A comprehensive analysis and resource to use CRISPR-Cas13 for broad-spectrum targeting of RNA viruses

Figure 1. Analysis of PAC-MAN for targeting broad-spectrum RNA viruses (A) An integrated pipeline for Cas13 crRNA analysis for virus-targeting coverage, efficiency, and specificity. (B) Targeting pandemic/epidemic-causing pathogenic RNA virus species using PAC-MAN. (C) Schematic of 10 RNA virus families that target different organs and systems in the human host. (D) The minimal number of crRNAs needed to target all sequenced human-infectious strains in each of 10 RNA virus families and HIV and IAV. (E) The minimal number of crRNAs needed to target 90% of sequenced human-infectious strains in each of 10 RNA virus families and HIV and IAV. (F) A cumulative curve showing the predicted minimum number of crRNAs to target all human-infectious strains in 10 RNA virus families in (C). (G) Coverage of pathogenic RNA viruses by the first 14 crRNAs in the minipool from (F). (H) A cumulative curve showing the predicted minimum number of crRNAs to target livestock-infectious strains in 10 RNA virus families in (C). Bovine, ovine, swine, feline, and canine viruses are included in the analysis. See also Figure S1 and Table S1.

Figure 2. Determinants of required crRNAs targeting broad-spectrum RNA virus families (A) Correlation between the number of human-infectious strains in each RNA virus family and the number of crRNAs needed for targeting those strains. (B) Correlation between the number of strains in each RNA virus family and the number of crRNAs needed for targeting all of the strains in that family. (C) The number of crRNAs needed to target all of the sequenced human-infectious strains in each of 6 RNA virus families with segmented genomes. (D) The number of crRNAs needed to target 90% of sequenced human-infectious strains in each of 6 RNA virus families with segmented genomes. (E) Correlation between the number of strains in the RNA virus family and the number of crRNAs needed for targeting all human-infectious viruses in 6 segmented RNA virus families.

Figure 3. CRIPSR-PACMAN as an online resource of using CRISPR-Cas13d for broad-spectrum antiviral targeting (A–C) An exemplar diagram showing the crRNAs designed to target SARS-CoV-2 and its alignment to the genes of SARS-CoV-2. These crRNAs are used for testing in Figure 4. The intensity of colors for each crRNA represents its predicted crRNA score. (D) An exemplar diagram showing the analysis of family-covering crRNAs targeting the Filoviridae family, including Ebola virus. See also Table S2.

Figure 4. Optimized crRNAs targeting SARS-CoV-2 sequences using CRISPR-PAC-MAN (A and B) Repression effects of the optimized minipool of crRNAs (crRNA-MP5) on SARS-CoV-2 GFP reporter (A) and RNA abundance (B). (A) GFP expression as measured by flow cytometry. p values for each group are included in Table S3. Lines indicate means; error bars indicate SDs (n = 3 independent biological replicates). (B) mRNA abundance as measured by quantitative real-time PCR. Relative RNA abundance is calculated by normalizing to the reporter-only sample. p values for each group are included in Table S3. Lines indicate means; error bars indicate SDs. The data represent 3 independent biological experiments performed in technical replicates (n = 9). (C) Bar graphs showing the percentage of genomes of SARS-CoV-2 isolates targeted by individual or multiple crRNAs in the optimized crRNA minipool. (D and E) Bar graphs showing the percentage of genomes of 3 new SARS-CoV-2 variants targeted by individual (D) or multiple crRNAs (E) in the optimized crRNA minipool. See also Figures S2 and S3 and Tables S3 and S4.