The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13’s potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13’s antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER’s potential broad utility for rapid diagnostic and antiviral drug development.

Here, we harness Cas13’s programmable RNA-targeting activity to develop an end-to-end technology platform called Cas13-assisted restriction of viral expression and readout (CARVER) that combines Cas13-mediated cleavage of viral RNA with a rapid, Cas13-based diagnostic readout using the SHERLOCK platform (). We first explored the broad utility of Cas13 for targeting mammalian ssRNA viruses by computationally analyzing >350 viral genomes from species known or predicted to infect humans. We then experimentally tested Cas13’s ability to inhibit viral replication in three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). To define crRNA design criteria, we performed a genome-wide LCMV screen. We further optimized this antiviral approach by investigating the importance of Cas13 localization and multiplexing crRNAs and compared the performance of Cas13 and short hairpin RNAs (shRNAs). Finally, we explored whether Cas13 targeting would result in mutations at the site of crRNA targeting. Together, these investigations establish CARVER as a powerful antiviral and diagnostic technology platform for a wide variety of ssRNA viruses.

A programmable antiviral technology would allow for the rapid development of antivirals that can target existing or newly identified pathogens. In the past 50 years, 90 clinically approved antiviral drugs have been produced, but these antivirals only treat nine viral diseases, only four of which are ssRNA viruses. Vaccines have emerged as the predominant approach to combating viral diseases, but only 16 viruses have FDA-approved vaccines (). Many new efforts have emerged to develop antiviral drugs, such as small-molecule screening approaches focused on identifying inhibitors of viral and host targets. Identifying potent inhibitors of these targets often requires biological or mechanistic understanding, which could slow antiviral development in the face of drug resistance or emerging pathogens. Furthermore, human viral pathogens are quite diverse and evolve rapidly, underscoring both the challenge of developing and great need for adaptable antiviral therapeutic platforms.

RNA-targeting CRISPR effectors could offer a promising alternative antiviral approach. Recent studies of the class 2 type VI CRISPR effector Cas13 have highlighted its ability to efficiently target and cleave RNA in several model systems, including mammalian cells (). Furthermore, Cas13 is able to process its CRISPR array and release individual CRISPR RNA (crRNAs) (), allowing for multiplexed targeting applications. In addition to CRISPR array processing activity, Cas13 has collateral cleavage activity that has been harnessed for diagnostic applications, such as specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) (). However, many Cas13 orthologs have minimal off-target effects on the host transcriptome in mammalian cells, as determined in several recent studies (). This suggests that Cas13 has potential as an antiviral platform if it can be programmed to efficiently target and destroy a wide variety of mammalian viruses. Establishing such a Cas13-based antiviral platform will require investigating the prevalence of target sites in ssRNA viral genomes, experimentally validating Cas13’s antiviral activity when directed to these target sites, and uncovering strategies to optimize this activity ( Figure 1 A).

(E) Effect of pooling 2 (gray) or 4 (black) crRNAs on the number of PspCas13b target sites identified by ADAPT for those viral species (n = 23) without targets as defined in (B).

CRISPR-Cas systems have revolutionized our ability to edit genes and modulate gene expression; however, these systems evolved in nature to defend bacteria against invading bacteriophages and other foreign nucleic acids (). DNA-targeting effectors like Cas9 provide protection against invading DNA bacteriophages, and type III and VI effectors with RNA-targeting activity enable defense against RNA pathogens. This suggests that CRISPR effectors could be repurposed to aid in defending mammalian cells against both DNA and RNA viruses. Indeed, recent applications of Cas9 have demonstrated that CRISPR effectors can inhibit replication of double-stranded DNA viruses or single-stranded RNA (ssRNA) viruses with DNA intermediates in mammalian cells (). However, about two-thirds of viruses that can infect humans have ssRNA genomes, and only 2.5% of those viruses have DNA intermediates that could be targeted with Cas9 (). Furthermore, many ssRNA viruses cause human disease and lack US Food and Drug Administration (FDA)-approved therapies (), underscoring the need for additional antiviral strategies. Recently characterized RNA- and DNA-targeting orthologs of Cas9 are less likely to be able to address this need, as they have low RNA-cleavage efficiency and could induce off-target effects on cellular DNA ().

