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. Author manuscript; available in PMC: 2020 Aug 12.
Published in final edited form as: Mol Cell. 2019 Oct 10;76(5):826–837.e11. doi: 10.1016/j.molcel.2019.09.013

Programmable inhibition and detection of RNA viruses using Cas13

Catherine A Freije 1,2,12,*, Cameron Myhrvold 1,3,12,13,*, Chloe K Boehm 1, Aaron E Lin 1,2, Nicole L Welch 1,2, Amber Carter 1, Hayden C Metsky 1,4, Cynthia Y Luo 1,3, Omar O Abudayyeh 1,5,6,7,8, Jonathan S Gootenberg 1,5,6,7,9, Nathan L Yozwiak 1,3, Feng Zhang 1,5,6,7,10, Pardis C Sabeti 1,2,3,10,11,*
PMCID: PMC7422627  NIHMSID: NIHMS1591539  PMID: 31607545

Summary

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.

Introduction

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 (Wright et al., 2016). DNA-targeting effectors like Cas9 provide protection against invading DNA bacteriophages while 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 (Liu et al., 2015; Ophinni et al., 2018; Price et al., 2015; Ramanan et al., 2015; Roehm et al., 2016; Wang et al. 2018; Yin et al., 2017). 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 (Brister et al., 2015; Woolhouse and Adair, 2013; Woolhouse et al., 2016). Furthermore, many ssRNA viruses cause human disease and lack US Food and Drug Administration (FDA)-approved therapies (De Clercq and Li, 2016), 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 (Price et al., 2015; Strutt et al., 2018).

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 (Abudayyeh et al., 2016, 2017; Aman et al., 2018a, 2018b; Bawage et al., 2018; Cox et al., 2017; Zhao et al., 2018). Furthermore, Cas13 is able to process its CRISPR array and release individual crRNAs (Shmakov et al., 2015), 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) (Gootenberg et al., 2017, 2018; Myhrvold et al., 2018). However, many Cas13 orthologs have minimal off-target effects on the host transcriptome in mammalian cells, as determined in several recent studies (Abudayyeh et al., 2017; Bawage et al. 2018 [preprint]; Cox et al., 2017; Konermann et al., 2018). 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 1A).

Figure 1. Potential Cas13 target sites are abundant in viral genomes.

Figure 1.

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(A) Overview of the strategy for identifying potent Cas13 target sites, which integrates computational screening and experimental validation to yield a combinatorial arsenal of crRNAs.

(B) Schematic of the approach for identifying conserved sites for Cas13 targeting.

(C) Fraction of human-associated ssRNA virome with at least 1, 10, 100, or 1,000 Cas13 target sites, based on known sequence diversity.

(D) Comparison of the number of LwaCas13a and PspCas13b target sites in the genomes of 396 human-associated ssRNA viruses.

(E) Effect of pooling 2 (grey) 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).

In (D) and (E), each point is a viral species; dashed lines indicate x=y.

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 (De Clercq and Li, 2016). 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.

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 (Gootenberg et al., 2017). 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.

Results

Computational Analysis Identifies Cas13 Sites in >350 Human-Associated Viral Genomes

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 (HAV). 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 (Brister et al., 2015; Woolhouse and Adair, 2013; Woolhouse et al., 2016), 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.

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 1A). 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 (Abudayyeh et al., 2017; Cox et al., 2017).

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 1B). We filtered potential target sites to ensure the presence of the PFS specific to each Cas13 ortholog (Abudayyeh et al., 2017; Cox et al., 2017). 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 1C; 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 (Figure 1C 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 1E; see STAR Methods for details). These results underscore that Cas13 can potentially be applied to target a wide range of mammalian ssRNA viruses.

Cas13 Has Potent Antiviral Activity Against Multiple ssRNA Viruses in Cell Culture

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 2A) 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 2B).

Figure 2. Cas13 efficiently reduces levels of viral RNA in mammalian cells.

Figure 2.

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(A) Schematic illustrating Cas13 targeting and cleavage of viral RNA (vRNA) to inhibit viral replication.

(B) Experimental setup for assays evaluating the targeting activity of LwaCas13a and PspCas13b against multiple ssRNA viruses. Red boxes, spacers. Grey diamonds, direct repeats.

