Programmable Nucleases Research Articles

Argonaute-Based Nucleic Acid Detection Technology: Advantages, Current Status, Challenges, and Perspectives.

Rapid and accurate detection is a prerequisite for precise clinical diagnostics, ensuring food safety, and facilitating biotechnological applications. The Argonaute system, as a cutting-edge technique, has been successfully repurposed in biosensing beyond the CRISPR/Cas system (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins), which has been extensively researched, but recognition of PAM sequences remains restricted. Argonaute, as a programmable and target-activated nuclease, is repurposed for fabricating novel detection methods due to its unparalleled biological features. In this comprehensive review, we initially elaborate on the current methods for nucleic acid testing and programmable nucleases, followed by delving into the structure and nuclease activity of the Argonaute system. The advantages of Argonaute compared with the CRISPR/Cas system in nucleic acid detection are highlighted and discussed. Furthermore, we summarize the applications of Argonaute-based nucleic acid detection and provide an in-depth analysis of future perspectives and challenges. Recent research has demonstrated that Argonaute-based biosensing is an innovative and rapidly advancing technology that can overcome the limitations of existing methods and potentially replace them. In summary, the implementation of Argonaute and its integration with other technologies hold promise in developing customized and intelligent detection methods for nucleic acid testing across various aspects.

CRISPRoffT: comprehensive database of CRISPR/Cas off-targets.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) programmable nuclease system continues to evolve, with in vivo therapeutic gene editing increasingly applied in clinical settings. However, off-target effects remain a significant challenge, hindering its broader clinical application. To enhance the development of gene-editing therapies and the accuracy of prediction algorithms, we developed CRISPRoffT (https://ccsm.uth.edu/CRISPRoffT/). Users can access a comprehensive repository of off-target regions predicted and validated by a diverse range of technologies across various cell lines, Cas enzyme variants, engineered sgRNAs (single guide RNAs) and CRISPR editing systems. CRISPRoffT integrates results of off-target analysis from 74 studies, encompassing 29 experimental prediction techniques, 368 guide sequences, 226164 potential guide and off-target pairs and 8840 validated off-targets. CRISPRoffT features off-target data from different CRISPR approaches (knockout, base editing and prime editing) applied under diverse experimental conditions, including 85 different Cas/guide RNA (gRNA) combinations used across 34 different human and mouse cell lines. CRISPRoffT provides results of comparative analyses for individual guide sequences, genes, cell types, techniques and Cas/gRNA combinations under different conditions. CRISPRoffT is a unique resource providing valuable insights that facilitate the safety-driven design of CRISPR-based therapeutics, inform experimental design, advance the development of computational off-target prediction algorithms and guide RNA design algorithms.

The role of prokaryotic argonautes in resistance to type II topoisomerases poison ciprofloxacin.

Argonaute proteins are programmable nucleases found in all domains of life. Eukaryotic argonautes (eAgos) participate in genetic regulation, antiviral response, and transposon silencing during RNA interference. Prokaryotic argonautes (pAgos) are much more diverse than eAgos and have been implicated in defense against invading genetic elements. Recently, it was shown that pAgos protect bacterial cells from a topoisomerase poison ciprofloxacin, raising a possibility that they may play a role in DNA replication and/or repair. Here, we discuss possible models of pAgo-mediated ciprofloxacin resistance. We propose that pAgos could (i) participate in chromosome decatenation as a backup to topoisomerases; (ii) participate in the processing of DNA repair intermediates formed after topoisomerase poisoning, or (iii) induce SOS response that generally affects DNA repair and antibiotic resistance. These hypotheses should guide future investigations of the involvement of pAgos in the emergence of resistance to ciprofloxacin and, possibly, other antibiotics.

Precision Genome Editing Techniques in Gene Therapy: Current State and Future Prospects.

Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.

Enrichment of Allelic Editing Outcomes by Prime Editing in Induced Pluripotent Stem Cells.

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.

Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Current Status of Biomedical Products for Gene and Cell Therapy of Recessive Dystrophic Epidermolysis Bullosa.

