A DNA-guided CRISPR platform could make RNA detection and control more stable, scalable, and precise, opening new routes for diagnostics, transcriptome engineering, and future therapeutic research.
Study: DNA-guided CRISPR–Cas12 for cellular RNA targeting. Image Credit: CI Photos / Shutterstock
In a recent study published in the journal Nature Biotechnology, researchers developed a new version of clustered regularly interspaced short palindromic repeats (CRISPR) technology. The system uses deoxyribonucleic acid (DNA)-based guides called ΨDNA to target ribonucleic acid (RNA), and can be paired with conventional CRISPR RNA guides to edit DNA using the same Cas12 enzyme. This approach improves upon existing systems that use fragile RNA guides. The system accurately detected hepatitis C virus (HCV) RNA in clinical samples after an amplification-based workflow. The DNA-guided systems also helped control and modify RNA molecules inside cells, highlighting the potential for more stable, scalable, and precise RNA diagnostics and experimental RNA-targeting applications.
DNA-Guided CRISPR RNA Targeting Background
CRISPR acts like a highly accurate ‘search-and-edit’ tool that scientists can program to find specific genetic material in cells. Different CRISPR systems can either edit DNA, target RNA, or help detect viruses and diseases with high precision. These systems typically depend on small RNA guides that direct the CRISPR proteins to the correct target. These guides, however, are expensive to make and difficult to store because they break down easily. Scientists have tried using more stable DNA guides, but most CRISPR systems do not perform well with DNA alone, warranting further research.
ΨDNA Cas12 Study Design
In the present study, researchers designed ΨDNA, a DNA-based guide that helps Cas12 systems find and interact with RNA, while conventional CRISPR RNA guides can still be used for DNA editing.
The team tested different CRISPR enzymes and identified two that worked best with ΨDNA. They then fine-tuned the system to recognize different types of RNA. These included small regulatory RNAs, viral RNA, and normal cellular RNA. To assess effectiveness, they performed several laboratory experiments to study binding interactions between CRISPR components and RNA.
To investigate whether the novel technology could work in real-world diagnostics, the researchers obtained blood samples from people with HCV infections and healthy individuals. They then tested the system using fluorescence-based assays after reverse transcription, polymerase chain reaction, and T7 transcription steps generated RNA targets for detection. Researchers subsequently assessed intracellular activity. To do so, they introduced the CRISPR enzyme AsCas12a along with ΨDNA guides into human embryonic kidney (HEK) cells in the laboratory. The guides were designed to target a red fluorescent molecule called mCherry, which acted like a visible marker. Successfully blocking the target RNA would reduce red fluorescence in the cells.
After these assessments, the team tested whether the same CRISPR enzyme could perform two tasks simultaneously. These included reducing specific RNA molecules while also editing DNA. This investigation would clarify whether combining ΨDNA with conventional CRISPR guides in a single Cas12a system could simultaneously silence gene expression at the RNA level and permanently edit genes at the DNA level.
HCV Detection and RNA Targeting Results
ΨDNA helped CRISPR enzymes locate and interact with RNA targets. The new DNA guides performed especially well with two CRISPR enzymes, AsCas12a and Cas12i1. They demonstrated high accuracy, activating only in the presence of the correct RNA target. The system remained robust across more than 14 different microRNAs tested. The optimized design achieved strong activity across a guide length of 16–28 nucleotides.
In addition to diagnostic accuracy, the system demonstrated high sensitivity. In clinical samples, involving 20 HCV-positive and 20 HCV-negative serum samples, the system achieved 100% diagnostic accuracy. Detection limits ranged from 1 to 10 picomolar. The platform also remained stable and effective under different laboratory conditions, suggesting promise for RNA-based diagnostic applications. The 5′ untranslated region guide detected all positive samples across HCV genotypes 1a and 1b, while the E2 guide was selective for genotype 1a.
The new system could effectively turn down disease-relevant or unwanted gene activity in living cells. ΨDNA guides reduced target RNA levels by 50-70% in standard experiments and by 80-95% in optimized cell systems. These included several human cell lines derived from cervical cancer (HeLa), liver cancer (HepG2), and breast cancer (MCF-7) cells. At a mechanistic level, the system blocked the cell’s protein-synthesizing machinery and triggered the cell’s own RNA degradation pathways to destroy the target RNA molecules, rather than directly cutting RNA itself.
There were far fewer unintended effects than the commonly used RNA-targeting enzyme RfxCas13d. The platform could also silence up to four RNA targets at once with over 70% efficiency. These findings highlight the system’s potential for improved specificity, although safety and therapeutic suitability would require further preclinical validation.
Dual RNA Control and DNA Editing
As an example of simultaneous RNA and DNA activity, the system edited the C-C chemokine receptor 5 (CCR5) gene while also reducing RNA levels in the same cells. This dual activity required co-delivery of ΨDNA for RNA targeting and of a conventional crRNA for DNA editing, using a single Cas12a effector. Researchers also added proteins, such as ribonuclease H1 (RNase H1) and methyltransferase-like protein 3 (METTL3). These proteins helped destroy RNA more effectively or chemically modify RNA in precise ways.
CRISPR Diagnostics and Gene Therapy Implications
The findings position ΨDNA-guided systems as cheaper, more stable, and versatile alternatives to existing RNA-guided technologies. By using DNA guides that are easier to prepare and more durable, the system offers a practical strategy to improve scalability for medical and research applications. Since the technology can control and modify RNA in addition to editing DNA, it could advance future gene-therapy research and personalized-medicine approaches to develop better treatments for infections, cancer, and genetic disorders.
Further research using animal and disease models is nevertheless required for validation and translation into clinical practice. The authors also note that ΨDNA guides cannot currently be genetically encoded or expressed from plasmids, which remains an important delivery consideration.
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