Journal: Computational and Structural Biotechnology Journal

Article Title: Next-Generation Sequencing Dataset Downloader and In Silico Sequence Mining: Graphical-User-Interface-Based Tools for Accessible, Multiprobe Target Mining in Next-Generation Sequencing Data

doi: 10.34133/csbj.0095

Figure Lengend Snippet: ISSM analysis of an NGS dataset obtained from CRFK cells infected with feline coronavirus (FCoV), using probes targeting various coronavirus groups, other viruses, CRFK cell sequences, and beta-actin. (A) Heatmap output generated by the ISSM. Probe sequences are color coded according to their target: SARS-CoV-2 (red), SARS-CoV (brown), Bovine CoV (yellow), Alphacoronavirus (blue), Universal coronavirus (green), Bat-CoV (purple), Feline coronavirus (gray), other viruses (black), CRFK cell (light orange), and beta-actin (pink). Color intensity indicates the relative number of matched reads, with deeper red representing higher read counts. (B) Relationship between extraction fraction and detected matched read proportion. Matched read counts obtained at 1%, 10%, 25%, and 50% extraction were expressed as percentages relative to the corresponding matched read counts obtained under 100% extraction, and a linear regression trendline was fitted to model this relationship. (C) Back-calculated 100% equivalent matched read counts derived from the regression equation shown in panel (B), compared with the actual matched read counts obtained under 100% extraction. The actual 100% extraction values used as reference are provided in Table . (D) Reliability assessment of downsampling-based back-calculation. The relationship between the log-transformed actual matched read counts obtained under 100% extraction and the relative error of the back-calculated 100% equivalent values is shown for each extraction setting. Trend equations were used to estimate approximate matched read ranges corresponding to selected relative error thresholds, which are summarized in Table .

Article Snippet: The feline coronavirus (FCoV) type 2 strain WSU 79-1683 (American Type Culture Collection VR-989) was obtained from the American Type Culture Collection and used in this study.

Techniques: Infection, Generated, Extraction, Derivative Assay, Transformation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Schematic representation of the SARS-CoV-2 genome and crRNA design (A) The SARS-CoV-2 genome is a single-stranded, positive-sense RNA. Its first two-thirds comprise overlapping open reading frames, ORF1a and ORF1b, which are translated into 16 nonstructural proteins (nsp1–nsp16). The remaining genome encodes structural proteins—spike (S), envelope (E), membrane (M), and nucleocapsid (N)—along with 8 accessory proteins: ORF3a, 3c, 6, 7a, 7b, 8, and 9b. (B) crRNA design. A highly conserved 26-nucleotide target region was identified within the viral genome of human coronaviruses. Based on this, four overlapping 23-nucleotides crRNAs were designed indicated in red. (C) The designed crRNAs were aligned to the genomes of all seven human coronavirus species. Mismatches relative to the SARS-CoV-2 reference sequence are highlighted in red. Each number on the right represents a single mismatch, with the value indicating its position within the target sequence. The Cas13d seed region, critical for target recognition and cleavage, is marked with a green rectangle.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Membrane, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Broad antiviral CRISPR-Cas13d-crRNA activity against human coronaviruses genome (A) Schematic diagrams illustrating the luciferase reporter system and the CRISPR-Cas13d expression cassette. The luciferase reporter gene is driven by the Pol II SV40 promoter, with the target sequence cloned downstream of the luciferase gene. The CRISPR-Cas13d expression cassette includes a U6 promoter for crRNA expression and an EF1α core promoter driving the expression of Cas13d and GFP. (B) Cas13d/crRNA construct was used to assess the inhibition of luciferase expression by various crRNAs. The tested crRNAs included those targeting the nsp12 genome (crRNA1-4) and a positive control Luc crRNA are shown relative to the Ctrl. Numbers above the bars indicate mismatch positions, with mismatches within the Cas13d seed region highlighted in green. Each number corresponds to a single mismatch. The data represent mean values (±SD) from three independent experiments each performed in duplicate. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. Significant differences are indicated (∗∗∗∗ p < 0.0001).