This detailed review describes innovative strategies and current products for gene and cell therapy at different stages of research and development to treat recessive dystrophic epidermolysis bullosa (RDEB) which is associated with the functional deficiency of collagen type VII alpha 1 (C7) caused by defects in the COL7A1 gene. The use of allogenic mesenchymal stem/stromal cells, which can be injected intradermally and intravenously, appears to be the most promising approach in the field of RDEB cell therapy. Injections of genetically modified autologous dermal fibroblasts are also worth mentioning under this framework. The most common methods of RDEB gene therapy are gene replacement using viral vectors and gene editing using programmable nucleases. Ex vivo epidermal transplants (ETs) based on autologous keratinocytes (Ks) have been developed using gene therapy methods; one such ET successively passed phase III clinical trials. Products based on the use of two-layer transplants have also been developed with both types of skin cells producing C7. Gene products have also been developed for local use. To date, significant progress has been achieved in the development of efficient biomedical products to treat RDEB, one of the most severe hereditary diseases.

Structural and mechanistic insights into a mesophilic prokaryotic Argonaute.

Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic Agos (pAgos) have gained increasing research interest due to their broad range of potential applications, yet their molecular mechanisms remain poorly understood. Here, we present seven cryo-electron microscopy structures of Kurthia massiliensis Ago (KmAgo) in various states. These structures encompass the steps of apo-form, guide binding, target recognition, cleavage, and release, revealing that KmAgo employs a unique DDD catalytic triad, instead of a DEDD tetrad, for DNA target cleavage under 5'P-DNA guide conditions. Notably, the last catalytic residue, D713, is positioned outside the catalytic pocket in the absence of guide. After guide binding, D713 enters the catalytic pocket. In contrast, the corresponding catalytic residue in other Agos has been consistently located in the catalytic pocket. Moreover, we identified several sites exhibiting enhanced catalytic activity through alanine mutagenesis. These sites have the potential to serve as engineering targets for augmenting the catalytic efficiency of KmAgo. This structural analysis of KmAgo advances the understanding of the diversity of molecular mechanisms by Agos, offering insights for developing and optimizing mesophilic pAgos-based programmable DNA and RNA manipulation tools.

An optimised CRISPR Cas9 and Cas12a mutagenesis toolkit for Barley and Wheat

BackgroundCRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community.ResultsWe identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately.ConclusionBased on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency.

Targeted mutagenesis in Arabidopsis and medicinal plants using transposon-associated TnpB.

The programmable nuclease TnpB is significantly smaller than Cas9, can edit genes in medicinal plants, including Artemisia annua, Salvia miltiorrhiza, Scutellaria baicalensis, Isatis indigotica, and Codonopsis pilosula, and has potential uses in molecular breeding to enhance crop yield and quality.

CRISPR/Cas9 opens new horizon of crop improvement under stress condition

Plants are exposed to a myriad of stresses, stemming from abiotic and biotic sources, significantly threatening agricultural productivity. The low crop yield, coupled with the global burden of population has resulted in the scarcity of quality food, exacerbating socio-economic issues like poverty, hunger, and malnutrition. Conventional breeding methods for the generation of stress-tolerant plants are time-consuming, limit genetic diversity, and are not sustainable for the consistent production of high-yielding crops. In recent years, the use of high-throughput, genome editing (GE) technique has revolutionized the crop-improvement paradigm, ushering greater prospects for agricultural progress. Among these tools, the Clustered regularly interspaced short palindromic repeat (CRISPR), and its associated nuclease protein Cas9, have appeared as a ground-breaking technology, allowing precise knockout (KO), upregulation, and downregulation of target gene expression. Apart from its high efficacy and speed, this programmable nuclease offers exceptional specificity with minimal off-target effects. Here in, we aim to review the latest findings on the application of the CRISPR/Cas9 genome editing tool for generating resilience in plants against environmental stresses.

Single-base tiled screen unveils design principles of PspCas13b for potent and off-target-free RNA silencing

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer–target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

Transposase-assisted target-site integration for efficient plant genome engineering

The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome ‘parasites’, transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3–5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6–9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10–12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean—a major global crop in need of targeted insertion technology. We have engineered a TE ‘parasite’ into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.

Hybrid Cas12a Variants with Relaxed PAM Requirements Expand Genome Editing Compatibility.

Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), which can efficiently recognize -2* A/G PAMs that are barely recognized by Cas12a-type proteins and their mutants. Finally, we demonstrated that the DNase-inactivated ehCas12a RRVR base editor (dehCas12a RRVR-BE) was capable of targeting noncanonical PAMs in vivo and disease-related loci for potential therapeutic applications. Overall, our findings highlight the modular design and reconfiguration of Cas proteins for enhanced functionality.

A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

Towards DNA-free CRISPR/Cas9 genome editing for sustainable oil palm improvement.

