Ultrasensitive Multi-Species Detection of CRISPR-Cas9 by a Portable Centrifugal Microfluidic Platform

The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitides, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.