In our experiments, we did not detect mutations within the crRNA target site or see evidence of elevated mutation rates in the secreted LCMV viral populations. The consensus LCMV sequences across all sample populations were identical ( Data S1 ), indicating that Cas13 targeting does not affect the majority population after 48 h of infection. We then investigated whether Cas13 targeting could lead to emergence of single-nucleotide minority variants (SNVs) that could cause crRNA resistance. We determined SNVs to be an allele present in both libraries, with an allele frequency >0.5%, and no strand bias (see STAR Methods for details). The number of detected SNVs and consequential mutation rate were similar between conditions with no Cas13, non-targeting crRNAs, and targeting crRNAs ( Figure 6 B; Table S3 ). Furthermore, no mutations were observed at the crRNA target sites in any of the conditions tested, despite high coverage (>400×) across all target sites and all conditions ( Figure 6 C).

The viral population secreted from cells following Cas13 treatment may have mutations within or near the crRNA binding site that could lead to crRNA resistance. To test for this possibility, we extracted viral RNA from the virus-containing supernatant of HEK293FT cells after 48 h of infection with LCMV (MOI = 1) for 5 LCMV-targeting crRNAs, 2 non-targeting controls, and mock transfected cells (no Cas13b; Figure 6 A). We prepared duplicate unbiased, metagenomic sequencing libraries from each of these samples. Mean LCMV genome coverage of each library ranged from 847 to 5,414 (mean: 2,606) across both segments of the LCMV genome.

(C) Mean sequencing coverage at each of the crRNA target sites for mock (black), non-targeting (gray), and targeting crRNA at the given site (red). Mean was calculated from the coverage across both library constructions.

Because IAV is highly diverse and rapidly evolves, multiplexed approaches could be extremely beneficial for CARVER’s ability to inhibit IAV replication. By pooling four crRNAs against NP, PspCas13b reduced IAV viral RNA by 8-fold, a modestly increased effect than induced by each individual NP-targeting crRNA alone ( Figure 5 D). Three of the four NP-targeting crRNAs we tested in the NP pool were already highly effective in MDCK cells (up to 8-fold knockdown of IAV viral RNA); thus we hypothesized whether this maximum observed effect is specific to measurement of IAV RNA levels following NP targeting and whether Cas13 cleavage of RNA could have even more drastic effects on resulting IAV infectivity. This could occur if partial genomic fragments were packaged and released into the cellular supernatant that would be detected by RT-qPCR measurements but would not produce infectious particles. Consistent with this hypothesis, we found that IAV infectivity was reduced by >300-fold for 2 of 5 crRNAs tested and >100-fold for 4 of 5 crRNAs tested 8 hpi ( Figure 5 E).

In order to explore additional strategies for enhancing Cas13’s antiviral activity, we tested the effects of PspCas13b localization, alternative cell types, and multiplexing approaches on this antiviral effect. We further investigated the IAV-targeting crRNAs (NP1–NP4 and M1) by electroporating PspCas13b and these crRNAs prior to infection and monitoring both viral RNA using RT-qPCR or IAV infectivity using median tissue culture infectious dose (TCID Figure 5 A). During the IAV viral life cycle, the RNA genome and cRNA genome reside in the nucleus of an infected cell and the viral mRNAs reside in the cytoplasm. We found that Cas13b localization to either the nucleus or the cytoplasm could impact crRNA activity, as crRNA NP1 only potently reduced IAV viral RNA levels when Cas13b was localized to the cytoplasm (i.e., targeting the mRNA) and there was no reduction in viral RNA when Cas13b was localized to the nucleus (i.e., targeting the complementary genomic RNA; Figure 5 B). Cas13b antiviral activity was just as potent in adenocarcinomic human alveolar basal epithelial (A549) cells, reducing viral RNA by up to 20.6-fold ( Figure 5 C).

Because Cas13’s targeting activity is highly specific, tolerating only a few mismatches for activation, CARVER can also be used to detect single-nucleotide changes in the resultant viral populations. To achieve this, we developed a SHERLOCK assay that identified the F260L mutation associated with the switch from acute to persistent infection by LCMV ( Figures 4 F, S5 D, and S5E). The turnaround time for both the bulk LCMV and F260L assays is <2 h, with limited equipment requirements, thereby providing rapid feedback about the effectiveness of treatment with Cas13 and information about specific viral mutations.

