CRISPR Technology as an Antiviral in dsDNA and ssRNA Viruses

The COVID-19 pandemic highlights the necessity of emergency response and pandemic preparedness, especially for emerging viral threats. Currently, virus-specific vaccines and antivirals are the primary tools to combat viral diseases; however, broad-spectrum antivirals that target more than one virus species could provide additional protection from emerging and re-emerging viral diseases (Andersen et al. 2020; Zhu et al. 2015; Hickman et al. 2022). Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated endonucleases have become recently utilized as potential antiviral strategies due to their high specificity, efficacy, and versatility (Najafi et al. 2022). While CRISPR-based antivirals have previously been used to target specific virus species, this current work ­­­leverages CRISPR endonuclease activity to develop antivirals at the viral family level, thereby establishing a more broad-spectrum antiviral tool. The overall objective of this work was to develop broad spectrum CRISPR-based antivirals that can cross-react among several species of a viral family. We demonstrated that this approach is extensible to both dsDNA viruses, as indicated by the success of this antiviral strategy in orthopoxviruses, as well as ssRNA viruses, as evidenced by the successful work with human coronaviruses (HCoV). Using vaccinia virus (VACV) as a model organism for other orthopoxviruses, CRISPR-Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92% for an individual target. Overall, this work identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro. Next, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different HCoVs. We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type VI CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments. Finally, we evaluated the efficacy of the pan-coronavirus CRISPR targets by testing their ability to reduce viable viral titer in three HCoVs, including HCoV-OC43, HCoV-229E, and SARS-CoV-2. In this work, we designed a CRISPR-CasRx effector system with 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 using Median Tissue Culture Infectious Dose assays (TCID50 assays). We determined that several CRISPR targets significantly reduce viral titer despite the presence of single nucleotide polymorphisms (SNPs) in the gRNA when compared to a non-targeting, negative control gRNA. CRISPR targets reduced viral titer between 85% - 100% in HCoV-OC43, 78% - 99% in HCoV-229E, and 70% - 94% in SARS-CoV-2 when compared to an untreated virus control. These data establish a proof-of-concept for a pan-coronavirus CRISPR effector system capable of reducing viable virus in both Risk Group 2 (RG-2) and Risk Group 3 (RG-3) HCoV pathogens. Together, the work presented here establishes a proof-of-concept for CRISPR-based, family-level antivirals in both dsDNA viruses and ssRNA viruses by identifying and targeting conserved regions of essential genes that are shared among viral families