Multiplex CRISPR Cas13a Assay

Droplets experiments were performed as previously described^{20 (link)} using the DropArray platform. Briefly, detection sets were prepared at 2.2X final concentration of 45 nM purified Leptotrichia wadei Cas13a, 22.5 nM total crRNA concentration (1.25 nM of each of 18 crRNA for pooled detection; 22.5 nM of one crRNA for individual detection), 500 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μl murine RNase inhibitor (New England Biolabs) in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, pH 7.3) with 1 mM NTPs and 0.6 μl T7 polymerase mix (New England Biolabs). Amplified samples were diluted 1:10 into nuclease-free water supplemented with 13.2 mM MgCl₂ prior to barcoding with fluorescent dyes. 20 µL of each sample and detection mix were then emulsified into droplets using a BioRad QX200 droplet generator using fluorous oil (3 M 7500, 70 µL) containing 2% 008-fluorosurfactant (RAN Biotechnologies.) Droplets were pooled and loaded into a DropArray chip, imaged for content identification by fluorescent barcode identification, droplet pairs merged and then incubated at 37 ˚C, and imaged for assay signal at 0, 1 h, and 3 h time points relative to the start of the incubation.