Guide RNA binding induces reverse transcription bias during RT-qPCR analysis of RNA-targeting CRISPR/Cas systems

Abstract

RNA-targeting CRISPR systems are commonly evaluated by RT-qPCR, but guide RNA binding can confound these measurements. We show that crRNA alone produces apparent knockdown without reducing target RNA abundance, whereas RNA sequencing remains unbiased and reveals guide-associated transcriptomic perturbations. A simple RNA denaturation step before reverse transcription restores accurate RT-qPCR quantification, providing practical guidance for RNA-targeting CRISPR analysis and guide design.