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: CRISPR, Activity Assay, Luciferase, Expressing, Sequencing, Clone Assay, Construct, Inhibition, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d broadly inhibits the replication of human coronavirus replicons (A) Genomic diagrams of the replicons for SARS-CoV-2, SARS-CoV, and MERS-CoV. The replicons contain the 5′ and 3′ cis -acting signals required for viral replication, the large ORFs 1a and 1b encoding the replicase non-structural proteins (nsps), and the N gene, which is essential for efficient coronavirus RNA synthesis. The SARS-CoV-2 and SARS-CoV replicons, generated by in vitro ligation, also include a mNeonGreen (mNG) or GFP gene, respectively, positioned downstream of ORF1b and regulated by the S gene transcription regulatory sequence (TRS-M). (B) Inhibition of human coronavirus replicons by different crRNAs. Subgenomic mRNA (sgmRNA) levels were measured by RT-qPCR 2 days after transfection. A non-targeting crRNA (Ctrl) was included as a negative control. The horizontal axis shows the tested crRNAs, and the vertical axis shows relative viral RNA levels normalized to Ctrl. Data are presented as mean values (±SD) from three independent experiments each performed in duplicate. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test, with significance levels indicated as ∗∗∗∗ p < 0.0001.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Generated, In Vitro, Ligation, Sequencing, Inhibition, Quantitative RT-PCR, Transfection, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d inhibits the replication of various human coronavirus species when applied prior and post-infection (A) Relative subgenomic mRNA (sgmRNA) levels at 2 days post-infection are shown as the ratio of viral RNA measured in the presence of different crRNAs relative to the control (Ctrl). (B) The antiviral effects of 4 crRNAs were evaluated by quantifying genomic RNA (gRNA) in the supernatant every 24 h, up to the peak of infection, with each crRNA represented by a different color. Viral gRNA levels, including those treated with the control crRNA, were quantified by RT-qPCR. (C) Relative sgmRNA levels are shown for cells treated with different Cas13d/crRNAs. A non-targeting crRNA (Ctrl) is included as a negative control and set to 100%. The horizontal axis indicates the tested crRNAs, and the vertical axis shows relative viral RNA expression. Data represent mean ± SD from three independent experiments. Statistical significance is indicated as ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Infection, Control, Quantitative RT-PCR, Negative Control, RNA Expression

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Cas13d-based SHERLOCK detection assay (A) Detection workflow. Infectious samples are collected, and RNA is extracted. The extracted RNA is reverse transcribed into DNA and then amplified using RPA. The amplified DNA is subsequently transcribed by T7 RNA polymerase to generate RNA targets. These RNA targets are recognized and cleaved by CRISPR-Cas13d guided by crRNA. Upon activation, Cas13d triggers collateral cleavage activity, degrading all surrounding RNAs, including fluorescent RNA reporters. Detection is performed using a lateral flow strip, where a single band indicates a negative result and two bands indicate a positive result. (B) Broad detection of human coronaviruses using Cas13d. Either crRNA2 or crRNA3 was used to broadly detect five human coronavirus species: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. C, control line; T, test line; NC, negative control. (C) Specificity testing against non-coronavirus respiratory viruses. Influenza A and Influenza B samples were tested under the same conditions used for SARS-CoV-2 detection. C, control line; T, test line; PC, positive control (SARS-CoV-2).

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Detection Assay, Reverse Transcription, Amplification, CRISPR, Activation Assay, Activity Assay, Stripping Membranes, Control, Negative Control, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Limit of detection of crRNA2 and crRNA3 for various human coronaviruses (A) Detection limit of crRNA2 and (B) crRNA3 for SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. CRISPR-Cas13d, guided by a crRNA, specifically recognizes and cleaves the target RNA. Upon activation, Cas13d exhibits collateral cleavage activity, degrading nearby RNAs, including fluorescent reporters. Detection is carried out on a lateral flow strip: one band indicates a negative result, while two bands indicate a positive result. The assay was performed with serial dilutions (10-fold) ranging from 100 RNA copies down to 1 RNA copy. C, control line; T, test line; NC, negative control.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: CRISPR, Activation Assay, Activity Assay, Stripping Membranes, Control, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Mismatches of Bat-CoV relative to SARS-CoV-2 genome Positions 15–21 of the crRNA correspond to the seed region (underlined in the SARS-CoV-2 reference sequence), which differs for each crRNA. Nucleotides conserved across all four crRNA seed regions are highlighted in green in the SARS-CoV-2 sequence, whereas mismatches present in Bat-CoV are shown in bold red. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; Bat-CoV, bat coronavirus.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Broad-spectrum CRISPR-Cas13d-mediated strategy for combating human coronaviruses

doi: 10.1016/j.omtn.2026.102888

Figure Lengend Snippet: Mismatches of other animal coronaviruses relative to SARS-CoV-2 genome Positions 15–21 of the crRNA correspond to the seed region (underlined in the SARS-CoV-2 reference sequence), which differs for each crRNA. Nucleotides conserved across all four crRNA seed regions are highlighted in green, whereas mismatches in the animal coronavirus sequences are shown in bold red. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; PRCV, porcine respiratory coronavirus; CCoV, canine coronavirus; PEDV, porcine epidemic diarrhea virus; SADS-CoV, swine acute diarrhea syndrome coronavirus; FCoV, feline coronavirus; FRCoV, ferret coronavirus; MHV, mouse hepatitis virus; RCoV, rat coronavirus; IBV, infectious bronchitis virus; PHEV, porcine hemagglutinating encephalomyelitis virus; CRCoV, canine respiratory coronavirus; BCoV, bovine coronavirus; ECoV, equine coronavirus; TCoV, turkey coronavirus; GCoV, goose coronavirus; DCoV, duck coronavirus; BWCoV, beluga whale coronavirus; WCoV, wigeon coronavirus; MHCoV, moorhen coronavirus; MCoV, munia coronavirus; THCoV, hrush coronavirus; PDCoV, porcine deltacoronavirus.

