Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations

Abstract

Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus (PfAgo) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow PfAgo selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process. The integrated single-tube system achieved great efficiency for enriching rare mutations compared with a divided system separating the cleavage and amplification. Thus, A-Star enables easy detection and quantification of 0.01% rare mutations with ≥5500-fold increase in efficiency. The feasibility of A-Star was also demonstrated for detecting oncogenic mutations in solid tumor tissues and blood samples. Remarkably, A-Star achieved simultaneous detection of multiple oncogenes through a simple single-tube reaction by orthogonal guide-directed specific cleavage. This study demonstrates a supersensitive and rapid nucleic acid detection system with promising potential for both research and therapeutic applications.

Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens

“…Unlike CRISPR‐Cas systems, pAgos do not require a specific motif for target recognition, which makes pAgos more versatile. Furthermore, pAgos with the precise guide‐directed endonuclease activity were also applied to enrich low‐frequency mutant alleles by specific degradation of wild‐type nucleic acids (Q. Liu, Guo, et al, 2021; Song et al, 2020). Besides nucleic acid detection, Pf Ago‐based platform could generate programmable DNA‐guided artificial restriction enzymes, which were capable of recognizing and cleaving DNA sequences at almost any sites with the assistance of designed DNA guides (Enghiad & Zhao, 2017).…”

A Hyperthermophilic Argonaute From Ferroglobus placidus With Specificity on Guide Binding Pattern

“…An artificial restriction enzyme platform based on Pyrococcus furiosus Argonaute ( Pf Ago) was developed to recognize and cleave DNA sequences at virtually any arbitrary site ( Enghiad and Zhao, 2017 ). Furthermore, thermophilic pAgos have been recently used as programmable tools for specific target detection and rare single nucleotide variant enrichment in molecular diagnosis applications ( He et al, 2019 ; Xun et al, 2019 ; Liu et al, 2021a ; Song et al, 2020 ).…”

Hairpin structure facilitates multiplex high-fidelity DNA amplification in real-time PCR

“…As the need to detect mutations with VAF low to or even lower than 0.01% VAF has increased for both basic research and clinical diagnosis, researchers and scientists have been exploring the solutions. Scientists have reported integrating CRISPR 31 or Argonaute-mediated 32,33 cleavage of targets with non-HiFi PCR to achieve rare mutation detection with improved sensitivity (LoD ≤0.01% VAF). Allele-specific BDA (As-BDA) 34 was also capable of detecting and profiling multiplexed mutations down to 0.01% VAF within the single tube using Taq DNA polymerases by implementing allele-specific TaqMan probes.…”