New Johns Hopkins Study Identifies Pervasive RT-qPCR Artifact in CRISPR Knockdown Studies and How UltraMarathonRT® Solves It

Guide RNAs from RNA-targeting CRISPR systems remain bound to their target mRNA after total RNA preparations and suppress conventional reverse transcription. uMRT's strand-displacing reverse transcriptase restores accurate knockdown quantification.

BRANFORD, Conn., Sept. 2, 2026 /PRNewswire/ -- RNAConnect, a life science company providing next-generation tools to the RNA community, today highlighted findings in Nature Biotechnology from the Wu Lab at Johns Hopkins University School of Medicine showing that a pervasive reverse transcription (RT) artifact inflates apparent knockdown (KD) efficiency in RNA-targeting CRISPR systems.

While investigating the RNA-targeting CRISPR system Csm, the Johns Hopkins team found that a catalytically inactive, RNase-dead Csm enzyme still produced strong apparent knockdown by RT-qPCR. After confirming that no RNA cleavage or protein-level knockdown was occurring, the researchers traced the false signal to the RT-qPCR kits used for the assay. The kits use conventional retroviral reverse transcriptase (e.g. MMLV RT) to copy RNA into cDNA, but these enzymes lack strand displacement activity, so cDNA synthesis was directly blocked by the tightly bound guide RNA (gRNA) itself. With bound gRNA preventing the enzyme from reading through the guide-binding region, any RT-qPCR amplicon spanning or sitting just upstream of the gRNA site was suppressed as a result, artificially indicating knockdown of the target gene. The effect held across every RNA-targeting CRISPR system tested, including Csm, PspCas13b, and CasRx. While no formal analysis is available, it is estimated that this artifact is pervasive throughout the literature as a cut-spanning RT-qPCR is a preferred method of quantitation.

The researchers also found a simple, direct fix. Because the artifact is a reverse transcription problem, not a biological one, replacing the retroviral RT with a highly processive, strand-displacing reverse transcriptase resolved the artifact. Using UltraMarathonRT (uMRT), a group II intron-encoded enzyme, the team saw no apparent knockdown in any of the RNase-dead controls, at any amplicon position, confirming that uMRT displaces and reads through the bound gRNA region.

"What began as a systematic comparison of RNA-targeting CRISPR systems led us to uncover a fundamental flaw in how the field has been measuring the efficiency of these enzymes," said Leslie Watkins, Johns Hopkins School of Medicine, Biochemistry, Cell and Molecular Biology. "We didn't set out to discover this quantification artifact, but we now know these inaccurate measurements have likely inflated reported knockdown efficiencies across the entire field. It turns out conventional RT kits aren't as reliable as many researchers thought, me included. UltraMarathonRT was just the solution we needed to get accurate measurements and move these technologies toward research and therapeutic applications"

uMRT produced consistent, orthogonally-validated knockdown values regardless of amplicon placement, restoring confidence in the workhorse RT-qPCR assays that researchers prefer for RNA-targeting CRISPR KO/KD experiments.

"It's inspiring to see a negative control experiment transform into this impactful key result for the whole RNA-targeting CRISPR field," Jason Underwood, PhD, Vice President, Technology Development at RNAConnect. "This group definitively shows that the guide RNA remains bound to its target mRNA even after an RNA prep and that stops a retroviral reverse transcriptase in its tracks. We were happy to see that uMRT's strong strand-displacement activity solve that directly and deliver accurate quantification."

About RNAConnect
RNAConnect is a life science company based in Branford, Connecticut, providing next-generation research tools for the RNA community. The UltraMarathonRT product line supports full-length cDNA synthesis, RT-PCR, RT-qPCR, and RNA sequencing workflows, with substantially improved detection and quantification of long and structurally complex transcripts. Learn more at www.rnaconnect.com.

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SOURCE RNAConnect