canada
Informations de copyright
Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.