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.