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.