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Many viral diagnostic approaches require nucleic acid extraction and amplification prior to detection. Here, the authors develop a method based on type III CRISPR systems which allows sequence specific capture, concentration, and detection of viral RNA directly from patient samples.

Background and Aims: Recent evidence suggests that the gut is an additional target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, whether SARS-CoV-2 spreads via gastrointestinal secretions remains unclear. To determine the prevalence of gastrointestinal SARS-CoV-2 infection in asymptomatic subjects, we analyzed gastrointestinal biopsy and liquid samples from endoscopy patients for the presence of SARS-CoV-2. Methods: We enrolled 100 endoscopic patients without known SARS-CoV-2 infection (cohort A) and 12 patients with a previous COVID-19 diagnosis (cohort B) in a cohort study performed at a regional hospital. Gastrointestinal biopsies and fluids were screened for SARS-CoV-2 by polymerase chain reaction (PCR), immunohistochemistry, and virus isolation assay, and the stability of SARS-CoV-2 in gastrointestinal liquids in vitro was analyzed. Results: SARS-CoV-2 ribonucleic acid was detected by PCR in the colonic tissue of 1/100 patients in cohort A. In cohort B, 3 colonic liquid samples tested positive for SARS-CoV-2 by PCR and viral nucleocapsid protein was detected in the epithelium of the respective biopsy samples. However, no infectious virions were recovered from any samples. In vitro exposure of SARS-CoV-2 to colonic liquid led to a 4-log–fold reduction of infectious SARS-CoV-2 within 1 hour (P ≤ .05). Conclusion: Overall, the persistent detection of SARS-CoV-2 in endoscopy samples after resolution of COVID-19 points to the gut as a long-term reservoir for SARS-CoV-2. Since no infectious virions were recovered and SARS-CoV-2 was rapidly inactivated in the presence of colon liquids, it is unlikely that performing endoscopic procedures is associated with a significant infection risk due to undiagnosed asymptomatic or persistent gastrointestinal SARS-CoV-2 infections.

In late December of 2019, high-throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we provide a short retrospective analysis of SARS-CoV-2 variants by analyzing a subset (n = 97,437) of all publicly available SARS-CoV-2 genomes (n = ~11.9 million) that were randomly selected but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron (BA.1) and Omicron sub-lineages (BA.2–BA.5) are significantly elevated compared to previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike and nucleocapsid proteins, while 24 other viral proteins—including those involved in SARS-CoV-2 replication—are generally conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered.

Summary: There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.

Genome editing with CRISPR RNA-guided endonucleases generates DNA breaks that are resolved by cellular DNA repair machinery. However, analogous methods to manipulate RNA remain unavailable. We show that site-specific RNA breaks generated with type-III CRISPR complexes are repaired in human cells and that this repair can be used for programmable deletions in human transcripts to restore gene function. Collectively, this work establishes a technology for precise RNA manipulation with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Selfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae , a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae , giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals. [ABSTRACT FROM AUTHOR]

Summary: SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.

The article discusses the history of DNA manipulation techniques, particularly recombinant DNA, and the development of tools for in vivo editing using site-specific nucleases. It highlights the limitations of current methods for manipulating RNA and introduces a new approach that combines type III CRISPR-based RNA cleavage with splinted RNA ligation to enable precise deletions and insertions in target RNA sequences, with potential applications in RNA virus genome engineering.

In late December of 2019, high-throughput sequencing technologies enabled rapid identification of SARS-CoV-2 as the etiological agent of COVID-19, and global sequencing efforts are now a critical tool for monitoring the ongoing spread and evolution of this virus. Here, we provide a short retrospective analysis of SARS-CoV-2 variants by analyzing a subset (n = 97,437) of all publicly available SARS-CoV-2 genomes (n = ~11.9 million) that were randomly selected but equally distributed over the course of the pandemic. We plot the appearance of new variants of concern (VOCs) over time and show that the mutation rates in Omicron (BA.1) and Omicron sub-lineages (BA.2–BA.5) are significantly elevated compared to previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike and nucleocapsid proteins, while 24 other viral proteins—including those involved in SARS-CoV-2 replication—are generally conserved. Collectively, this suggests that the genetic distinction of Omicron primarily arose from selective pressures on the spike, and that the fidelity of replication of this variant has not been altered. [ABSTRACT FROM AUTHOR]

Immune systems generate diverse alarm signals in response to invading pathogens. In a recent Nature paper, Leavitt et al. identified a family of phage-encoded "codebreakers" that intercept nucleotide-derived immune signals and render the cell defenseless to viral infection. [ABSTRACT FROM AUTHOR]

Over 950,000 whole-genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been determined for viruses isolated from around the world. These sequences are critical for understanding the spread and evolution of SARS-CoV-2. Using global phylogenomics, we show that mutations frequently occur in the C-terminal end of ORF7a. We isolate one of these mutant viruses from a patient sample and use viral challenge experiments to link this isolate (ORF7aΔ115) to a growth defect. ORF7a is implicated in immune modulation, and we show that the C-terminal truncation negates anti-immune activities of the protein, which results in elevated type I interferon response to the viral infection. Collectively, this work indicates that ORF7a mutations occur frequently, and that these changes affect viral mechanisms responsible for suppressing the immune response. [Display omitted] • ORF7a mutations are found in SARS-CoV-2 genomes isolated from around the globe • The ORF7aΔ115 isolate displays a replication defect • An ORF7a mutation limits viral suppression of the interferon response Nemudryi et al. use global SARS-CoV-2 phylogenomics to identify mutations that frequently occur in the C-terminal end of ORF7a accessory protein. They isolate one of these mutant viruses from a patient sample and demonstrate that ORF7a truncation affects viral mechanisms responsible for suppressing host immune response. [ABSTRACT FROM AUTHOR]

[Display omitted] • Reliable and rapid direct to consumer diagnostics are necessary to stop the spread of highly contagious infectious agents. • Programable diagnostics that rely on base-pairing are emerging as disruptive new alternatives to PCR. • Nucleic acid-guided proteins are being coupled to reliable and easy to read detection devices. • Standardized reporting metrics will facilitate objective comparisons between platforms. Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics. [ABSTRACT FROM AUTHOR]