Figure S1.

Individual crRNAs quantitatively detect SARS-CoV-2 RNA, related to Figure 1

(A) Cas13a RNPs made individually with each N gene crRNA (final RNP complex concentration of 100 nM) were tested against extracted SARS-CoV-2 viral RNA. Background fluorescence by the individual RNP in the absence of target RNA is shown as “RNP Only.” Raw fluorescence values over 2 h is shown. Data are represented as mean ± SD of three technical replicates.

(B and C) Cas13a reaction rate is linearly proportional to the target RNA concentration.

(B) The reaction rate of Cas13a was measured by adding a range of concentrations of IVT N gene RNA to reactions that contain 100 nM of Cas13a RNP with crRNA 2 and 400 nM of polyU reporter (error bars indicate the standard deviation of triplicate measurements). The reaction rate is determined by fitting a linear curve to the data (black curves).

(C) The reaction rate of Cas13a for a range of IVT N gene RNA concentrations as measured in Figure S1B but with crRNA 4 used in place of crRNA 2.

(D) Cas13a reaction rate – either with crRNA 2 or crRNA 4 – scales linearly with the target IVT RNA concentration (R² = 0.990 for crRNA 2 and 0.996 for crRNA 4). Assuming that Cas13a enzymatic activity can be described by the Michaelis-Menten kinetics model, and that the amount of IVT RNA sets the upper limit of active Cas13a RNP, we predict the ratio of active Cas13a to IVT RNA in for K_cat = 600/s and K_M = 1 μM or 3 μM, which is the range of K_M previously found for Cas13b (Slaymaker et al., 2019).