Unlike other nucleic-acid-based approaches and quite remarkably, Cas13 can be applied in both viral detection and knockdown contexts, creating a potential for an end-to-end platform for diagnosis and treatment of infectious diseases. Although we can measure the response to Cas13-crRNA expression using recombinant GFP-expressing LCMV in cell culture, rapid measurement of wild-type viral RNA levels would be required in clinical settings. To address this, we developed CARVER as a platform for measuring viral RNA levels following Cas13 targeting in real time using the Cas13-based nucleic acid detection technology SHERLOCK ( Figure 4 E). SHERLOCK uses isothermal amplification with recombinase polymerase amplification (RPA) followed by in vitro transcription and Cas13 detection with a cleavage reporter. We paired Cas13-based targeting with a SHERLOCK assay that measured LCMV RNA levels in cell culture supernatant following Cas13 treatment. We isolated LCMV RNA in the supernatant using HUDSON, which rapidly inactivates the nucleases present in complex samples with a combination of heat and chemical treatment, and used SHERLOCK for RNA detection ( Figures S5 A and S5B). To highlight the utility of CARVER, we targeted LCMV with a selection of crRNAs identified in the LCMV full-genome screen and rapidly detected LCMV levels; SHERLOCK fluorescence was reduced by >15-fold for 4 out of 5 of the crRNAs tested ( Figure S5 C).

Delivery of multiple crRNAs or CRISPR arrays can be used to further enhance Cas13’s antiviral effect. To measure the effect of multiplexing, we collected virus-containing supernatant 24 hpi and measured bulk levels of LCMV using the rapid viral inactivation method, HUDSON (heating unpurified diagnostic samples to obliterate nucleases) and Cas13-based detection platform, SHERLOCK ( Figure 4 B). Pooling multiple validated LwaCas13a crRNAs successfully reduced viral replication, with higher amounts of pooling enhancing this effect ( Figure 4 C). Furthermore, because Cas13 is able to process its CRISPR array and release individual crRNAs (), we showed that PspCas13b transfected with a CRISPR array can deliver up to four crRNAs simultaneously for viral RNA targeting ( Figure 4 D). Multiplexing is therefore an inherent and powerful advantage of Cas13-based therapies.

The promise of Cas13 as a new platform for antiviral therapy would require its activity to be competitive with current gold standards of nucleic-acid-based therapeutics. To test this, we compared the performance of Cas13 crRNAs side by side with location-matched shRNAs. We found that PspCas13b-mediated inhibition of LCMV was comparable to that of shRNAs; tested crRNAs reduced viral RNAs between 4- and 10-fold compared to non-targeting crRNAs as measured by RT-qPCR ( Figure 4 A).

For (A), (C), and (D), n = 3–5. S, LCMV S segment; t, tiled. Pooled or single targeting crRNAs, red; CRISPR arrays, purple; red boxes, spacers; gray diamonds, direct repeats. All time points were 24 hpi except (F), which is 48 hpi. For (A), (C), (D), and (F), error bars represent SD.

(F) SHERLOCK fluorescence following a 1-h incubation using a two-color F260L SNP detection reaction combining LwaCas13a and PsmCas13b, SNP-specific crRNAs and two cleavage reporters with fluorescein amidite (FAM) and ATTO fluorescence are shown (see STAR Methods for details). SHERLOCK fluorescence measured from cell culture supernatant following infection with mixed populations of LCMV Armstrong (A) and Clone 13 (C) strains is shown. Supernatant was collected 48 hpi.

Delivery of Cas13 ribonucleoprotein complexes (RNPs) offers an alternative approach to plasmid expression with faster kinetics, which could be useful in specific therapeutic contexts. As has been demonstrated with Cas13, Cas12, and Cas9, there are multiple expression modalities for Cas13 differing in expression kinetics and required dosages, which include plasmids, mRNAs, and RNPs (). Although alternative nucleic-acid-based therapeutics, like RNAi, would only require delivery of nucleic acid, these approaches have limitations largely due to off-target effects and limited on-target activity (). We explored the RNA targeting efficiency of LwaCas13a and Cas13b from Prevotella sp. MA2016 (PsmCas13b) RNPs against both luciferase and a selection of LCMV-directed crRNAs ( Figures S4 A and S4B). Both LwaCas13a and PsmCas13b RNPs, transfected 4 h prior to infection, reduced viral RNA levels, demonstrating that multiple expression modalities can be used for Cas13-based viral RNA targeting applications ( Figures S4 C and S4D).

