Online Methods

Polyvalent Guide RNAs for CRISPR Antivirals

CODE AVAILABILITY

Our computational tools for designing pgRNAs for Cas9 and Cas13 are available at https://www.github.com/ejosephslab/pgrna.

ONLINE METHODS

Design of polyvalent guide RNAs (pgRNAs)

The pgRNA design algorithm was implemented in MATLAB (Mathworks, Inc.) or Python using code written in-house and made available at: https://github.com/ejosephslab/pgrna. Viral genome sequences used to identify targets are listed in Supplementary Table S9. See Supplementary note 3 for detailed description of the design algorithm.

Generation of Cas9 target sequences

Cas9 DNA targets in Table 4 were generated by either (1) using plasmids containing targets listed in Table S5 or PCR amplification of targets in plasmids using primers listed in Table S9.

Plasmids were purified using Monarch Plasmid Miniprep Kit following standard protocols (NEB, New England Biolabs, Ipswich, MA). PCR amplification was carried out using 4.5 ng of plasmid DNA, downstream and upstream PCR primers (IDT, Integrated DNA Technologies, Coralville, Iowa, United States) at a final concentration of 0.2uM, and Taq 2x Master Mix (NEB, New England Biolabs, Ipswich, MA, United States) following standard thermocycling protocols. Amplified PCR targets were purified using a Monarch PCR and DNA cleanup kit (NEB) following standard protocols. DNA oligonucleotides were hybridized to form duplex DNA targets by using equal molar concentration of oligos (IDT, Integrated DNA Technologies, Coralville, IA) to a final concentration of 10uM in nuclease-free IDT Duplex buffer. Reactions were heated to 95°C for 2 min and allowed to cool to room temperature prior to the reaction assembly.

in vitro transcription of Cas9 gRNAs

Single guide RNA (sgRNA) was synthesized by using the EnGen sgRNA synthesis Kit (NEB, New England Biolabs, Ipswich, MA, United States) following standard protocols. DNA oligos (IDT) were designed to contain a T7 promoter sequence upstream of the target sequences with an initiating 5’-d(G), as well as overlapping tracrRNA DNA sequence at the 3’ end of the target. The sgRNA was purified using Monarch RNA Cleanup Kit (NEB) and quantitated using standard protocols.

Duplex gRNA generation

Duplex CRISPR gRNAs (cRNA:tracrRNA) was generated by hybridizing synthetic RNA oligos listed in Table S9 to a universal synthetic tracer RNA oligo (IDT). To hybridize oligos, equal molar concentration of oligos were combined in IDT duplex buffer to a final concentration of 10uM. Reactions were heated to 95°C for 2 min and allowed to cool to room temperature prior to the reaction assembly.

Cas9 cleavage reactions

Cas9 Nuclease from S. pyogenes (NEB) was diluted in 1x NEB Buffer 3.1. prior to the reaction assembly. Cas9 cleavage activity was performed using either PCR-amplified targets, whole plasmid, or hybridized DNA oligos containing desired targets using standard methods. Briefly, Cas9 was preincubated with either a sgRNA or duplex gRNA (crNA:tracRNA) for 5 min at equal molar concentrations in 1x NEB Buffer 3.1 (NEB) in a volume total of 10 ul. Reactions were incubated for 5-10 min at room temperature. Target DNA was then added to the reactions, NEB Buffer 3.1 was added back to a final concentration of 1x, and nuclease-free water was added bringing the final volume to 20 ul. The final reaction contained 100nM Cas9-CRISPR complex and 10nM of target DNA. Similar reactions without the addition of gRNAs to Cas9 were used as a control for uncut DNA. Reactions were incubated at 37°C for 1 hour, followed by the addition of 1 unit of Proteinase K and further incubation at 56°C for 15 min. Reactions were stopped by the addition of one volume of purple Gel Loading dye (NEB).

Fragments were separated and analyzed using a 1.5% Agarose gel in 1xTAE and 1X SYBR Green 1 Nucleic Acid Gel Stain (Thermo Fisher Scientific; Waltham, MA), and fluorescence was photographed and measured (AmershamTM Imager 600; GE Life Sciences, Piscataway, NJ, United States).

Construction of RfxCas13d for in planta expression

The DNA sequences of the plant codon optimized Cas13d-EGFP with the Cas13d from Ruminococcus flavefaciens (RfxCas13d) flanked by two nuclear localization signal (NLS) was amplified from plasmid pXR001 (Addgene #109049) using Q5 high fidelity of DNA polymerase (NEB). Similarly, overlap extension PCR was performed to amplify plant expression vector pB35S/mEGFP (Addgene #135320) with ends that matched the ends of the Cas13 product so RfxCas13d expression would be under the control of 35S Cauliflower mosaic virus promoter. The PCR products were treated with DpnI (NEB), assembled together in a HiFi DNA assembly reaction (NEB), transformed into NEB10b cells (NEB), and grown overnight on antibiotic selection to create plasmid pB_35S/RfxCas13. Successful clones were identified and confirmed by sequencing followed by transformation into electro-competent _Agrobacterium tumefaciens strain GV3101 (pMP90).