(C) Normalized LCMV vRNA levels 48 hpi when transfection of LwaCas13a and pilot L-targeting or non-targeting control crRNA plasmids is performed 24 h prior to infection (MOI=5).

(D) Normalized IAV vRNA levels 24 hpi when transfection of PspCas13b and pilot NP-targeting, M-targeting, or non-targeting control crRNA plasmids is performed 24 h prior to infection (MOI=0.01).

(E) Normalized VSV vRNA levels 48 hpi when transfection of PspCas13b and pilot VSV-targeting crRNA plasmids is performed 24 h prior to infection (MOI=1).

In (C-E) individual points are biological replicates, n=5–6. Red, p ≤0.01 compared to all non-targeting crRNAs. vRNA, viral RNA. NT, non-targeting. p, pilot. L, LCMV L segment. See STAR Methods for normalization details.

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, ~7.3kb; small: S, ~3.5kb), 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 (Geisbert, 2018), making them a prime candidate for development of new antiviral approaches. We designed 11 pilot crRNAs (pL1-pL6, pS1-pS5), with spacers 28 nt in length (Abudayyeh et al., 2016), to target various locations along the L and S segments.

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 hours post infection (hpi) (Figure 2C). L-targeting and S-targeting crRNAs mediated a reduction in LCMV viral RNA when transfection of LwaCas13a and these crRNAs was performed prior or post LCMV infection at high MOI (MOI=5; Figures 2C, S1A, and S1B). LwaCas13a’s antiviral activity, when Cas13 is expressed prior to infection, persisted at low MOIs (Figure S1C); and LwaCas13a’s catalytic activity and cytoplasmic localization were required for antiviral activity against LCMV (Figure S1D). Furthermore, LwaCas13a targeting in the context of LCMV infection did not lead to changes in cell viability (Figure S1E).

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 – 2.3kb), and IAV’s viral RNA and complementary viral RNA remain in the nucleus while 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 2B). PspCas13b reduced IAV viral RNA levels by 7- to 22-fold 24 hpi (Figure 2D).

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 2B 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.

A Genome-wide Screen against LCMV Reveals Viral RNA Targeting Principles for Cas13

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 3A and S2A). 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 (Ngo et al., 2015). We monitored GFP expression post infection, which strongly correlated with viral RNA levels (Figures S2B-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 S3A).

Figure 3. Identification of Cas13 target sites and crRNA design criteria.

Figure 3.

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(A) Schematic of LCMV full genome screen using LwaCas13a crRNA design and experimental set up. 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.

(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 log2FC ≥2 and FDR ≤0.05, black.

(C) Log2FC 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 log2FC ≥2 and FDR ≤0.05, blue.

(D) Frequency as a percentage of PspCas13b crRNAs tested that were active binned by mean nt identity score above or below 0.8.

(E) Log2FC 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 log2FC ≥2 and FDR ≤0.05, black.

(F) Percentage of PspCas13b crRNAs tested that were active binned by relative A frequency of the gene’s target site.

(G) Log2FC 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 log2FC ≥2 and FDR ≤0.05, blue.

In (B,C,E,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 value ≤0.05 compared to all non-targeting controls.

The genome-wide screen identified 124 active crRNA target sites (44% of tested sites; Figure 3B), many of which have high nucleotide sequence conservation. While nearly all anticoding-sense sites were active (92%), the coding sense (31%) had far fewer active sites (Figure S3B). Active coding-sense sites clustered into targeting “hotspots” along the viral genome, which were closer than expected by chance (p < 10˗4; Figures S3C 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 < 10−15; Figure S3E; see STAR Methods for details), had a higher frequency of active sites (43% of all S target sites vs. 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 3C). 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 (von Eije et al., 2008).

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 3D). 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 S3F).

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, were anticorrelated, showing preferential activity for one of the senses (Figure S3G). 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 (Abudayyeh et al., 2016; Gootenberg et al., 2018). 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 3E). PspCas13b, in contrast, cleaves at As (Gootenberg et al., 2018), 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 3F). 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 (van Hemert et al., 2016). 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 3G).

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 (Siegfried et al., 2014) to characterize the RNA secondary structure of in vitro transcribed vRNA, vcRNA, and 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 (Perez and de la Torre, 2003; Salvato and Shimomaye, 1989; Figures S3H 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 S3J).

