mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA

Abstract

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.

Fig 1. Experimental workflow and crRNA assay optimization analysis.

(A) Schematic representation of the experimental workflow. (B) Schematic representation of Influenza PB2 gene (3’ to 5’) with the positions of the qPCR primer/probe Sets 1–5 (dark blue) and crRNA biding site (light grey). (C) Fold change of PB2 RNA levels normalized to the NTCR condition in A549 cells infected with A/H1N1/California/04/09 MOI 0.001 and treated with Cas13 mRNA and the indicated guide using primer/probe Sets 1–5. (D) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 MOI 0.001 and treated with Cas13 mRNA and the indicated guide using primer/probe Set 5. (E) Fold change of PB2 RNA levels normalized to NTCR condition for the data in part (B). In parts (C-E), bars represent mean ± s.d. n = 6 per condition and ****p< 0.0001 (Two-way ANOVA with Tukey’s multiple comparisons). p-values for each comparison in part (E) are listed in S4 Table. (F) Coverage of RNA-Seq reads for the A/H1N1/California/04/09 PB2 segment normalized to the coverage of the virus only condition at each position. The sequence amplified by all primer/probe sets is indicated in light grey. (G) Detail of RNA-Seq reads between positions 2100 and 2280. The sequence amplified by primer/probe set 5 is indicated in light grey and the crRNA’s binding site is indicated in dark grey. (H) Counts for all A/H1N1/California/04/09 segments normalized to the virus only condition. The error bands represent mean± SEM (n = 3 per condition). p-values for each comparison are listed in S7 Table. Fig 1A and 1B were created in part using BioRender.com.

Fig 2. Screening of genome targeting guides across PB2.

(A) Sequence of crRNA spacer sequences (5’-3’, S2 Table) tiled across the conserved PB2 genomic sequence. (B) Schematic representation of the cell-free detection assay used to screen the Influenza genome guides in (A) and determine cutting kinetics. (C) Half-time analysis of the raw fluorescence reads generated for each candidate guide by the cell-free assay. The bars represent mean ± s.d. n = 3 per condition. *p = 0.0199, **p =  0.0055, ****p< 0.0001 (Two-way ANOVA with Dunnett’s multiple comparisons) (D) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 MOI 0.01 and treated with Cas13 mRNA and the indicated guide using primer/probe Set 5. (E) Fold change of PB2 RNA levels normalized to the NTCR condition for the data in part (D). The bars represent mean ± s.d. n = 6 per condition. p-values for each comparison in parts (D and E) are listed in S8 Table. Fig 2B was created in part using BioRender.com.

Fig 3. Potency of PB2 mRNA or genome targeting guides across multiplicity of infection.

(A) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 at the indicated MOIs and treated with Cas13 mRNA and the PB2_m4 mRNA targeting guide using primer/probe Set 5. (B) Fold change of PB2 RNA levels normalized to the NTCR condition for the data in part (A). (C) Viral titer of A/H1N1/California/04/09 after infection at MOI 0.001 and treatment with Cas13a mRNA and guide PB2_m4 or NTCR. The bars represent mean ± s.d. n = 3 per condition. ***p 0.0003 (Unpaired t test comparison). (D) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 at the indicated MOIs and treated with Cas13 mRNA and the PB2_g1 genome targeting guide using primer/probe Set 5. (E) Fold change of PB2 RNA levels normalized to the NTCR condition for the data in part (D). (F) Viral titer of A/H1N1/California/04/09 after infection at MOI 0.001 and treatment with Cas13a mRNA and guide PB2_g1 or NTCR. The bars represent mean ± s.d. n = 3 per condition. **p 0.0084 (Unpaired t test comparison). In parts (A,B,D,E) bars represent mean ± s.d. n = 6 per condition. ***p 0.0001 and ****p < 0.0001 (Two-way ANOVA with Šídák’s multiple comparisons).

Fig 4. Cas13’s enzymatic activity is needed for potent mitigation of infection.

(A) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 at MOI 0.1 and treated with Cas13, dCas13 or GFP mRNA and guide PB2_g1 using primer/probe Set 5. (B) Fold change of PB2 RNA levels normalized to the virus only condition for the data in part (A). The bars represent mean ± s.d. n = 6 per condition. ****p < 0.0001 (One-way ANOVA with Tuckey’s multiple comparisons) (C) Coverage of RNA-Seq reads for the A/H1N1/California/04/09 PB2 segment normalized to the coverage of the virus only condition at each position. (D) Detail of RNA-Seq reads between positions 2100 and 2280. The sequence amplified by primer/probe set 5 is indicated in light grey and the crRNA’s binding site is indicated in dark grey. (E) Absolute counts for indicated A/H1N1/California/04/09 segments. (F) Counts normalized to the virus only condition. The error bands represent mean± SEM (n = 3 per condition). p-values for each comparison are listed in S11 Table.

