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CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes -mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1–10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26–50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11–25 bp) to moderate deletions (26–50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs. Key message We demonstrate that engineered exonuclease-fused CRISPR systems enable efficient, target-specific large deletions in soybean, providing a valuable tool to enhance functional genomics and accelerate trait discovery in crops.

A fluorescence biosensor for determination of aflatoxin B.sub.1 (AFB.sub.1) based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB.sub.1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB.sub.1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with a determination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheat samples. The stability, accuracy, and specificity of the method demonstrated the applicability for real sample analysis. Graphical abstract

In this study, we developed an aptamer-based fluorescent sensing platform for the detection of ochratoxin A (OTA) based on RecJf exonuclease-assisted signal amplification and interaction between graphene oxide (GO) and the OTA aptamer (OTA-apt). After optimizing the experimental conditions, the present aptamer-based sensing system can exhibit excellent fluorescent response in the OTA assay, with a limit of detection of 0.07 ng/mL. In addition to signal amplification, this strategy is also highly specific for other interfering toxins. Furthermore, this aptasensor can be reliably used for assessing red wine samples spiked with different OTA concentrations (2.4, 6 and 20 ng/mL). The proposed assay plays an important role in the field of food safety and can be transformed for detecting other toxins by replacing the sequence that recognizes the aptamer.

DNA helicases and exonucleases play essential roles in genome maintenance; however, little is known about bacterial helicase-exonuclease fusion proteins. This study examines DNA helicases and exonucleases that play essential roles in genome maintenance; however, little is known about bacterial helicase-exonuclease fusion proteins. This study provides the first structural and functional characterization of Staphylococcus aureus DinG (SaDinG), a unique enzyme that combines 5′-3′ helicase and 3′-5′ exonuclease activities. Our findings resolve previous uncertainties about SaDinG's function and reveal an ATP-dependent regulatory mechanism that modulates its activity. Additionally, we demonstrate that SaDinG is critical for bacterial resistance to DNA crosslinking agents. These insights not only expand our understanding of bacterial DNA repair but also suggest potential avenues for targeting DinG-like enzymes in antimicrobial strategies. Given the growing concerns over antibiotic resistance, understanding how bacteria maintain genome integrity under stress conditions is crucial. This work lays the foundation for further exploration of bacterial helicase-exonuclease systems and their role in genome stability and adaptive survival.

MicroRNAs (miRNAs) are about 22-nucleotide (nt) noncoding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3′→5′ exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3′hExo), capable of trimming AGO-associated full-length miRNAs to 14-nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

Traditional methods for detecting antibiotic and mycotoxin residues rely on large-scale instruments, which are expensive and require complex sample pretreatment processes and professional operators. Although aptamer-based electrochemical sensors have the advantages of simplicity, speed, low cost, and high sensitivity, most aptamer-based sensors lack a signal amplification strategy due to their direct use of aptamers as probes, resulting in insufficient sensitivity. To solve the sensitivity problem in the electrochemical detection process, a novel electrochemical sensing strategy was established for ultrasensitive zearalenone (ZEN) detection on the basis of exonuclease I (Exo I) and branched hybridization chain reaction (bHCR) to amplify the signal. The amplification strategy showed excellent analytical performance towards ZEN with a low detection limit at 3.1×10 −12 mol/L and a wide linear range from 10 −11 to 10 −6 mol/L. Importantly, the assay was utilized in the corn powder samples with satisfactory results, holding promising applications in food safety detection and environmental monitoring.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. In this paper, the hepatitis C virus inhibitors Pibrentasvir and Ombitasvir are found to inhibit the SARS-CoV-2 exonuclease and are shown to have therapeutic potential when combined with SARS-CoV-2 polymerase inhibitors in viral cell cultures.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer’s disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5′ exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids. Nemazee and colleagues show that PLD3 and PLD4 are endolysosomal exonucleases that digest ingested nucleic acids and thereby prevent activation of endosomal TLRs. Mice that lack PLD3 and PLD4 develop autoinflammatory disease.

CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity 1 . CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient 2 – 5 . In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation 6 , 7 , but many CRISPR–Cas systems lack Cas4 8 . Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited 9 , 10 exonucleases for faithful acquisition of new CRISPR immune sequences. CRISPR systems lacking Cas4 can use fused or recruited exonucleases for faithful acquisition of new CRISPR immune sequences.