A pervasive RT-qPCR artifact inflates RNA knockdown by RNA-targeting CRISPR.

Guide RNAs co-purify during RNA extraction and cause a significant overestimation of RNA knockdown efficiency in RT-qPCR measurements across all tested CRISPR systems.

• Why it matters: Accurate assessment of RNA knockdown is crucial for evaluating CRISPR system performance, but the presence of guide RNAs introduces artifacts that can mislead interpretations and hinder progress.
• What they did: The study identified that guide RNAs inhibit RT-qPCR when amplicons overlap or are upstream of their binding sites, and recommends using processive reverse transcriptases with strand-displacing activity along with orthogonal validation methods.
• The result: Implementing these recommendations will improve the accuracy of RNA quantification, enabling more reliable evaluation of CRISPR-mediated knockdown and advancing gene editing research.