Dataset Information

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.

ABSTRACT: CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We determined that several CRISPR targets significantly reduce viral titer, despite the presence of single nucleotide polymorphisms in the gRNA when compared with a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% and >99% in HCoV-OC43, between 78% and >99% in HCoV-229E, and between 70% and 94% in SARS-CoV-2 when compared with an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system that is capable of reducing viable virus in both Risk Group 2 and Risk Group 3 HCoV pathogens.

SUBMITTER: Mayes CM

PROVIDER: S-EPMC10457650 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.

Mayes Cathryn M CM Santarpia Joshua L JL

The CRISPR journal 20230313 4

CRISPR-based technology has become widely used as an antiviral strategy, including as a broad-spectrum human coronavirus (HCoV) therapeutic. In this work, we have designed a CRISPR-CasRx effector system with guide RNAs (gRNAs) that are cross-reactive among several HCoV species. We tested the efficacy of this pan-coronavirus effector system by evaluating the reduction in viral viability associated with different CRISPR targets in HCoV-OC43, HCoV-229E, and severe acute respiratory syndrome coronav ...[more]

PMID: 36912815

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Project description:Four coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E) are endemic in human populations. All these viruses are seasonal and generate short-term immunity. Like the highly pathogenic coronaviruses, the endemic coronaviruses have zoonotic origins. Thus, understanding the evolutionary dynamics of these human viruses might provide insight into the future trajectories of SARS-CoV-2 evolution. Because the zoonotic sources of HCoV-OC43 and HCoV-229E are known, we applied a population genetics-phylogenetic approach to investigate which selective events accompanied the divergence of these viruses from the animal ones. Results indicated that positive selection drove the evolution of some accessory proteins, as well as of the membrane proteins. However, the spike proteins of both viruses and the hemagglutinin-esterase (HE) of HCoV-OC43 represented the major selection targets. Specifically, for both viruses, most positively selected sites map to the receptor-binding domains (RBDs) and are polymorphic. Molecular dating for the HCoV-229E spike protein indicated that RBD Classes I, II, III, and IV emerged 3-9 years apart. However, since the appearance of Class V (with much higher binding affinity), around 25 years ago, limited genetic diversity accumulated in the RBD. These different time intervals are not fully consistent with the hypothesis that HCoV-229E spike evolution was driven by antigenic drift. An alternative, not mutually exclusive possibility is that strains with higher affinity for the cellular receptor have out-competed strains with lower affinity. The evolution of the HCoV-OC43 spike protein was also suggested to undergo antigenic drift. However, we also found abundant signals of positive selection in HE. Whereas such signals might result from antigenic drift, as well, previous data showing co-evolution of the spike protein with HE suggest that optimization for human cell infection also drove the evolution of this virus. These data provide insight into the possible trajectories of SARS-CoV-2 evolution, especially in case the virus should become endemic.

Project description:Human seasonal coronaviruses (hCoVs) are a group of viruses that affect the upper respiratory tract. While seasonal patterns and the annual variability of predominant hCoV species are well-documented, their genetic and species diversity in St. Petersburg and across Russia remains largely unexplored. In this study, we developed a two-pool, long-amplicon (900-1100 bp) PCR primer panel for the whole-genome sequencing of four seasonal hCoV species. The panel was validated using nasopharyngeal swab samples collected within the Global Influenza Hospital Surveillance Network (GIHSN) project. Over a period of six epidemiological seasons from 2017 to 2023, we retrospectively analyzed 14,704 nasopharyngeal swabs collected from patients hospitalized in St. Petersburg clinics. Of these samples, 5010 (34.07%) tested positive for respiratory viruses, with 424 (2.88% of all samples) identified as seasonal human coronaviruses. The assessment of species diversity showed that predominant hCoV species alternate between seasons. Whole-genome sequences for 85 seasonal human coronaviruses (hCoVs) with >70% genome coverage were obtained, including 23 hCoV-OC43, 6 hCoV-HKU1, 39 hCoV-229E, and 17 hCoV-NL63. These represent the first near-complete genomes of seasonal hCoVs from the Russian Federation, addressing a significant gap in the genomic epidemiology of these viruses. A detailed phylogenetic analysis of the sequenced genomes was conducted, highlighting the emergence of hCoV-229E subclades 7b.1 and 7b.2, which carry numerous substitutions in the Spike protein. Additionally, we sequenced a historical hCoV-229E isolate collected in the USSR in 1979, the oldest sequenced 229E virus from Eurasia, and demonstrated that it belongs to Genotype 2. The newly developed PCR-based sequencing protocol for seasonal hCoVs is straightforward and well-suited for genomic surveillance, providing a valuable tool to enhance our understanding of the genetic diversity of human seasonal coronaviruses.

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Dataset Information

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.

Publications

Pan-Coronavirus CRISPR-CasRx Effector System Significantly Reduces Viable Titer in HCoV-OC43, HCoV-229E, and SARS-CoV-2.

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