Fig 5. Efficacy of guide PB2_g1 across H1N1 and H3N2 Influenza A strains.

(A) Fold change of PB2 RNA levels normalized to the NTCR condition in A549 cells infected with the indicated Influenza A strains and treated with Cas13 mRNA and guide PB2_g1 guide using the primer/probe set in S2 Table. *p 0.000005 (Multiple unpaired t test comparisons on log-transformed data). (B) Fold change of PB2 RNA levels normalized to the NTCR condition in HBEC3-KT cells infected with the indicated Influenza A strains and treated with Cas13 mRNA and guide PB2_g1 guide using the primer/probe set in S2 Table. *p 0.000008 (Multiple unpaired t test comparisons on log-transformed data). The bars represent mean ± s.d. n = 4 per condition.

Fig 6. Screening of PB2_g1 chemically modified guides.

(A) Details of the chemical modifications designed for guide PB2_g1. (B) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 at MOI 0.01 and treated with Cas13 mRNA and PB2_g1 genome targeting guides with the indicated chemical modifications using primer/probe Set 5. (C) Fold change of PB2 RNA levels normalized to the NTCR condition for the data in part B. (D) Copy number of PB2 RNA in A549 cells infected with A/H1N1/California/04/09 at MOI 0.01 and treated with Cas13 mRNA and PB2_g1 genome targeting guides with the indicated chemical modifications using primer/probe Set 5. Cells were processed for qPCR at the indicated hours post-transfection. (E) Fold change of PB2 RNA levels normalized to the NTCR condition for the data in part D. In all parts bars represent mean ± s.d. n = 6 per condition. p-values for each comparison are listed in S12 Table.

Fig 7. Nebulization of mRNA-expressed Cas13a and guide PB2_g1 against Influenza infection.

(A) Schematic representation of the in vivo experimental design for mitigation of A/H1N1/California/04/09 infection after nebulization of Cas13a and guide PB2_g1 formulated with polymer P76 and delivered prophylactically. (B) Copy number of PB2 RNA in lungs of hamsters infected with A/H1N1/California/04/09 10⁵ PFU and untreated (Cal/04/09) or treated 24h before infection with Cas13 mRNA and guide PB2_g1. The bars represent mean ± s.d. n = 8 per condition. **p 0.0013 (Unpaired t test comparison on log-transformed data). (C) Lung viral titers for experiments in part A. The bars represent mean ± s.d. n = 8 per condition. **p 0.0047 (Unpaired t test comparison on log-transformed data). (D) Schematic representation of the in vivo experimental design for mitigation of A/H1N1/California/04/09 infection after nebulization of Cas13a and guide PB2_g1 formulated with polymer P76 and delivered as treatment 24h post infection. (E) Copy number of PB2 RNA in lungs of hamsters infected with A/H1N1/California/04/09 10⁵ PFU and untreated (Virus only) or treated 24h before infection with Cas13 mRNA and guide PB2_g1. The bars represent mean ± s.d. n = 8 per condition. **p 0.0236 (Unpaired t test comparison on log-transformed data). (F) Lung viral titers for experiments in part E. The bars represent mean ± s.d. n = 8 per condition. **p 0.0125 (Unpaired t test comparison on log-transformed data). (G) Schematic representation of the in vivo experimental design for mitigation of A/H1N1/California/04/09 infection after nebulization of Cas13a and guide PB2_g1 formulated with polymer P76 and delivered before and after infection. (H) Copy number of PB2 RNA in lungs of hamsters infected with A/H1N1/California/04/09 10⁵ PFU and untreated (Virus only) or treated before and after infection with Cas13 mRNA and guide PB2_g1. The bars represent mean ± s.d. n = 7 per condition. ****p<0.0001 (Unpaired t test comparison on log-transformed data). (I) Lung viral titers for experiments in part H. The bars represent mean ± s.d. n = 7 per condition. **p 0.0006 (Unpaired t test comparison on log-transformed data). Fig 7A and 7D and 7G were created in part using BioRender.com.