CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy

The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.