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, messenger RNAs (mRNAs), and RNPs (Bawage et al., 2018; Kim et al., 2014; Strecker et al., 2019; Zhao et al., 2018). 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 (Abudayyeh et al., 2017; Cox et al., 2017). 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 S4A and S4B). Both LwaCas13a and PsmCas13b RNPs, transfected four hours prior to infection, reduced viral RNA levels, demonstrating that multiple expression modalities can be used for Cas13-based viral RNA targeting applications (Figures S4C and S4D).

Optimizing Cas13 Targeting Using Multiplexing

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 4A).

Figure 4. Enhanced Cas13-based antivirals through multiplexing and paired diagnostics.

Figure 4.

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(A) Normalized vRNA levels comparing PspCas13b crRNAs to position-matched shRNAs. PspCas13b NT crRNA, NT3.

(B) Experimental setup for testing CARVER using LCMV.

(C) SHERLOCK fluorescence 24 hpi and 48 hours post transfection (hpt) of LwaCas13a and single or pooled crRNAs.

(D) SHERLOCK fluorescence with cells expressing PspCas13b and pooled crRNAs or CRISPR array.

(E) CARVER schematic of HUDSON-SHERLOCK readout paired with Cas13 viral RNA targeting.

(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 FAM and ATTO fluorescence (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. Supernatant collected 48 hpi.

For (A,C,D), n=3–5. NT, non-targeting. t, tiled. L, LCMV L segment. S, LCMV S segment. Pooled or single targeting crRNAs, red. CRISPR arrays, purple. Red boxes, spacers. Grey diamonds, direct repeats. All timepoints were 24 hpi except F which is 48 hpi. For (A,C,D,F), error bars, s.d.

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 (Gootenberg et al., 2017; Myhrvold et al., 2018) (Figure 4B). Pooling multiple validated LwaCas13a crRNAs successfully reduced viral replication, with higher amounts of pooling enhancing this effect (Figure 4C). Furthermore, because Cas13 is able to process its CRISPR array and release individual crRNAs (Shmakov et al., 2015), we showed that PspCas13b transfected with a CRISPR array can deliver up to four crRNAs simultaneously for viral RNA targeting (Figure 4D). Multiplexing is therefore an inherent and powerful advantage of Cas13-based therapies.

Development and Testing of CARVER as an End-to-End Platform for Detecting and Destroying Viral RNA

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. While 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 (Gootenberg et al., 2017, 2018; Myhrvold et al., 2018; Figure 4E). 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 (Myhrvold et al., 2018; Figures S5A 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 S5C).

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 (Sullivan et al., 2011; Figures 4F, S5D, 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.

Additional Factors Mediating Cas13-based Targeting Effects against IAV

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, 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 (TCID50; Figure 5A). During the IAV viral life cycle, the RNA genome and complementary RNA genome reside in the nucleus of an infected cell while 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 5B). 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 5C).

Figure 5. Additional considerations for improving Cas13-based antivirals.

Figure 5.

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(A) Experimental schematic of additional testing of PspCas13b’s antiviral activity against IAV with plasmids introduced by electroporation and reduction of IAV post infection measured by RT-qPCR or TCID50.

(B) Normalized IAV vRNA 24 hpi with electroporation of NP-targeting or non-targeting crRNAs and NES- or NLS-PspCa13b encoding plasmids in MDCK cells.

(C) Normalized IAV vRNA 24 hpi with electroporation of IAV-targeting or non-targeting crRNAs and NLS-PspCa13b encoding plasmids in A549 cells.

(D) Normalized IAV vRNA 24 hpi with electroporation of a pool of NP-targeting or of non-targeting crRNAs and NLS-PspCa13b encoding plasmids in MDCK cells.

(E) IAV focus forming units/mL (FFU/mL) as measured TCID50 8 hpi with electroporation of IAV-targeting or non-targeting crRNAs and NES-PspCa13b encoding plasmids in MDCK cells.

For (B-D) n=3; error bars, s.d; red, p value ≤0.01 using Student’s t-test.

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 5D). 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 5E).

