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

Authors

Huang, B., Orosco, C., Stewart, E., Balaraju, M., Elhabashy, Y. B., Jain, P. K.

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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 19 Aug 2026.

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