A pervasive RT–qPCR artifact inflates RNA knockdown by RNA-targeting CRISPR

Knockdown efficiency of RNA-targeting CRISPR systems is commonly measured by quantitative polymerase chain reaction with reverse transcription (RT–qPCR). Here we discovered that guide RNAs copurify during RNA extraction and inhibit RT–qPCR for amplicons spanning or upstream of the guide RNA binding site, resulting in overestimation of knockdown efficiency across all CRISPR systems tested. We recommend using a processive reverse transcriptase with strong strand-displacing activity, with orthogonal methods, to ensure accurate quantification when using RT–qPCR. A quantification artifact confounds CRISPR-mediated RNA knockdown.