Mutations at the crRNA target site are not detected in viral populations following Cas13 treatment

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 hours of infection with LCMV (MOI=1) for 5 LCMV-targeting crRNAs, 2 non-targeting controls and mock transfected cells (no Cas13b; Figure 6A). 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.

Figure 6. Analysis of viral mutations following Cas13 treatment.

Figure 6.

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(A) Schematic of the sequencing experiments where transfection is followed by LCMV infection (MOI=1) and virus-containing supernatant is collected 48 hpi.

(B) Frequencies of bases with >0.5% minor allele variants normalized by genome length 48 hpi, as measured by next-generation sequencing (NGS) of virus-containing supernatants, for mock (black), non-targeting (grey), and targeting (red) conditions.

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

For (B) and (C), data from n=2 independent experiments (circles and squares) are shown.

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 File 1) indicating that Cas13 targeting does not affect the majority population after 48 hours 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 6B; 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 6C).

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.

LCMV, IAV, and VSV are optimal test cases for CARVER, as clinically or scientifically impactful viruses in need of effective, alternative antiviral technologies. LCMV is a model viral system for studying mammarenaviruses, many of which cause hemorrhagic fever in humans, including Lassa fever virus. Lassa fever virus lacks a virus-specific, FDA-approved vaccine or antiviral and remains challenging to diagnose due to its high sequence diversity (Andersen et al., 2015; Geisbert, 2018; Raabe and Koehler, 2017). Approaches like CARVER offer a promising new solution to this unmet need. Similarly, IAV is a very diverse viral pathogen with high seasonal prevalence and pandemic potential. IAV’s rapid evolution and propensity for recombination are obstacles for the development of both antivirals and vaccines and readily allow for the emergence of drug resistance. Lastly, we highlight Cas13’s utility against an additional ssRNA virus, VSV, which could enable high-throughput studies of crRNA targeting properties because productive VSV infection results in cell death. Because recombinant forms of VSV can express alternative viral glycoproteins, successful testing of Cas13 would enable screening of glycoprotein-targeting crRNAs for numerous highly pathogenic RNA viruses that would otherwise require a biosafety level 4 facility. Despite different localization, sequence content and replication biology, Cas13-based targeting potently inhibited all three ssRNA viruses tested.

Our results are striking in light of the challenges associated with targeting replicating viruses. Unlike previous applications of Cas13-based targeting of mRNA (Abudayyeh et al., 2017; Cox et al., 2017; Konermann et al., 2018; Zhao et al., 2018), viral RNA is actively replicating and evolving, and thus Cas13 must act at high enough efficiency to overcome this replication rate. Our LCMV cell culture experiments were performed at high MOI in cell lines that lack a functioning innate immune system, thus providing a stringent test of Cas13’s ability to inhibit viral RNA (see Table S4). In contrast, infections in animal models occur at low MOIs as viral particles spread within tissues, and a functional immune system is present. Measurements of viral infectivity following Cas13 targeting also indicated that Cas13 can have even stronger effects on viral infectivity. In an in vivo context, infectivity most accurately represents the amount of functional virus that can continue the infection.

Target site mutations can reduce the potency of many antiviral approaches, and thus the propensity for these mutations to rise following treatment should be monitored. Previous studies of CRISPR-based antivirals using Cas9 reported mutations at target sites as early as 40 hpi (Wang et al. 2018) or within 3–7 days post infection (Liu et al. 2015; Ophinni et al. 2018; Roehm et al. 2016). However, we did not observe crRNA target site mutations following Cas13 treatment over the course of 48 hours. This suggests (1) that Cas13 targeting does not lead to high mutation rates and (2) the resulting mutation rates are similar to those of small-molecule antivirals, where resistance does not emerge after a few rounds of viral replication (De Clercq and Li, 2016; Tang and Shafer, 2012). Future work will be required to evaluate the time-scale and the degree to which viral RNAs can evolve resistance to Cas13 targeting.

These results highlight Cas13’s potential for rapid antiviral design, as one does not need to test a large number of crRNA sequences to identify ones with potency. Previous studies have reported particular RNA structures are preferred cleavage sites for Cas13 (Abudayyeh et al., 2016) and target RNA structure has also been shown to impact other nucleic-acid based antiviral approaches like RNAi (Low et al., 2012; Westerhout et al., 2005). The lack of correlation between RNA accessibility and Cas13 activity in our data could be related to the presence of viral or host RNA binding proteins (e.g. LCMV nucleoprotein which coats the vRNA, vcRNA, but not the mRNAs) or a more complex relationship between secondary structure and sequence content. Therefore, in vivo determination of RNA structure would be necessary to fully understand its impact on Cas13 targeting, and the optimal viral genomic regions to pursue.

