Expanding Cas12a Activity Control with an RNA G‐Quadruplex at the 5′ end of CRISPR RNA DOI Creative Commons
Wenjuan Huang,

Jiaqi Wang,

Cheng Wang

et al.

Advanced Science, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 25, 2024

Precise control of Cas12a activity is essential for the improvement detection limit clinical diagnostics and minimization errors. This study addresses challenge controlling activity, especially in context nucleic acid where inherent incompatibility between isothermal amplification CRISPR reactions complicates accurate diagnostics. An RNA G-quadruplex (RG4) structure at 5' end crRNA introduced to modulate accurately without need chemical modifications. The results indicate that presence RG4 does not significantly impact Cas12a's cleavage but can be controlled by stabilizers, enabling suppression subsequent restoration with potential precise control. Moreover, use expanded incorporating it into split crRNA, introducing directly direct repeat (DR) region, tailored regulation different targets matching various Spacer regions. Additionally, a light-controlled one-pot method activating developed, thereby enhancing accuracy sensitivity samples. showcases pioneering manipulating streamlining diagnostics, paving way advances testing.

Language: Английский

Direct repeat region 3′ end modifications regulate Cas12a activity and expand its applications DOI Creative Commons
Wei Zhang, Yulong Zhong, Jiaqi Wang

et al.

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(3)

Published: Jan. 24, 2025

Abstract CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3′ end modifications and de-modifications, which can regulate LbCas12a’s cis- trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA at DR on functionality. find that temporary inhibitory function Cas12a be reactivated by modification corresponding substances, such alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), exonucleases, ultraviolet radiation, glycosylases, greatly expand scope application Cas12a. Clinical demonstrated promising results in ALP, AFP, trace Epstein–Barr virus detection compared gold standard methods. Our research provides valuable insights into regulating activity significantly expands potential clinical targets, paving way future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.

Language: Английский

Citations

0
RNA aptamer-based CRISPR-Cas12a system for enhanced small molecule detection and point-of-care testing DOI
Yuanfang Liu,

Jiaqi Wang,

Gangfeng Cui

et al.

International Journal of Biological Macromolecules, Journal Year: 2025, Volume and Issue: 303, P. 140675 - 140675

Published: Feb. 4, 2025

Language: Английский

Citations

0
Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation DOI
Guangrong Zou,

Penglong Si,

Jiaqi Wang

et al.

ACS Sensors, Journal Year: 2025, Volume and Issue: 10(3), P. 2073 - 2079

Published: March 1, 2025

5-Hydroxymethylcytosine (5hmC) plays a key role in the DNA demethylation process and serves as stable epigenetic marker human genome which is closely associated with disease progression, particularly diabetes, colorectal cancer, liver cancer. However, convenient sensitive methods for detecting quantifying 5hmC are scarce, especially complex biological environments. Herein, novel attempt at hypersensitive quantitative detection of was presented. A multifunctional photosensitive probe therefore introduced specific labeling, enrichment, elution 5hmC-DNA. Combining isothermal assay leveraging rolling circle amplification Cas12a accurate recognition, we achieved trace amounts level 11 fM. Global measured various samples using little 10 ng input by real-time PCR instrument. The reported approach imposed no sequence restrictions, demonstrating promising potential modified bases nucleic acids within environments, such blood, urine, saliva samples.

Language: Английский

Citations

0
Expanding Cas12a Activity Control with an RNA G‐Quadruplex at the 5′ end of CRISPR RNA DOI Creative Commons
Wenjuan Huang,

Jiaqi Wang,

Cheng Wang

et al.

Advanced Science, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 25, 2024

Precise control of Cas12a activity is essential for the improvement detection limit clinical diagnostics and minimization errors. This study addresses challenge controlling activity, especially in context nucleic acid where inherent incompatibility between isothermal amplification CRISPR reactions complicates accurate diagnostics. An RNA G-quadruplex (RG4) structure at 5' end crRNA introduced to modulate accurately without need chemical modifications. The results indicate that presence RG4 does not significantly impact Cas12a's cleavage but can be controlled by stabilizers, enabling suppression subsequent restoration with potential precise control. Moreover, use expanded incorporating it into split crRNA, introducing directly direct repeat (DR) region, tailored regulation different targets matching various Spacer regions. Additionally, a light-controlled one-pot method activating developed, thereby enhancing accuracy sensitivity samples. showcases pioneering manipulating streamlining diagnostics, paving way advances testing.

Language: Английский

Citations

3