We hypothesized that RNA accessibility would also influence which regions of the LCMV vRNA, vcRNA, and viral mRNAs had effective viral knockdown. To examine this relationship, we used SHAPE-MaP () to characterize the RNA secondary structure of in vitro transcribed vRNA, vcRNA, and nucleoprotein (NP)-mRNA of the LCMV S segment. SHAPE-MaP reactivity scores were reproducible across replicates and confirmed the presence of secondary structure of LCMV’s intergenic region (IGR) and the 5′ and 3′ ends of the genome ( Figures S3 H and S3I). However, comparing the reactivity scores of non-active target sites and active target sites, we did not observe a relationship within this dataset ( Figure S3 J).

Further analysis of the genome tiling results showed that target RNA nucleotide content, but not experimentally derived RNA secondary structure, influences crRNA activity. Targeting efficiency of overlapping crRNAs, designed against either the coding or anticoding sense, was anticorrelated, showing preferential activity for one of the senses ( Figure S3 G). We hypothesized that this anticorrelation could be related to the frequency of Us flanking target sites given that LwaCas13a cleaves at Us outside the crRNA binding site (). In concordance with this hypothesis, a greater percentage of target sites near regions with above-average gene-level U frequency were active compared to sites near regions with below-average U frequency (46% versus 39%; Figure 3 E). PspCas13b, in contrast, cleaves at As (), and we observed that a greater percentage of sites near regions with above-average gene-level A frequency were active target sites compared to those with below-average A frequency (80% versus 55%; Figure 3 F). Together, these results suggest that nucleotide context near the target site, specifically the presence of cleavage nucleotides, influences the likelihood a crRNA will be active and is an important consideration due to the biased nucleotide composition of viral genomes (). When considering both conservation and target RNA nucleotide content, we found that nine of ten of sites with >15-fold knockdown possessed both high levels of nucleotide conservation and high U content neighboring the crRNA target site ( Figure 3 G).

We hypothesized that PspCas13b would similarly have increased knockdown efficiency if directed toward conserved regions of the LCMV genome. To test this, we designed 23 crRNAs targeting the L gene vRNA in regions with high- or low-nucleotide-level sequence conservation. Indeed, when crRNAs targeted highly conserved regions, a higher proportion were active target sites compared to poorly conserved regions (63.6% versus 33.3%; Figure 3 D). Further, crRNAs designed against regions conserved among all strains of LCMV were more likely to be active than those targeting conserved regions specific to the LCMV strain being tested (88.8% versus 43.75%; Figure S3 F).

The genome-wide screen identified 124 active crRNA target sites (44% of tested sites; Figure 3 B), many of which have high-nucleotide sequence conservation. Although nearly all anticoding-sense sites were active (92%), the coding sense (31%) had far fewer active sites ( Figure S3 B). Active coding-sense sites clustered into targeting “hotspots” along the viral genome, which were closer than expected by chance (p < 10 Figures S3 C and S3D), prompting us to evaluate the sequence conservation in these regions. The S segment, which has higher levels of nucleotide conservation across all strains of LCMV compared to the L segment (p < 10 Figure S3 E; see STAR Methods for details), had a higher frequency of active sites (43% of all S target sites versus 18% of all L target sites). A high proportion of active coding-sense sites were conserved (78.6%), and 30% of tested conserved sites were active compared to 24% of non-conserved sites ( Figure 3 C). These results suggest that selecting conserved regions for experimental validation of Cas13 antiviral activity could increase the probability of identifying active sites, consistent with previous observations for other nucleic-acid-based approaches ().

