Type III CRISPR-based RNA editing for programmable control of SARS-CoV-2 and human coronaviruses

Figure 1.. TEAR-CoV approach inhibits SARS-CoV-2 using a simplified type III CRISPR-Csm system. (A) Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) derived from Streptococcus thermophilus type III CRISPR-Cas system to limit SARS-CoV-2 replication and infection by directly targeting and degrading the viral RNA genome and reduction of viral mRNAs. (B) Conserved regions of SARS-CoV-2 sequences with SARS-CoV and MERS-CoV. Top: gRNA abundance in the highly conserved regions that target SARS-CoV-2 sequences and SARS or MERS. Bottom: percentage of conservation between aligned viral genomes. (C) A collection of gRNAs targeting SARS-CoV-2 strains derived from Asia, South America (SA), North America (NA), Oceania, Africa, and Europe. The Y axis represents the number of gRNAs targeting SARS-CoV-2. Upset plot of each area overlaps across the six continents. (D) Cleavage assay of the SARS-CoV-2 RNA genome by TEAR-CoV in vitro. gRNA1 and gRNA2 target the RdRP sequence; gRNA3 and gRNA4 target the S sequence; and not-target represents a gRNA not targeting the SARS-CoV-2 RNA genome. (E) Transcription-translation (TXTL) reaction assessing TEAR-CoV in repression of S fragment-deGFP…

Figure 2.. TEAR-CoV permits RNA-Guided RNA cleavage in human cells. (A) Procedure of RNA cleavage experiments in HEK293T cells. TEAR-CoV activity by gRNA against Gaussia. (B) Quantification of RNA cleavage of Gaussia luciferase using the TEAR-CoV in human cells HEK193T. Dots represent six biological replicates, error bars indicate SEM, one-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant. (C) Optimization of TEAR-CoV of RNA targeting efficiency. Increasing amounts of StCsm complex were used to evaluate the ratio of Gaussia luciferase knockdown by TEAR-CoV (as measured by luciferase activity).

Figure 3.. TEAR-CoV inhibits SARS-CoV-2 gene expression in mammalian cells. (A) Schematic illustrating TEAR-CoV targeting and cleavage of C-terminal domain (CTD) of SARS-CoV-2 S mRNA and RdRP of SARS-CoV-2-RdRP-GFP lentivirus. (B) qRT-PCR detecting expression levels of the CTD of S1 subunit (called RBD domain) of SARS-CoV-2 S when TEAR-CoV with gRNA5 targeting CTD sequence was delivered. Relative RNA expression was calculated by normalizing to GAPDH. Bars indicate mean SEM, one-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant. (C) TEAR-CoV affecting mRNA abundance as measured by qRT-PCR when SARS-CoV-2-RdRP-GFP was delivered via lentiviral transduction. One-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant. (D, E) GFP expression as measured by western blot (D) and flow cytometry (E) when SARS-CoV-2-RdRP-GFP was delivered via lentiviral transduction. One-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant.

Figure 4.. TEAR-CoV possesses antiviral activity against live IAV infection in human cells and in mice. (A) Schematic illustrating the use of TEAR-CoV for the defense of influenza A virus (IAV) infection. (B) Normalized IVA RNA levels when transfection of TEAR-CoV with pilot non-targeting control gRNA or gRNA6 and gRNA7 targeting the IVA mRNA and complementary viral RNA that is nucleocapsid protein (NP) gene in genomic segment 5. Boxplots: center line represents median, whiskers represent minimum to maximum, and dots represent six independent replicates; one-way ANOVA plus Tukey test. (C) Comparison of viral replication when TEAR-CoV were delivered at MOI = 0.01. One-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant. (D) Schematic depiction of TEAR-CoV treatment for IAV post-infection mouse model. (E) Normalized IAV RNA levels after transfection of TEAR-CoV into IAV-infected mice. (F) H&E staining assessing lung injury in mice with IAV infection with or without TEAR-CoV treatment.

Figure 5.. TEAR-CoV enables rapid design of prophylactics and therapeutics targeting any coronaviruses. (A) Phylogenetic tree of human coronaviruses constructed by the complete reference sequences. The outer ring shows coverage by each of gRNAs (hCoV-gRNA1 to hCoV-gRNA5) targeting almost all human coronaviruses’ genomes. The inner ring shows current species of human coronaviruses that infect humans, including SARS-CoV-2. (B) Prediction of minimal numbers of hCoV-gRNAs targeting the human coronaviruses reference genomes. (C) qRT-PCR detecting expression levels of the SARS-CoV-2 envelope when TEAR-CoV with hCoV-gRNA pool was delivered. Relative RNA expression was calculated by normalizing to GAPDH. Bars indicate mean SEM, one-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant. (D–H) qRT-PCR detecting expression levels of human coronaviruses by TEAR-CoV with hCoV-gRNA pools. Human coronaviruses: SARS-CoV (D), MERS-CoV (E), CoV KHU1 (F), CoV 229E (G) and CoV NL63 (H). Relative RNA expression was calculated by normalizing to GAPDH. Bars indicate mean SEM, one-way ANOVA plus Tukey post hoc test and P < 0.05 was considered statistically significant.