The CRISPR/Cas9 genome editing system has been in the spotlight compared to programmable nucleases such as ZFNs and TALENs due to its simplicity, versatility, and high efficiency. CRISPR/Cas9 has revolutionized plant genetic engineering and is broadly used to edit various plants' genomes, including those transformation-recalcitrant species such as oil palm. This review will comprehensively present the CRISPR-Cas9 system's brief history and underlying mechanisms. We then highlighted the establishment of the CRISPR/Cas9 system in plants with an emphasis on the strategies of highly efficient guide RNA design, the establishment of various CRISPR/Cas9 vector systems, approaches of multiplex editing, methods of transformation for stable and transient techniques, available methods for detecting and analyzing mutations, which have been applied and could be adopted for CRISPR/Cas9 genome editing in oil palm. In addition, we also provide insight into the strategy of DNA-free genome editing and its potential application in oil palm.

Prokaryotic Argonaute nuclease cooperates with co-encoded RNase to acquire guide RNAs and target invader DNA.

Argonautes are an evolutionary conserved family of programmable nucleases that identify target nucleic acids using small guide oligonucleotides. In contrast to eukaryotic Argonautes (eAgos) that act on RNA, most studied prokaryotic Argonautes (pAgos) recognize DNA targets. Similarly to eAgos, pAgos can protect prokaryotic cells from invaders, but the biogenesis of guide oligonucleotides that confer them specificity to their targets remains poorly understood. Here, we have identified a new group of RNA-guided pAgo nucleases and demonstrated that a representative pAgo from this group, AmAgo from the mesophilic bacterium Alteromonas macleodii, binds guide RNAs of varying lengths for specific DNA targeting. Unlike most pAgos and eAgos, AmAgo is strictly specific to hydroxylated RNA guides containing a 5'-adenosine. AmAgo and related pAgos are co-encoded with a conserved RNA endonuclease from the HEPN superfamily (Ago-associated protein, Agap-HEPN). In vitro, Agap cleaves RNA between guanine and adenine nucleotides producing hydroxylated 5'-A guide oligonucleotides bound by AmAgo. In vivo, Agap cooperates with AmAgo in acquiring guide RNAs and counteracting bacteriophage infection. The AmAgo-Agap pair represents the first example of a pAgo system that autonomously produces RNA guides for DNA targeting and antiviral defense, which holds promise for programmable DNA targeting in biotechnology.

Abstract 7246: Enhancing PDAC therapy through in vivo correction of KRAS G12D using enIscB-directed base editor combined with ICB

Abstract In this study, we aim to investigate the therapeutic potential of in vivo genetic therapy using AAV-mediated adenine base editor (ABE) delivery for pancreatic ductal adenocarcinoma (PDAC) carrying the KRASG12D mutation (PDACG12D), and further explore its potential when combined with immune checkpoint blockade (ICB). The KRASG12D mutation is a dominant genetic alteration representing a highly sought-after and effective therapeutic target in PDAC. However, the in vivo genetic therapy for KRASG12D in PDAC has been rarely explored. Programmable base editors have been shown to install genetic mutations precisely and have significant therapeutic value, providing a promising strategy for genetic therapy of PDACG12D. Here, we engineered ABEs derived from various programmable nucleases, considering the protospacer adjacent motif (PAM) or target adjacent motif (TAM) sequence. The hypercompact A8e-enIscB demonstrated the highest editing efficiency in HEK293TG12D and PANC-1 cell lines. Subsequently, A8e-enIscB was packaged into a single-AAV system and delivered to humanized mice subcutaneously transplanted with patient-derived organoidG12D (PDOG12D). We found the delayed tumor progression with BE-based genetic therapy. Additionally, a substantial editing efficiency in tumor tissues and lower off-target editing in normal tissues was observed. By comparing single-cell transcriptome sequencing data between BE-treated and non-treated samples, we observed a reprogrammed immune microenvironment characterized by elevated infiltration of cytotoxic CD8+ T cells and NK cells, as well as decreased recruitment of suppressive PMN-MDSCs, Tregs and TAMs upon correction of KRASG12D. Furthermore, AAV-directed ABE genetic therapy could significantly sensitize PDAC to ICB therapy (anti-PD-1, anti-CTLA4) in humanized PDAC PDO/PDX mouse models. Accordingly, an ongoing work in which the genetic therapy combined with ICB phase IA investigator-initialized trial will be designed to evaluate the safety of this regimen. In summary, our study demonstrates the efficiency and safety of innovative genetic therapy for correcting the KRASG12D mutation in PDAC. Furthermore, we demonstrated the therapeutic potential of combining this genetic therapy with ICB. Citation Format: Chao Yang, Qingxiao Fang, Tianxing Zhou, Yongjie Xie, Jun Yu, Jihui Hao. Enhancing PDAC therapy through in vivo correction of KRAS G12D using enIscB-directed base editor combined with ICB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7246.

