Lwacas13a

The assay mix contained LwaCas13a (GenScript) and on occasion LbaCas12a (New England Biolabs). Concentration varied with experiment: 1× Assay Loading Reagent (Fluidigm), 69U T7 RNA Polymerase mix (Lucigen) and crRNA concentration varied with experiment for a total volume of 16 μl per reaction. See below for details pertaining to each mCARMEN panel.

Assay mixes were prepared in 16 μL volumes for each unique crRNA target consisting of 1 μL of 50 U/μL NxGen T7 Polymerase (Biosearch Technologies 30223–1), 2 μL of 800 nM LwaCas13a (Genscript), 1 μL of a 1.6 μM target-specific Cas13 crRNA, 8 μL of 2X Fluidigm Assay Loading Buffer, and 4 μL of nuclease-free water. 1.5X sample premixes were created with 2.4 μL of 10X T7 Buffer (Biosearch Technologies F88905–1, Hoddesdon, United Kingdom), 3.2 μL of 7.5X sample buffer, 0.2 μL of murine RNase inhibitor (NEB M0314S, Ipswich, MA), 0.96 μL of 25 mM rNTP mix (NEB N0466S, Ipswich, MA), 0.12 μL of 100 μM 6U FAM reporter (IDT), 0.48 μL of 50X ROX reference dye (Thermo Fisher Scientific 12223012, Waltham, MA), 1.20 μL of 20X GE buffer, and 7.44 μL of nuclease-free water. 7.5X Sample Buffer solutions were made in 5 mL aliquots with 375 μL of Tris-HCl, 75 μL of 5M NaCl, 37.5 μL of 1M MgCl₂, 750 mg of PEG-8000, and 4512.5 μL of nuclease-free water. Sample premixes were added to pre-amplified sample mixtures at a 2:1 ratio of premix to sample.

The 0.4 μl HEPES (1 M; Gibco, cat. no. 15630–106), 0.2 μl MgCl₂ (1 M; Invitrogen, cat. no. AM9530G), 2 μl Ribonucleotide Solution Mix (25 mM; NEB, cat. no. N0466L), 2 μl LwaCas13a (1 μM; Genscript), 1 μl Cas13a-crRNA (1 μM), 0.1 μl T7 RNA polymerase (50 U/μl, Lucigen, cat. no. 30223–1), 1 μl Murine RNase inhibitor (40 U/μl; NEB, cat. no. M0314L), and 0.2 μl ssRNA reporter (100 μM, FAM-BHQ) were added to 20 μl reaction mixture. The reaction mixture was gently mixed and immediately placed on ice until use. Next, a 2 μl amplification product was added to an 18 μl reaction mixture for CRISPR/Cas13a detection assay. The detection assay was performed at 37°C for 20 min, while the fluorescence was monitored as described above. The fluorescence images were acquired every 2.5 min.