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

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

doi:10.64898/2026.08.16.745121

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bioRxiv · 10.64898/2026.08.16.745121

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

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

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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.

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Huang, B., Orosco, C., Stewart, E., Balaraju, M., Elhabashy, Y. B., Jain, P. K.. 2026-08-19. Guide RNA binding induces reverse transcription bias during RT-qPCR analysis of RNA-targeting CRISPR/Cas systems. https://doi.org/10.64898/2026.08.16.745121

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