High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis.
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.
['Pham|Quang T|QT|', 'Raad|Suleen|S|', 'Mangahas|Chenicka-Lyn|CL|', "M'Callum|Marie-Agnès|MA|", 'Raggi|Claudia|C|', 'Paganelli|Massimiliano|M|']
D000818:Animals D064113:CRISPR-Cas Systems D002460:Cell Line D004247:DNA D000072669:Gene Editing D057166:High-Throughput Screening Assays D006801:Humans D057026:Induced Pluripotent Stem Cells D051379:Mice D009154:Mutation D009691:Nucleic Acid Denaturation D020641:Polymorphism, Single Nucleotide
genome editing induced pluripotent stem cells CRISPR-Cas9 human iPSCs