Abstract 5768: Proximity labeling with molecular signal sensing of oncogenic PIK3CA

Abstract PIK3CA mutations are major oncogenic drivers for oncogenesis. Mutant PIK3CA continuously activates the PI3K pathway, transmitting oncogenic signals within the transformed cells. Despite decades of efforts focused on targeting oncogenic PIK3CA for cancer therapy, the precise molecular and functional machinery remains incomplete due to the molecular complexity of the PI3K pathway. Mapping of proximity proteins in the oncogenic PI3K pathway and identifying mutant signals will signify a deep understanding of oncogenesis and a broad range of therapeutic applications. In this study, we hypothesize that PIK3CA mutant cancer cells establish a unique molecular program networking with distinct proteins, which differs from that of cancer cells with wild-type (WT) PIK3CA. To test this hypothesis, we have developed a novel bioengineering approach to decode protein-protein interactions of oncogenic PIK3CA and identify key molecular partners, potentially providing therapeutic vulnerabilities in cancer cells. First, we applied the proximity labeling technique, based on the fusion of a promiscuous protein ligase to a targeting protein of interest (POI), featuring proximity-dependent biotinylation of interacting and neighboring proteins. The promiscuous biotin ligases, BioID and BioID2, were utilized to integrate WT PIK3CA or gain-of-function hotspot mutations in at E545K or H1047R residues. To optimize the labeling protocol and biotinylation conditions, the BioID and BioID2 plasmids were fused with each POI, incorporating multiple combinations of protein tags, including myc, HA, and red fluorescence protein. Lamin A was used as the positive control, and a total of 15 plasmids were constructed in pcDNA3.1 mammalian expression vector. The sizes of the plasmids and the gene sequences were validated by PCR amplification and Sanger sequencing. Next, we employed a programmable nuclease, the prokaryotic Argonaute (pAgo), assisted by fluorescence resonance energy transfer technology, to detect oncogenic signals derived from mutant PIK3CA in cell-free conditions. To test our methods, we engineered two cancer cell lines (DHSA-1426 and COSB) derived from naturally occurring hemangiosarcoma in dogs, to induce PIK3CA H1047R mutations using CRISPR/Cas9. Our technical approach enables temporal and spatial molecular sensing of oncogenic PIK3CA and its functional interactome in living cancer cells by integrating high-resolution molecular mapping with a pAgo-based detection system. Our ongoing work involves the standardization and optimization of proximity labeling for oncogenic PIK3CA across ontogenetically distinct cancer cells. Furthermore, we will determine the sensitivity and specificity of the integrated approach to achieve proximity labeling with programmable detection of molecular signals from oncogenic PIK3CA. Citation Format: Rong Li, Donghee Lee, Md Abdullah, Jong Hyuk Kim. Proximity labeling with molecular signal sensing of oncogenic PIK3CA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5768.

Thermal activation of Argonaute nuclease enables one-pot multiplex detection of viruses

Advances in programmable nucleases such as CRISPR-associated proteins (Cas) and Argonaute (Ago), combined with isothermal amplification, have made point-of-care testing more accessible than before. However, the non-specific binding of the nuclease to nucleic acid results in incompatibility issues between the amplification and nuclease systems, substantially limiting the feasibility of one-step workflow. Herein, we propose a temperature control solution based on thermotolerant Pyrococcus furiosus Ago (PfAgo) beads. The use of immobilized PfAgo can effectively prevent interference with loop-mediated isothermal amplification at 65 °C and accurately identifies amplicons when activated at 95 °C. Following optimization, a sensitivity of 0.6 copies/μL was achieved within 45 min, and the system demonstrated high specificity with no cross-reactivity with 21 other pathogens. Additionally, multiplex detection was designed for herpesvirus sensing, with 86.4% and 100% agreement for positive and negative samples, respectively. Our research presents an effective one-pot method for combining amplification and cleavage through the use of thermo-controllable nucleases, significantly improving the clinical applicability of diagnostic techniques dependent on programmable nucleases.