To increase our understanding of the design criteria for Cas13 targeting of viral RNAs, we designed a set of crRNAs tiling the entire LCMV genome. These covered the L and S genome segments (vRNA), both antigenomic segments (viral complementary RNA [vcRNA]), as well as the corresponding mRNAs, resulting in 283 crRNAs that target both negative-sense and positive-sense RNAs ( Figures 3 A and S2 A). We transfected HEK293FT cells with a construct expressing cytoplasmic-localized LwaCas13a fused to blue fluorescent protein (BFP), followed by infection (MOI = 1) with recombinant LCMV expressing GFP (). We monitored GFP expression post-infection, which strongly correlated with viral RNA levels ( Figures S2 B–S2F). We defined active crRNA target sites as regions where LwaCas13a significantly reduced GFP levels (false discovery rate [FDR] ≤ 0.05) with ≥2-fold reduction in GFP fluorescence as compared to non-targeting crRNA controls ( Figure S3 A).

In (B), (C), (E), and (G), all tiled crRNAs had n = 3; p value was calculated using a Student’s t test, and FDR was corrected using the Benjamini-Hochberg method. For (D) and (F), PspCas13b crRNA n = 9. PspCas13b active sites had measured viral RNA fold change of ≥1.5, and p ≤ 0.05 compared to all non-targeting crRNAs using a Student’s t test.

(G) Log 2 FC of all coding-sense targeting tiled LwaCas13 crRNAs binned by whether both the mean nt identity score and relative U frequency are high or not. Tiled coding-sense targeting crRNAs with log 2 FC ≥ 2 and FDR ≤ 0.05, blue.

(E) LogFC of all targeting tiled LwaCas13 crRNAs binned by whether the relative U frequency flanking the target site is above or below the average U frequency of gene’s target site (see STAR Methods for details). Tiled targeting crRNAs with logFC ≥ 2 and FDR ≤ 0.05, black.

(C) LogFC of all coding-sense targeting tiled LwaCas13 crRNAs binned by whether the mean nt identity score is above or below 0.8 (see STAR Methods for details). Tiled coding-sense targeting crRNAs with logFC ≥ 2 and FDR ≤ 0.05, blue.

(B) Volcano plot of the results of the LCMV genome screen. Each point represents a single tiled crRNA. To calculate fold changes, each tiled crRNA was compared to all non-targeting controls. Tiled crRNAs with log 2 FC ≥ 2 and FDR ≤ 0.05, black.

(A) Schematic of LCMV full-genome screen using LwaCas13a, crRNA design, and experimental setup. Tiled crRNAs were designed to target the coding (50-nt spacing) and anticoding sense (150-nt spacing) strands of both segments of LCMV. Cas13 activity was assessed by measuring GFP expression 48 hpi with a recombinant LCMV expression GFP (rLCMV-GFP) with inoculation occurring post LwaCas13a and crRNA plasmid transfection.

To further demonstrate the generalizability of Cas13 targeting, we evaluated whether PspCas13b could inhibit the neurotropic RNA virus VSV, which has a negative sense ssRNA genome (∼11 kb) composed of a single linear segment. VSV is a prototype for studies of nonsegmented, negative-strand ssRNA viruses, making it a great candidate for measuring Cas13’s antiviral activity against this class of RNA viruses that make up 37.8% of the human-associated RNA virome. We designed crRNAs against conserved regions of the two main VSV serotypes, Indiana and New Jersey, and selected two target sites for each gene. HEK293FT cells expressing PspCas13b and VSV-specific crRNAs had between 7.8- and 43.3-fold decreased levels of secreted VSV viral RNA 48 hpi (MOI = 1; Figures 2 B and 2E). Together, our results demonstrate that both LwaCas13a and PspCas13b can efficiently and specifically cleave viral RNA in 3 distinct ssRNA viruses, highlighting Cas13’s future potential as a potent inhibitor of viral RNA for a wide variety of human ssRNA pathogens.

We then designed a set of crRNAs to target another high priority virus, IAV, which has a different replication strategy and cellular localization than LCMV. IAV is a negative sense ssRNA virus with eight genomic segments of varying length (ranging from 0.89 to 2.3 kb), and IAV’s viral RNA and complementary viral RNA remain in the nucleus although the mRNAs reside in the cytoplasm. We designed five IAV-targeting crRNAs to target both the mRNA and the complementary viral RNA. We tested PspCas13b’s antiviral activity against IAV in Madin-Darby canine kidney (MDCK) epithelial cells by electroporating plasmids expressing individual crRNAs and PspCas13b and measuring IAV viral RNA levels in the cell culture supernatant by RT-qPCR ( Figure 2 B). PspCas13b reduced IAV viral RNA levels by 7- to 22-fold 24 hpi ( Figure 2 D).

