bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.03.27.645718

A Rapid and Highly Sensitive CRISPR Assay Utilizing Cas12a Orthologs for the Detection of Novel Duck Reovirus

Wang, Y.·Fu, l.·Li, s.·Tao, d.·Gong, P.·Yang, Y.·Ruan, J.·Xie, S.·Wang, C.·He, D.
View original source ↗Export metadata ↓

Abstract

The novel duck reovirus (NDRV) disease presents a significant threat to the poultry industry due to the absence of effective therapeutic measures. As a result, there is an urgent need to develop innovative rapid diagnostic methods for early virus detection. In this study, we developed a Rapid Visual CRISPR Assay to detect the NDRV S3 gene using novel Cas12a orthologs. Specifically, we compared the performance of two candidates, Gs12-16 and Gs12-18, in detecting the NDRV S3 gene to identify a highly sensitive and efficient CRISPR-based diagnostic method. Our results demonstrated that both Gs12-16 and Gs12-18 exhibited strong cis- and trans-cleavage activities for classical "TTTV" protospacer adjacent motif (PAM)-containing targets in vitro, although they required different reaction temperatures. Notably, Gs12-18 showed relatively higher activity for dsDNA targets compared to Gs12-16, indicating that Gs12-18 is more suitable for CRISPR-based nucleic acid detection applications. To leverage these properties, we integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to establish a LAMP-CRISPR/Gs12-18-mediated method for detecting the NDRV S3 gene. This approach enables highly sensitive and visually detectable on-site identification of the NDRV S3 gene, achieving a sensitivity of 38 copies per reaction. Our LAMP-CRISPR/Gs12-18-based method can be utilized for highly sensitive detection of NDRV nucleic acids.

Source connections

Explore related subjects

synthetic biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Wang, Y., Fu, l., Li, s., Tao, d., Gong, P., Yang, Y., Ruan, J., Xie, S., Wang, C., He, D.. 2025-03-30. A Rapid and Highly Sensitive CRISPR Assay Utilizing Cas12a Orthologs for the Detection of Novel Duck Reovirus. https://doi.org/10.1101/2025.03.27.645718

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Reassessing the contribution of the histone H3 tail to KRAB-DNMT3L-mediated epigenetic silencing

Neumann et al. introduced CHARM, a compact epigenetic silencer in which a histone H3 tail fused to DNMT3L was proposed to recruit and stimulate endogenous DNMT3A, enabling durable gene repression without a fused DNMT3A catalytic domain. Here, we evaluated the contribution of the H3 tail in independent reporter and endogenous-gene contexts. In an SNRPN reporter system, a KRAB-DNMT3L-dCas9 construct lacking the H3 tail displayed silencing kinetics comparable to CRISPRcharm Kv2, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this silencing. Similarly, after transient delivery of editor mRNAs to HEK293T cells, CRISPRcharm Kv2 did not consistently outperform the corresponding H3-tail-free construct at three endogenous loci, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this activity. These observations suggest that the engineered H3 tail does not confer a general functional advantage within the KRAB-DNMT3L-dCas9 architecture under the conditions tested.

synthetic biology↗
bioRxiv2026

Coupling a developmental promoter to CRISPR interference for Wnt pathway regulation in human pluripotent stem cells

While directed differentiation of human pluripotent stem cells commonly relies on the timed delivery of extracellular factors, these uniform treatments often yield heterogeneous responses across cell populations. Linking intracellular gene regulation directly to an emerging developmental state offers a complementary strategy to coordinate these differentiation signals from within the cell. Here, we explored this approach by coupling a T/Brachyury promoter to CRISPR interference targeting CTNNB1, which encodes the canonical Wnt signaling mediator {beta}-catenin. A T-promoter EGFP reporter line exhibited transiently increased activity during early differentiation, supporting the use of this promoter as a developmentally responsive input. We then combined the promoter with dCas9-KRAB and a CTNNB1-targeting guide RNA. Cas9-mediated integration was accompanied by indels at the CTNNB1 target site, whereas a Cas12a-mediated integration strategy yielded clones with no indels detected by ICE analysis. During differentiation, the selected T-dCas9 CTNNB1 clone showed reduced CTNNB1 expression and attenuated induction of Wnt associated genes. Together, these findings provide a proof of concept for combining a developmental promoter with a programmable intracellular regulator and identify a strategy for separating circuit integration from unintended target-site editing. This modular approach provides a foundation for developing genetic interventions whose expression is linked to developmental state.

synthetic biology↗
bioRxiv2026

Thermodynamic, Electrochemical and Practical Constraints on Electromicrobial Formate Assimilation

Electromicrobial production (EMP) technologies aim to combine renewable electricity, CO2, and engineered microbes to make energy-dense molecules at efficiencies exceeding photosynthesis. CO2 can be electrochemically reduced to formate, which is far easier to handle at the bench than H2 or an electrode, but formate carries only two electrons per carbon against the six in a biofuel. The remaining electrons must come from oxidizing additional formate, from H2 oxidation, or from extracellular electron uptake (EEU), and no rigorous comparison of these options coupled to the choice of carbon assimilation pathway currently exists. We calculate upper-limit efficiencies for butanol production by six carbon assimilation pathways, each paired with all three electron delivery mechanisms, using electrochemical parameters drawn from a survey of the recent literature. Electrical to butanol energy conversion efficiencies range from 35.5 to 51.7%, corresponding to solar-to-fuel efficiencies of 11.7 to 17%, so even the least efficient route exceeds the 8% theoretical ceiling of algal photosynthesis. The serine variant of the reductive glycine pathway reaches an electrical energy conversion efficiency of when using H2 oxidation, within 1.9 points of the most efficient pathway, and is the only high-efficiency option that tolerates O2. This makes an EMP system that combines electron delivery by formate coupled with the serine variant of reductive glycine pathway highly attractive, as it presents few barriers to rapid, iterative engineering in the lab, and a high theoretical ceiling. Drawing both carbon and electrons from formate costs 6.2 points against H2 at a state-of-the-art whole-cell voltage (2.2 V). However, this small penalty is amplified three-fold by any rise in the CO2-to-formate cell voltage, and reaches 13.5 points at the highest whole-cell voltages reported for scaled-up CO2-to-formate electrolyzers, where formate-only operation falls to 11.2 electrical-to-fuel and 3.7% solar-to-fuel efficiency, below the ceiling of photosynthesis, against 24.7 and 8.1% for H2 (only just above algal photosynthesis). Our choice between a formate-only system and one coupled to H2 oxidation or EEU therefore depends on our belief about the trajectory of CO2 reduction technology. If whole-cell voltages continue to fall at the rate of the past decade, formate alone is the right target, and the simplicity of its workflow is bought at low cost. However, if that improvement plateaus, the electron delivery mechanism must be swappable, and a system should be designed from the outset so that it can be. At the US Department of Energy SunShot target of 2 cents per kilowatt hour, the electricity to make a US gallon of butanol costs $1.40 for a formate-only system at the state of the art, rising to $5.45 at the highest scaled-up electrolyzer voltage reported, against $1.23 and $2.47 for formate and H2 system.

synthetic biology↗