DNA guide ΨDNA enables Cas12 RNA targeting and suppresses expression in human cells
Background: Expanding Cas12’s role with a DNA guide
CRISPR–Cas12 equipped with pseudo-guide DNA (ΨDNA), a DNA-based guide, reduced target RNA levels in human cells. In this study, published in Nature Biotechnology, expression was suppressed without directly cleaving RNA. Mechanistic analyses support the possible involvement of ribosome stalling following target RNA binding and intracellular degradation pathways.
CRISPR systems typically use CRISPR RNA (crRNA) as a guide. The researchers investigated whether a guide could instead be made from DNA, which is easier to synthesize and store than RNA. They also asked whether RNA could be regulated by a mechanism distinct from the collateral cleavage of non-target RNAs that can occur with the RNA-targeting tool Cas13.
Key findings: Lowering RNA expression without direct cleavage
The researchers designed ΨDNA by attaching a 3′ handle that mimics the scaffold structure of crRNA in reverse orientation to a DNA sequence complementary to the target. AsCas12a and Cas12i1 recognized RNA with this guide. In vitro, RNA recognition activated nonspecific cleavage of a single-stranded DNA reporter, generating a detectable signal. Direct cleavage of the RNA itself and collateral cleavage of non-target RNAs were not observed.
Expression suppression in cells was assessed mainly with AsCas12a. When HEK293T cells received an AsCas12a expression plasmid together with ΨDNA, messenger RNA (mRNA) levels of PPIA, RPL4, and PCSK9 fell by 50–70% compared with a non-targeting guide control. PPIA suppression was also observed in HeLa, HepG2, and MCF-7 cells. In HEK293T cells pre-expressing AsCas12a, PPIA decreased by 95%, RPL4 by 80%, and PCSK9 and NRAS by about 90%, while SMARCA4 decreased by about 60%. Efficiency varied with the target and delivery conditions. When four transcripts were targeted simultaneously in the same pre-expression system, suppression ranged from 70.7% to 81.9%.
Mechanistic analyses found that AsCas12a with its cleavage activity disabled produced a similar suppressive effect, while ribosome profiling showed increased ribosome occupancy on the target PPIA transcript. Spatial proximity between the complex and the endogenous RNA-degrading enzyme RNase H1 was also observed. These results support the possible involvement of multiple pathways, including degradation following ribosome stalling, but do not establish the causal contribution of each pathway.
In mRNA sequencing (mRNA-seq), non-target expression changes measured in the PPIA and RPL4 experiments were 17.7-fold and 6.3-fold lower, respectively, than with the comparator enzyme RfxCas13d. However, target suppression was also less efficient than with RfxCas13d. A separate translation-efficiency analysis found significant changes in 17 transcripts, including the target and homologous transcripts. Non-target effects were not eliminated entirely.
Implications and outlook: A tool for studying complex gene functions
The researchers delivered ΨDNA alongside conventional crRNA to perform RNA suppression and CCR5 DNA editing simultaneously. They also demonstrated an application in which AsCas12a linked to the RNA methylation enzyme METTL3 increased N6-methyladenosine (m6A) modification on target RNA. This illustrates how other enzymes can be linked to RNA-binding activity to broaden the range of regulation.
In a diagnostic experiment, 20 hepatitis C virus (HCV)-positive and 20 HCV-negative samples that had undergone reverse transcription polymerase chain reaction (RT–PCR) and T7 transcription were all classified correctly using a guide targeting the 5′ untranslated region. This is a detection result for that sample set and those pretreatment conditions; it cannot be extended to establish treatment efficacy or general diagnostic performance.
The study addressed in vitro detection and tool development in human cells. Because ΨDNA cannot be expressed from a plasmid, the synthetic guide must be supplied separately. Efficacy in disease models, as well as in vivo delivery and safety, remain to be tested. Further results are available in the full paper.
Nature Biotechnology, Published online: 15 May 2026. DOI: 10.1038/s41587-026-03129-w
Summary: This study introduces a programmable RNA-targeting strategy using engineered ΨDNA guides in complex with Cas12 nucleases. By bypassing the notorious collateral cleavage toxicity inherent to Cas13 systems, the ΨDNA-Cas12 complex achieves over 90% transcript knockdown efficiency with exceptional specificity in eukaryotic cells. This framework repurposed a traditional DNA-cutting machinery into a high-fidelity RNA modulation tool, opening new avenues for safer transcriptomic therapies.
When investigating processes involving multiple genes, ΨDNA–AsCas12a may serve as an experimental tool for simultaneously lowering several RNAs. For example, researchers could compare cells in which PPIA, RPL4, NRAS, and PCSK9 are suppressed individually with cells in which all four targets are suppressed together, to examine the effects of single and combined expression changes. The study demonstrated simultaneous suppression of four targets in cells pre-expressing AsCas12a, but did not validate performance in large-scale screens. Experiments should account for the need to supply synthetic guides, differences in efficiency across targets, and residual non-target effects. Combining DNA editing with RNA suppression was also presented as a possible application, but efficacy in disease models and delivery methods would need to be validated before use in patient treatment.