Article Snippet: Complete genome sequences of coronaviruses from 23 animal species were obtained from the National Center for Biotechnology Information (NCBI) database.

Techniques: Sequencing, Virus

Journal: bioRxiv

Article Title: Proof-of-concept of targeted degradation of p38α/β MAPK host-kinase as a potent inhibitor of coronaviruses

doi: 10.64898/2026.04.29.721712

Figure Lengend Snippet: (a) PROTACs are bifunctional molecules, consisting of a target protein-binding ligand and an E3 ligase ligand attached via a linker. Upon binding of both targets, the E3 ligase catalyses ubiquitination of the target protein, inducing its degradation. (b) Structure of p38α/β-targeting PROTAC NR-7h. The shaded regions indicate the kinase-binding ligand (based on PH-797804) and the CRBN E3 ligase ligand. A549 cells treated with 10 μM NR-7h for 24 h show significant degradation of p38 kinase (c) but no effects on cell viability, as measured by MTS assay (d) . A549 cells infected with OC43 (MOI=0.1) and treated with 10 μM NR-7h showed significant reduction of viral infectivity (e) and viral RNA (f) after 72 h. (g) NR-7h showed greater antiviral efficacy compared to two p38 kinase small molecule inhibitors (NR-7h, IC 50 1.0 nM; PH-797804, IC 50 >10 µM; LY2228820, IC 50 648.4 nM). Graphs show mean ± SEM (n=3); *, p≤0.05; **, p≤0.01; ***, p≤0.001.

Article Snippet: Human coronaviruses OC43 (ATCC VR-1558) stocks were grown in HCT-8 cells, and human coronavirus 229E (ATCC VR-740) stocks were grown and titred in MRC-5 cells.

Techniques: Protein Binding, Binding Assay, Ubiquitin Proteomics, MTS Assay, Infection

Journal: bioRxiv

Article Title: Proof-of-concept of targeted degradation of p38α/β MAPK host-kinase as a potent inhibitor of coronaviruses

doi: 10.64898/2026.04.29.721712

Figure Lengend Snippet: BHK-21 cells treated with 10 μM NR-7h for 24 h show significant degradation of p38 kinase (a) but no effects on cell viability, as measured by MTS assay (b) . BHK-21 cells infected with OC43 (MOI=0.1) and treated with 10 μM NR-7h showed significant reduction of viral infectivity (c) and viral RNA (d) after 72 h. (e) NR-7h showed greater inhibition in BHK-21 cells compared to two p38 kinase small molecule inhibitors (NR-7h, IC 50 9.9 nM; PH-797804, IC 50 3.5 µM; LY2228820, IC 50 69.3 nM). Graphs show mean ± SEM (n=3); *, p≤0.05; **, p≤0.01; ***, p≤0.001. Graphs show mean ± SEM (n=3); *, p≤0.05.

Article Snippet: Human coronaviruses OC43 (ATCC VR-1558) stocks were grown in HCT-8 cells, and human coronavirus 229E (ATCC VR-740) stocks were grown and titred in MRC-5 cells.

Techniques: MTS Assay, Infection, Inhibition

Journal: bioRxiv

Article Title: Proof-of-concept of targeted degradation of p38α/β MAPK host-kinase as a potent inhibitor of coronaviruses

doi: 10.64898/2026.04.29.721712

Figure Lengend Snippet: (a) BHK-21 cells were infected with OC43, and treated with 10 µM NR-7h at 0, 1, 3, or 6 h post-infection, and quantified 14 h post-infection. (b) Quantification of infection was normalised to DMSO-treated cells. Inhibition of infection was not observed for any of these conditions. Graphs show mean ± SEM (n=3).

Article Snippet: Human coronaviruses OC43 (ATCC VR-1558) stocks were grown in HCT-8 cells, and human coronavirus 229E (ATCC VR-740) stocks were grown and titred in MRC-5 cells.

Techniques: Infection, Inhibition

Journal: bioRxiv

Article Title: Proof-of-concept of targeted degradation of p38α/β MAPK host-kinase as a potent inhibitor of coronaviruses

doi: 10.64898/2026.04.29.721712

Figure Lengend Snippet: Cell viability of A549 cells treated with (a) MG132 or (b) MLN4924, no cytotoxicity was observed up to 10 µM for both inhibitors. A549 cells treated with 10 µM NR-7h and (c) MG132 (5 µM) or (d) MLN4924 (3 µM) showed no degradation of p38 kinase compared to when no inhibitors were present. A549 cells infected with OC43 (MOI=0.1) showed significant reduction in infection 72 h after treatment in presence of NR-7h, but infectivity was recovered in presence of (e) MG132 (5 µM) or (f) MLN4924 (3 µM). Graphs show mean ± SEM (n=3); **, p≤0.01; ***, p≤0.001.

Article Snippet: Human coronaviruses OC43 (ATCC VR-1558) stocks were grown in HCT-8 cells, and human coronavirus 229E (ATCC VR-740) stocks were grown and titred in MRC-5 cells.

Techniques: Infection