LwaCas13a efficiently reduced LCMV viral RNA (vRNA) levels for a majority of pilot crRNAs tested, even at high MOI. We tested the pilot crRNAs against LCMV at a high MOI (MOI = 5) in HEK293FT cells and observed a 2- to 14-fold reduction in viral RNA with five of six L-segment-targeting crRNAs (83.33%) as measured by RT-qPCR 48 h post infection (hpi) ( Figure 2 C). L-targeting and S-targeting crRNAs mediated a reduction in LCMV viral RNA when transfection of LwaCas13a and these crRNAs was performed prior to or post-LCMV infection at high MOI (MOI = 5; Figures 2 C, S1 A, and S1B). LwaCas13a’s antiviral activity, when Cas13 is expressed prior to infection, persisted at low MOIs ( Figure S1 C), and LwaCas13a’s catalytic activity and cytoplasmic localization were required for antiviral activity against LCMV ( Figure S1 D). Furthermore, LwaCas13a targeting in the context of LCMV infection did not lead to changes in cell viability ( Figure S1 E).

We initially tested the antiviral activity of LwaCas13a against the model mammarenavirus LCMV. Mammarenaviruses, within family Arenaviridae, are enveloped viruses with a bi-segmented (large: L, ∼7.3 kb; small: S, ∼3.5 kb), ambisense ssRNA genome with the life cycle, RNA genome, and RNA replication intermediates restricted to the cytoplasm of infected cells. Several mammarenaviruses cause hemorrhagic fever disease in humans (e.g., Lassa fever virus) and pose important public health problems in their endemic regions. These viruses lack virus-specific, FDA-approved vaccines or therapeutics (), making them a prime candidate for development of new antiviral approaches. We designed 11 pilot crRNAs (pL1–pL6 and pS1–pS5), with spacers 28 nt in length (), to target various locations along the L and S segments.

To evaluate Cas13’s potential to serve as a programmable antiviral platform for targeting ssRNA viruses, we measured Cas13’s antiviral effects against three ssRNA viruses with distinct sequence diversity and replication strategies. We hypothesized that Cas13 crRNAs, when directed to cleave viral RNA, would potently inhibit viral replication ( Figure 2 A) and subsequently tested whether expression of Cas13 and virus-specific crRNAs in cell culture would reduce viral RNA following infection with LCMV, IAV, or VSV ( Figure 2 B).

To identify potential Cas13 target sites, we aligned viral genomes and defined a potential target site as a 28- or 30-nt window where most or all positions are conserved, allowing only 2 or fewer positions to have an allele frequency <95% ( Figure 1 B). We filtered potential target sites to ensure the presence of the PFS specific to each Cas13 ortholog (). We determined that 94.6% of the 396 ssRNA HAVs have ≥10 putative Cas13a target sites and 86.3% have ≥10 putative Cas13b target sites, based on known sequence diversity ( Figure 1 C; Table S2 ). Due to Cas13b’s PFS requirements, Cas13b has fewer potential target sites than Cas13a. However, potential Cas13 target sites are quite numerous overall: 86% of ssRNA HAVs have ≥100 Cas13a target sites and 76% have ≥100 Cas13b target sites ( Figures 1 C and 1D). Furthermore, the number of potential Cas13b target sites that can cover >95% of available genome sequences, as measured by ADAPT (H.C.M. and P.C.S, unpublished data), can be enhanced by pooling up to four crRNAs; this allows sites with higher sequence diversity to be targeted ( Figure 1 E; see STAR Methods for details). These results underscore that Cas13 can potentially be applied to target a wide range of mammalian ssRNA viruses.