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 (Wilson and Gilbert, 2018), Cas13 was discovered recently and is only beginning to be characterized and applied (Abudayyeh et al., 2016; East-Seletsky et al., 2016). 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 (Ma et al., 2019). 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.

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 (Abudayyeh et al., 2017, 2019; Cox et al., 2017). 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.

Supplementary Material

Table S3

Table S3. Summary of sequencing results from virus-containing cell supernatant, related to Figure 6.

Table S2

Table S2. Number of potential Cas13 target sites by viral species, related to Figure 1.

Table S6

Table S6. Names and sequences of oligos used in this study, related to STAR Methods.

Table S7

Table S7. Summary of full-genome screen results and analyses, related to Figure 3.

Data S1

Data File 1. Fasta file of LCMV consensus sequences for each sample and library preparation, related to Figure 6.

Table S8

Table S8. Summary of SHAPE-MaP results, related to STAR Methods and Figure S3.

Supplemental Figures and Methods, as word doc
Supplemental Figures and Methods, as pdf

Acknowledgements

We thank J. de la Torre for providing the BHK-21 cell line and rLCMV-GFP viral stocks, protocols, and thoughtful discussions and reading of the manuscript. We thank D. Lingwood and M. Sangesland for providing the MDCK cell line and IAV viral stocks and protocols. We thank S. Whelan and L. Gehrke for providing rVSV-GFP viral stocks, BsrT7 cell line and protocols. We thank M. Tabebordbar, A. Piantadosi, K. Barnes, K. Siddle, S. Wohl, E. Brown, and other Sabeti lab members for discussions and thoughtful manuscript feedback. Funding was provided by HHMI, NIH grant U19AI110818, and Defense Advanced Research Projects Agency (DARPA) grant D18AC00006. The views, opinions, and/or findings expressed should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. government. This study has been approved for public release; distribution is unlimited. F.Z. is supported by NIH grants 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201; HHMI; the New York Stem Cell, Allen, and Vallee foundations; the Tan-Yang Center at MIT; and J. and P. Poitras and R. Metcalfe. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. All data are available in the article or the supplementary materials.

Footnotes

Declaration of Interests

C.A.F., C.M., P.C.S., O.O.A., J.S.G., and F.Z. are co-inventors on patent applications filed by the Broad Institute relating to work in this study. O.O.A., J.S.G., F.Z., and P.C.S are co-founders of Sherlock Biosciences.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S3

Table S3. Summary of sequencing results from virus-containing cell supernatant, related to Figure 6.

NIHMS1591539-supplement-Table_S3.xlsx (14.5KB, xlsx)
Table S2

Table S2. Number of potential Cas13 target sites by viral species, related to Figure 1.

NIHMS1591539-supplement-Table_S2.xlsx (25.1KB, xlsx)
Table S6

Table S6. Names and sequences of oligos used in this study, related to STAR Methods.

NIHMS1591539-supplement-Table_S6.xlsx (32.8KB, xlsx)
Table S7

Table S7. Summary of full-genome screen results and analyses, related to Figure 3.

NIHMS1591539-supplement-Table_S7.xlsx (73.9KB, xlsx)
Data S1

Data File 1. Fasta file of LCMV consensus sequences for each sample and library preparation, related to Figure 6.

NIHMS1591539-supplement-Data_S1.zip (44.9KB, zip)
Table S8

Table S8. Summary of SHAPE-MaP results, related to STAR Methods and Figure S3.

NIHMS1591539-supplement-Table_S8.xlsx (443.2KB, xlsx)
Supplemental Figures and Methods, as word doc
NIHMS1591539-supplement-Supplemental_Figures_and_Methods__as_word_doc.docx (3MB, docx)
Supplemental Figures and Methods, as pdf
NIHMS1591539-supplement-Supplemental_Figures_and_Methods__as_pdf.pdf (3MB, pdf)

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