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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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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