Figure 4.

CRISPRD orthogonality. (a) Orthogonality of the CRISPR/Cas activation. Each Cas effector was tested for its ability to form an RNP with gRNAs from other effectors. (b) Orthogonality and specificity of the target. Each RNP was incubated with all three different targets to test whether targets could activate incorrect RNPs. (c) Orthogonality of the excitation and emission of fluorophores. Three different CRISPRD reactions were carried out to detect RNase P and the cleavage of reporters carrying three different fluorophores. Each reaction was read through three different channels in the qPCR machine (Bio-Rad). (d) Orthogonality of the reporter preference. Activated Cas effectors were incubated with different reporters to check if there was any aberrant cleavage of reporters. All reactions were conducted in triplicate, and the mean signal is visualized by heat maps. Panels (a–c) had 1 ng of template per reaction, while panel (d) had 4 ng of template per reaction.