Argonaute-mediated system for supersensitive and multiplexed detection of rare mutations

The ability to detect rare mutations has revolutionized diagnosis and monitoring of tumors, but is limited by the shortage of sensitive, cost-effective and high coverage methods for identification of extremely low abundant mutations. Here, we establish a single-tube multiplex PCR system by employing thermophilic Argonaute-derived DNA-guided nuclease for highly efficient rare mutation detection, referred to as A-Star (Argonaute-directed specific target enrichment and detection), that combines the selective cleavage of the wild type DNA in the DNA denaturation step and the followed amplification of mutant DNA during PCR. A-Star enables easy detection and quantitation of rare mutations originally as low as 0.01% in allele frequency with a ⩾ 5500-fold efficiency. We also demonstrate the feasibility of A-Star for detecting oncogenic mutations in complex biological systems such as solid tumors tissues and blood samples. Remarkably, A-Star could achieve the detection of multiple oncogenic genes simultaneously through a simple single-tube reaction. Taken together, our work illustrates a supersensitive and rapid nucleic acid detection system, thereby extending the utility for both research and therapeutic applications. The detection of rare polymorphic alleles is becoming increasingly relevant for the early diagnosis and monitoring of a variety of tumors1,2. The clinical detection methods for rare mutations, including single nucleotide variations (SNVs) and insertion/deletion (indel) mutations, that are currently available involve either sequencing or PCR3, 4. Compared with authentication by sequencing DNA fragments, allele-specific diagnostic PCR is not only simpler and timesaving but also practical and effective5-7. Therefore, the detection of extremely rare variant alleles within a complex mixture of DNA molecules has attracted increasing attention aimed at solving the technical problems associated with the strict requirements for both a precise single-nucleotide resolution and simple multiplex detection, especially in the detection of circulating tumor DNA in the circulated tumor DNA of patients8-10. Recently, a variety of rare mutation detection methods based on endonucleases with sequence-specific catalytic capabilities11,12, particularly the enzymes of the CRISPR/Cas systems, have been developed13-15. These strategies are primarily based on Cas9 cleavage activity16,17 or the collateral cleavage activity of Cas12 or Cas13a effector18,19. Once combined with the pre-enrichment of recombinase polymerase amplification (RPA), 5% SNV allele can be achieved in the Cas12/13-enzymes20-22. However, the major limitations of these approaches are different cutting preferences needed for the vicinity of target sequence cleavage and the designed reporter system20-24, which restricts the utility of these methods for the detection of a wide spectrum of target genes, also poses a great challenge in the multiplexed detection that needed tedious screening for orthogonal CRISPR enzymes in targeting multiple genomic loci simultaneously24. Additionally, the system including the synthesized guide RNA, the target RNA converted from the pre-amplified ctDNA by T7 transcription, and Cas enzymes reaction causes the quite high-cost and technical inconvenience. One potential means of circumventing these limitations is to take advantage of the unique properties of thermophilic Argonaute (Ago) proteins, which do not require a prerecognition motif and instead specifically cleave target nucleic acids via the base-pairing with the small guide nucleic acids25,26, such as reported from Pyrococcus furiosus, Thermus thermophiles, Methanocaldococcus jannaschii and Methanocaldococcus fervens, etc27-30. The revealed capacity of Agos from hyperthermophile Pyrococcus furiosus (PfAgo), which had been biochemistry characterized and designed as a programmable DNA-guided artificial restriction enzyme27,31, caught our great attention with functioning precise cleavage of the target DNA (tDNA) between positions 10 and 11 of the pairing guide DNA (gDNA) at temperatures as high as 95 °C. We assume that the powerful catalytic ability of PfAgo for the single strand DNA (ssDNA) may allow it to cleave the unwinding double strands DNA (dsDNA) during the first denaturing step of PCR. A deep understanding and diligent exploration of PfAgo, through how gDNA-directed Ago discriminates the wild-type (WT) DNA and rare variant allele precisely, how to combine PfAgo into a PCR reaction to enrich rare mutations efficiently, and whether PfAgo can deal with multiplex detection rapidly, etc., may facilitate the development of new protocols that enable the rare mutation detection with great precision and efficiency. Here, we report the establishment of A-Star (Ago-directed Specific Target enrichment and detection), a simple but supersensitive single-tube PCR system that takes advantage of PfAgo to specifically cleave WT sequences in the denaturing step of PCR, leading to progressive and rapid enrichment of rare variant-containing alleles during the PCR process. As a proof of concept, the compatibility of PfAgo with PCR is evaluated by introducing the well-designed gDNA directly in a single tube, which demonstrate the highly efficiency by orders of magnitude compared with a divided system between the WT cleavage and variant alleles amplification. The mocked cell-free DNAs (cfDNAs) and clinical human samples are tested for target oncogene enrichment. Furthermore, a multiplexed variant alleles system for one-pot reliable detection with single-digit copies is demonstrated by multi-gDNA directed PfAgo system in the orthogonal manner. Our investigation indicates that the A-Star method we have developed provides a novel, supersensitive, rapid and inexpensive way in rare variant alleles detection, which exhibits great potential from applications in basic research to potential new diagnostic tools and clinical utility.