We started with a computational analysis of viral genome sequences to identify highly conserved potential target sites for Cas13, followed by experimental validation in cell culture models, to yield an arsenal of antiviral crRNAs that can be multiplexed in a combinatorial fashion ( Figure 1 A). For computational screening and experimental validation, we used Cas13a from Leptotrichia wadei (LwaCas13a) and Cas13b from Prevotella sp. P5-125 (PspCas13b). Both of these Cas13 orthologs efficiently knockdown mRNA in mammalian cells but could have varying optimal target sites due to differences in cleavage activity and protospacer flanking site (PFS) requirements ().

To evaluate the future prospects of Cas13 as a comprehensive antiviral strategy, we explored the abundance of Cas13 target sites in the genomes of human-associated viruses (HAVs). HAVs are defined as viral species that can infect humans or with close relatives that infect humans. There are 396 ssRNA viral species annotated as human associated in the NCBI genome neighbors database (), at least 20 of which are high-risk human pathogens ( Table S1 ). We began by generating an alignment for each genome segment of each of the 396 species (see STAR Methods for details). Given the extreme diversity of ssRNA viruses, a key challenge is to identify enough sites for Cas13 targeting within these alignments to cover the total diversity for a given species and to be deemed functional following experimental validation.

Discussion

Here, we showed that the CRISPR-Cas effector Cas13 can effectively target multiple distinct mammalian ssRNA viruses and highlighted CARVER’s broader potential for diagnosis and treatment of ssRNA viruses. Our computational analysis uncovered numerous potential Cas13 target sites within >300 ssRNA HAV genomes, and subsequent experimental testing demonstrated that targeting conserved regions and sites with a high frequency of cleavage nucleotides influences Cas13’s targeting activity against viral RNAs. Future testing of Cas13’s antiviral activity against other viral species, including DNA viruses with RNA intermediates, could expand upon viral crRNA design principles. Such advances would aid in the full realization of CARVER as a platform that enables informed Cas13-based antiviral treatment strategies.

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Chen J. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components. As with many CRISPR-based technologies, in vivo delivery to cell types of interest remains a challenge. Indeed, our study suggests that, in the context of viral infection, delivery of Cas13 is especially important, as it sets a ceiling on the effect size that can be observed. Although in vivo Cas9 demonstrations have become quite numerous (), Cas13 was discovered recently and is only beginning to be characterized and applied (). Thus, in vivo applications for targeting mammalian RNA with Cas13 are in their infancy and will be demonstrated at scale in the coming years for programmable viral RNA targeting (). Our study represents an essential first step in the repurposing of Cas13 for targeting mammalian viruses, setting the foundation for testing these applications in vivo.

Abudayyeh et al., 2017 Abudayyeh O.O.

Gootenberg J.S.

Essletzbichler P.

Han S.

Joung J.

Belanto J.J.

Verdine V.

Cox D.B.T.

Kellner M.J.

Regev A.

et al. RNA targeting with CRISPR-Cas13. Abudayyeh et al., 2019 Abudayyeh O.O.

Gootenberg J.S.

Franklin B.

Koob J.

Kellner M.J.

Ladha A.

Joung J.

Kirchgatterer P.

Cox D.B.T.

Zhang F. A cytosine deaminase for programmable single-base RNA editing. Cox et al., 2017 Cox D.B.T.

Gootenberg J.S.

Abudayyeh O.O.

Franklin B.

Kellner M.J.

Joung J.

Zhang F. RNA editing with CRISPR-Cas13. Beyond potential antiviral applications, Cas13 targeting and viral knockdown can be used as a research tool to investigate viral replication, localization, and evolution. Specifically, previously developed catalytically dead mutants of Cas13 (dCas13) could be used to study viral RNA localization, and dCas13-fusion proteins with RNA-editing capabilities could be used to functionally characterize specific viral polymorphisms (). Such tools will allow researchers to both visualize and perturb viral replication with a high degree of precision.

It is remarkable that the same protein, Cas13, has the potential to be used for both treatment and diagnosis of a broad range of viral diseases as part of an end-to-end platform. With CARVER, the CRISPR-Cas effector Cas13 can target multiple mammalian viruses as well as measure the effects of targeting and the viral response. Such technological flexibility is unprecedented for a single protein, and it underscores the power and promise of programmable nucleases, such as Cas13. Thus, Cas13 could enable the rapid development of antivirals for a wide variety of human pathogens, both known and newly identified, with substantial implications for the diagnosis and treatment of infectious diseases.