First ddPCR™ –Based Assays to Quickly Quantify CRISPR Efficiency
Bio-Rad Laboratories has announced the launch of ddPCR™ Genome Edit Detection Assays, the first tests to characterize edits generated by CRISPR-Cas9 or other genome editing tools using Bio-Rad’s Droplet Digital PCR (ddPCR) technology. Users can specify their sequences and order assays through Bio-Rad’s Digital Assay Site.
Successful genome editing is a rare outcome that depends substantially on experimental conditions including type of cells used, transfection method, target sequence, and many other factors. Editing stem cell genomes, for example, typically succeeds in less than 5% of cells.
Current methods for assessing genome edit efficiency, including next-generation sequencing (NGS) and high resolution melt analysis, present drawbacks in cost, time, simplicity, and sensitivity. With NGS, for example, turnaround time is often several weeks and analysis is complex and costly, making NGS less than optimal for routine screening. Additionally, maximum sensitivity requires researchers to sequence the same DNA many times, which can greatly inflate time and expense.
Bio-Rad’s ddPCR technology is well suited for the task of genome editing, empowering scientists to precisely evaluate the efficiency of their experiment in less time and at lower cost than with any other method. By partitioning samples into thousands of droplets, ddPCR technology increases the signal-to-noise ratio, which allows users to quantify extremely rare edits — even frequencies of 0.5% and from as little as 5 ng of genomic DNA — and still obtain the results within one day.
Edit Detection Enables New Applications for Gene Editing
Bio-Rad’s offering follows a wave of research in ddPCR methods to measure genome-editing efficiency. In 2015, Science published a ddPCR-based method used at Duke University to detect edits designed to treat Duchenne muscular dystrophy in mice. The following year, research papers in Nature Protocols and Scientific Reports further detailed more ddPCR strategies for assessing genome editing outcomes. In a study released this year, researchers at the Broad Institute of MIT and Harvard used ddPCR to verify the sensitivity of an innovative CRISPR-based nucleic acid detection platform.
“Genome editing holds great promise not only in basic and applied science, but particularly in the area of gene therapy,” said Boris Fehse, a professor of cell and gene therapy in Hamburg, Germany, and an author of the Nature Protocols paper. “Therapeutic applications require reliable, highly sensitive, and easy-to-perform assays to monitor efficiency as well as potential side effects. In my opinion, digital PCR represents an ideal tool fulfilling these requirements.”