Characterizing SaCas9 and SaCas9[D10E] tolerance to mismatches using Directed Evolution Using Fluorescence Activated Cell Sorting (DEUFACS)
Abstract
CRISPR-Cas9 nucleases are commonly used for genome editing but are limited by their off- target effects, which must be reduced for therapeutic use. Current strategies for engineering higher fidelity variants do not directly compare on- versus off-target activity. I establish Directed Evolution Using Fluorescence Activated Cell Sorting (DEUFACS), a bacterial dual- fluorophore system for characterizing and altering the sequence preferences of nucleases. I aimed to use DEUFACS to identify SaCas9 variants with increased on-target and decreased off-target activity. I found that D10E substitution in the RuvC domain of SaCas9 improved discrimination and was superior to other RuvC active site variants containing substitutions with similar properties. Using tiled transversion substrates, SaCas9[D10E] preferentially cleaved the on-target versus most transversions. However, D10E did not show enriched on-target activity using a D10 site-saturation library. My results suggest DEUFACS is a powerful tool for the simultaneous profiling of nuclease activity on desired on-target versus unwanted off- target substrates.