Construction of crRNA expression vector

Single stranded oligonucleotides corresponding to “monovalent”, non-targeting (NT), and “polyvalent” gRNAs were purchased from Integrated DNA Technologies (Coralville, IA), phosphorylated, annealed, and ligated into binary vector SPDK3876 (Addgene #149275) that had been digested with restriction enzymes XbaI and XhoI (NEB) to be expressed under the pea early browning virus promoter (pEBV). The binary vector containing the right constructs were identified, sequenced and finally transformed into Agrobacterium tumefaciens strain GV3101.

Agroinfiltration of Nicotiana benthamiana (tobacco) leaves

In addition to pB35S/RfxCas13 and the SPDK3876’s harboring gRNA sequences (TRV RNA2), PLY192 (TRV RNA1) (Addgene #148968) and RNA viruses TRBO-GFP (Addgene # 800083) were individually electroporated into _A. tumefaciens strain GV3101. Single colonies were grown overnight at 28 degrees in LB media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl; pH 7). The overnight cultures were then centrifuged and re-suspended in infiltration media (10mM MOPS buffer pH 5.7, 10mM MgCl2, and 200 μM acetosyringone) and incubated to 3-4 hours at 28 degrees. The above cultures were mixed to a final OD600 of 0.5 for CasRX-NLS-GFP-pB35, 0.1 for PLY192 (TRV RNA1), 0.1 for RNA2-crRNAs and 0.005 for TRBO-GFP and injected into healthy leaves of five to six-week-old N. benthamiana plants grown under long-day conditions (16 h light, 8 h dark at 24 °C). A total of four leaves for each gRNA were infiltrated. Three days post-transfection, leaves were cut out and photographed under a handheld UV light in the dark, and stored at −80°C before subsequent analysis.

Quantitative RT-PCR

Total RNA was extracted from infiltrated leaves using RNeasy Plant Mini Kit (Qiagen) and the yield was quantified using nanaodrop. A total of 1ug RNA from control (NT gRNAs) and experimental samples were used for DNase I treatment (Ambion, AM2222) followed by reverse transcription using a poly-dT primer and the Superscript III First Strand cDNA Synthesis System for RT–PCR (Invitrogen). Quantitative PCR was performed on Quant studio 3 Real-Time PCR System from Applied Biosystem using iTaq PowerUP™ SYBR Green pre-formulated 2x master mix (Applied Biosystems). Relative expression levels based on fold changes were calculated using the ddCT method. Cycle 3 GFP mRNA expression levels from the TRBO-GFP replicon were normalized against transcripts of the tobacco PP2A. The samples were performed in three biological replicates.

Prevalence of pgRNA target pairs in viral genomes

All complete sequences of all RNA viruses with human, mammal, arthropoda, aves, and higher plant hosts found in the NCBI Reference Sequence database were subjected to a brute force direct (nucleotide-by-nucleotide, no gaps) alignment for each of their 23 nt sequence targets to each other, considering only sequence polymorphisms at the same site. We considered only the (+) strand, as even for (-) and dsRNA viruses these sequences would match the vast majority of mRNA sequences. Only targets lacking polynucleotide repeats (4 consecutive rU’s, rC’s, rG’s, or rA’s) were considered viable targets. Targets derived from different segments or cDNAs of the same viral strain were considered together. In total: arthropoda (1074 viral species), aves (111), mammal (496), higher plant / embrophyta (691), and human (89) - hosted viruses were considered (Supplementary Tables S1 and S2).

Estimation of SARS-CoV-2 target sequence conservation

All complete SARS-CoV-2 genomic sequences available from the NCBI Virus database were downloaded on November 23, 2020 (29,123 sequences). For each of the 205 target pairs possessing biophysically feasible pgRNA candidates, we aligned (no gaps) each target sequence to each genome to determine the closest matching sequence. Alignments containing ambiguous nucleotide calls were not included. Sequence variants were grouped together, with a minimum prevalence of 0.1%, with the fraction of hits by the most prevalent group being considered the sequence conservation reported.

Article TitlePolyvalent Guide RNAs for CRISPR Antivirals

Abstract

CRISPR biotechnologies, where CRISPR effectors recognize and degrade specific nucleic acid targets that are complementary to their guide RNA (gRNA) cofactors, have been primarily used as a tool for precision gene editing1 but possess an emerging potential for novel antiviral diagnostics, prophylactics, and therapeutics.25 In gene editing applications, significant efforts are made to limit the natural tolerance of CRISPR effectors for nucleic acids with imperfect complementarity to their gRNAs in order to prevent degradation and mutation at unintended or “off-target” sites; here we exploit those tolerances to engineer gRNAs that are optimized to promote activity at multiple viral target sites, simultaneously, given that multiplexed targeting is a critical tactic for improving viral detection sensitivity,3 expanding recognition of clinical strain variants,6 and suppressing viral mutagenic escape from CRISPR antivirals.7 We demonstrate in vitro and in higher plants that single “polyvalent” gRNAs (pgRNAs) in complex with CRISPR effectors Cas9 or Cas13 can effectively degrade pairs of viral targets with significant sequence divergence (up to 40% nucleotide differences) that are prevalent in viral genomes. We find that CRISPR antivirals using pgRNAs can robustly suppress the propagation of plant RNA viruses, in vivo, better than those with a “monovalent” gRNA counterpart. These results represent a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and highlight the need for specific approaches and tools that can address the differential requirements of precision gene editing vs. CRISPR antiviral applications in order to mature these promising biotechnologies.


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