Guide RNAs inflated measurements of target RNA knockdown in human cells

Guide RNAs inflated measurements of target RNA knockdown in human cells

On September 1, Nature Biotechnology published a paper describing an error in experiments with RNA-targeting CRISPR systems. The authors studied HEK293T, a laboratory human cell line, using three such systems: Csm, PspCas13b, and CasRx. The guide RNA that directs CRISPR to its target could interfere with RT-qPCR, an assay that estimates RNA abundance from DNA copies synthesized from the RNA.

In these experiments, researchers extract RNA from cells. Reverse transcriptase uses the RNA to produce DNA copies, which qPCR then counts. Fewer copies usually indicate that the amount of target RNA has decreased.

The authors identified the problem by comparing conventional Csm with a variant that could no longer cleave RNA. When qPCR measured a region spanning the guide RNA binding site, the inactive variant also appeared to cause a substantial reduction. However, the amount of the fluorescent protein mCherry remained unchanged in the same cells.

After purification, the guide RNA can remain bound to its target. If reverse transcriptase must cross the binding site or begin synthesis nearby and upstream of it, the bound guide RNA can prevent the enzyme from completing the DNA copy. qPCR then detects fewer copies and overestimates the extent of RNA knockdown.

When qPCR measured an RNA region downstream of the binding site, the reduction persisted only with active enzymes. The authors observed this pattern with Csm and both Cas13 systems.

Researchers often measure a region spanning the target site to avoid counting RNA fragments that remain after cleavage. A 2011 study had already identified a similar problem involving short interfering RNAs. In a 2023 study of Csm, some targets were also assessed by imaging RNA within cells and by RNA sequencing.

For measurements using a conventional reverse transcriptase, the authors recommend assaying a region downstream of the guide RNA binding site. If the assay must span the binding site, researchers can use uMRT, an enzyme that displaces molecules bound to RNA. Comparing the result with protein abundance or with RNA visualized inside cells can help distinguish genuine loss of the target from a measurement error.