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CRISPR RNA-guided endonucleases have enabled precise editing of DNA. However, options for editing RNA remain limited. Here, we combine sequence-specific RNA cleavage by CRISPR ribonucleases with programmable RNA repair to make precise deletions and insertions in RNA. This work establishes a new recombinant RNA technology with immediate applications for the facile engineering of RNA viruses.One-Sentence SummaryProgrammable CRISPR RNA-guided ribonucleases enable recombinant RNA technology.

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.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.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 (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.

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.

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.

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 analyze a subset (n=83,204) of all publicly available SARS-CoV-2 genomes (n=~5.6 million) that were randomly selected, but equally distributed over the course of the pandemic. We plot the emergence and extinction of new variants of concern (VOCs) over time and show how this corresponds to the ongoing accumulation of mutations in SARS-CoV-2 genomes and individual proteins. While the accumulation of mutations generally follows a linear regression, non-synonymous mutations are significantly greater in Omicron viruses than in previous variants–especially in the spike and nucleoproteins–and these differences are more pronounced in a recently identified sub-lineage (BA.2) of Omicron.ImportanceOmicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike and nucleoproteins have accumulated the most mutations in Omicron variants, but that the accessory proteins of SARS-CoV-2 sequences are changing most rapidly relative to their size. Collectively, this “Observation” provides a concise overview of SARS-CoV-2 evolution, reveals mutational differences between two Omicron lineages, and highlights changes in the SARS-CoV-2 proteome that have been under reported.

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 analyze a subset (n=87,032) of all publicly available SARS-CoV-2 genomes (n=∼5.6 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 viruses are significantly greater than those in previously identified SARS-CoV-2 variants. Mutations in Omicron are primarily restricted to the spike protein, while 25 other viral proteins— including those involved in SARS-CoV-2 replication—are highly 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.ImportanceOmicron is the fifth SARS-CoV-2 variant to be designated a Variant of Concern (VOC) by the World Health Organization (WHO). Here we provide a retrospective analysis of SARS-CoV-2 variants and explain how the Omicron variant is distinct. Our work shows that the spike protein is a ‘hotspot’ for viral evolution in all variants, suggesting that existing vaccines and diagnostics that target this protein may become less effective against Omicron and that our therapeutic and public health strategies will have to evolve along with the virus.

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.

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.

Over 200,000 whole genome sequences of SARS-CoV-2 have been determined for viruses isolated from around the world. These sequences have been 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 demonstrate that this mutation results in a growth defect. ORF7a has been implicated in immune modulation, and we show that the C-terminal truncation results in distinct changes in interferon stimulated gene expression. Collectively, this work indicates that ORF7a mutations occur frequently and that these changes affect viral mechanisms responsible for suppressing the immune response. Declaration of Interest: B.W. is the founder of SurGene LLC, and VIRIS Detection Systems Inc. B.W., A. Nemudryi, and A. Nemudraia are inventors on patents related to CRISPR-Cas systems and applications thereof.

To combat viral pandemics, there is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 in an assay that can be performed in one hour or less. RNA recognition by type III systems triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that amplified products of the Cas10-polymerase are detectable using colorimetric or fluorometric readouts.

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., Graphical Abstract, Highlights SARS-CoV-2 RNA concentrations in wastewater correlate with COVID-19 epidemiology SARS-CoV-2 RNA levels in wastewater follow symptom onset by 5–8 days SARS-CoV-2 RNA levels in wastewater precede clinical PCR test results by 2–4 days SARS-CoV-2 genome from wastewater can trace phylogenetic origin, Nemudryi et al. demonstrate that wastewater can be used to monitor the progression and abatement of SARS-CoV-2 spread at the community level. The authors show a correlation between epidemiological indicators and viral concentrations measured in wastewater. In addition, they infer viral ancestry using a phylogenetic analysis of sequenced SARS-CoV-2 genome(s) from wastewater.

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., Graphical abstract, Recognition of a complementary target RNA by the type III CRISPR systems uniquely triggers the activation of a CRISPR-associated polymerase domain in Cas10. The polymerase generates oligoadenylates, protons, and pyrophosphates. Santiago-Frangos et al. repurposed the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2 by developing three different detection methods that rely on each of these products.

The comparison of the Km and Vmax values for the primers was carried out. The primers were either completely complementary to the template or contained non-complementary bases at different positions with respect to the 3'-end. The addition of NaF, selectively inhibiting 3'----5'-exonuclease activity of the enzyme, was shown to result in the increase of Vmax values by 10% and 30% for complementary and partially complementary primers, respectively, Km values of the latters being unchanged. Km values for d[(pT)10pC] is about 146-fold greater than that for d[(pT)11]. Km values for d[(pT)7pC(pT)2] (20 microM) and d[[(pT)2pC]3pT] (20 microM); d[(pT)4pC(pT)5] (5.0 microM); d[(pC)(pT)7] (1.3 microM) and d[(pT)2pC(pT)7] (1.2 microM) are comparable with those for d[(pT)2] (22 microM), d[(pT)5] (4.1 microM) and d[(pT)7] (1.2 microM), respectively, but not with the decathymidylate d[(pT)10] (0.2 microM). We suggest that it is not the length of the primers but the number of bases in the fragment beginning with the first nucleotide from the 3'-end and ending in the non-complementary base, that determines the efficiency of interaction of the primers containing non-complementary bases with the enzyme. The addition of one link to d(pT)n (n less than or equal to 10) resulted in a 1.8-fold increase in the affinity. When 11 less than n less than 25 the affinity is decreased so that d(pT)22-23 have minimal affinity to the enzyme. The primers containing more than 50 units were found to have about the same affinity (calculated on base concentration) as d(pT)10-11.