Phosphate‐Engineered DNA Reporters Boost CRISPR‐Cas12a Trans‐Cleavage for Rapid and Sensitive Nucleic Acid Detection
4 affiliations
- 1Department of Biomedical Engineering University of Connecticut Health Center Farmington Connecticut USA
- 2Department of Biomedical Engineering University of Connecticut Storrs Connecticut USA
- 3Department of Pathology and Laboratory Medicine University of Connecticut Health Center Farmington Connecticut USA
- 4Department of Medicine Division of Infectious Diseases University of Connecticut Health Center Farmington Connecticut USA
Abstract
CRISPR–Cas12a molecular diagnostics rely on target‐activated collateral cleavage of single‐stranded DNA (ssDNA) reporters to generate fluorescence signals, yet the mechanisms governing reporter substrate processing remain poorly understood. Here we show that Cas12a trans‐cleavage involves both endonucleolytic cleavage and exonucleolytic trimming. We further demonstrate that terminal phosphate modification of ssDNA substrates accelerates substrate cleavage activity and modulates trans‐cleavage kinetics, thereby enhancing fluorescence output. Guided by this mechanistic insight, we engineered fluorophore–quencher ssDNA reporters bearing terminal phosphate modifications and identified a 3′‐phosphate‐engineered reporter that enables faster signal generation and higher fluorescence intensity. Incorporation of the engineered reporter into the CRISPR–Cas12a system enabled the development of Phos‐CRISPR (Phosphate‐engineered DNA reporters boost CRISPR), a rapid, sensitive, and specific nucleic acid detection platform that achieves results within 20 min, approximately threefold faster than conventional CRISPR assays. Applied to herpes simplex virus 1 detection, the Phos‐CRISPR combined with recombinase polymerase amplification achieved a limit of detection of 20 aM and accurately identified clinical swab samples. These findings provide a general framework for understanding Cas12a trans‐cleavage activity and establish a mechanistically informed strategy for enhancing next‐generation CRISPR diagnostics.
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BibTeX
@article{yang2026phosphateengineered,
title = {{Phosphate‐Engineered DNA Reporters Boost CRISPR‐Cas12a Trans‐Cleavage for Rapid and Sensitive Nucleic Acid Detection}},
author = {Rui Yang and Chong Guo and Minjie Pei and Jiongyu Zhang and Xin Guan and Shuo Zhang and Xingye Liu and Kurt T. Schalper and Lori Avery and Rocco LaSala and David Banach and Changchun Liu},
journal = {Advanced Science},
year = {2026},
month = oct,
doi = {10.1002/advs.78445},
url = {https://doi.org/10.1002/advs.78445},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Phosphate‐Engineered DNA Reporters Boost CRISPR‐Cas12a Trans‐Cleavage for Rapid and Sensitive Nucleic Acid Detection
AU - Rui Yang
AU - Chong Guo
AU - Minjie Pei
AU - Jiongyu Zhang
AU - Xin Guan
AU - Shuo Zhang
AU - Xingye Liu
AU - Kurt T. Schalper
AU - Lori Avery
AU - Rocco LaSala
AU - David Banach
AU - Changchun Liu
PY - 2026
DA - 2026/10/25
JO - Advanced Science
DO - 10.1002/advs.78445
UR - https://doi.org/10.1002/advs.78445
AB - CRISPR–Cas12a molecular diagnostics rely on target‐activated collateral cleavage of single‐stranded DNA (ssDNA) reporters to generate fluorescence signals, yet the mechanisms governing reporter substrate processing remain poorly understood. Here we show that Cas12a trans‐cleavage involves both endonucleolytic cleavage and exonucleolytic trimming. We further demonstrate that terminal phosphate modification of ssDNA substrates accelerates substrate cleavage activity and modulates trans‐cleavage kinetics, thereby enhancing fluorescence output. Guided by this mechanistic insight, we engineered fluorophore–quencher ssDNA reporters bearing terminal phosphate modifications and identified a 3′‐phosphate‐engineered reporter that enables faster signal generation and higher fluorescence intensity. Incorporation of the engineered reporter into the CRISPR–Cas12a system enabled the development of Phos‐CRISPR (Phosphate‐engineered DNA reporters boost CRISPR), a rapid, sensitive, and specific nucleic acid detection platform that achieves results within 20 min, approximately threefold faster than conventional CRISPR assays. Applied to herpes simplex virus 1 detection, the Phos‐CRISPR combined with recombinase polymerase amplification achieved a limit of detection of 20 aM and accurately identified clinical swab samples. These findings provide a general framework for understanding Cas12a trans‐cleavage activity and establish a mechanistically informed strategy for enhancing next‐generation CRISPR diagnostics.
SN - 2198-3844
ER -