science.sciencemag.org/cgi/content/full/science.abb6151/DC1

Supplementary Materials for

A phage-encoded anti-CRISPR enables complete evasion of

type VI-A CRISPR-Cas immunity

Alexander J. Meeske*, Ning Jia*, Alice K. Cassel, Albina Kozlova, Jingqiu Liao,

Martin Wiedmann, Dinshaw J. Patel†, Luciano A. Marraffini†

*These authors contributed equally to this work.

†Corresponding author. Email: marraffini@rockefeller.edu (L.A.M.); pateld@mskcc.org (D.J.P.)

Published 28 May 2020 on Science First Release

DOI: 10.1126/science.abb6151

This PDF file includes:

Materials and Methods

Figs. S1 to S12

Tables S1 to S7

References

MDAR Reproducibility Checklist

Other Supplementary Material for this manuscript includes the following:

(available at science.sciencemag.org/cgi/content/full/science.abb6151/DC1)

MDAR Reproducibility Checklist (PDF)

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Materials and Methods Bacterial strains and growth conditions All genetically modified L. seeligeri strains generated in this study are derived from L. seeligeri SLCC3954 (19). Environmental L. seeligeri isolates and L. monocytogenes strains are listed in Table S2. Unless otherwise stated, L. seeligeri and L. monocytogenes strains were cultured in Brain Heart Infusion (BHI) medium at 30°C. Where appropriate, BHI was supplemented with the following antibiotics for selection: nalidixic acid (50 µg/mL) chloramphenicol (10 µg/mL), erythromycin (1 µg/mL), or kanamycin (50 µg/mL). For cloning, plasmid preparation, and conjugative plasmid transfer, E. coli strains were cultured in Lysogeny Broth (LB) medium at 37°C. Where appropriate, LB was supplemented with the following antibiotics: ampicillin (100 µg/mL), chloramphenicol (25 µg/mL), kanamycin (50 µg/mL). For conjugative transfer of E. coli – Listeria shuttle vectors, plasmids were purified from Turbo Competent E. coli (New England Biolabs) and transformed into the E. coli conjugative donor strains SM10 λpir or S17 λpir (30). Phage isolation and propagation Temperate listeriaphages were isolated by prophage induction via stimulation of the SOS response with the DNA-damaging agent mitomycin C, followed by plaque isolation on the L. seeligeri ∆RM Dspc indicator strain. Each strain of L. seeligeri and L. monocytogenes was cultured overnight and diluted to OD600 = 0.1, then treated with 1 µg/mL mitomycin C to activate the phage lytic cycle. Prophage induction was carried out overnight at 30°C, then culture supernatants were passed through 0.45 µm filters. Each filtrate was screened for phages by infection of ∆RM Dspc using the top agar overlay method: 100ul of serially diluted induction filtrate was used to infect 100 µL of saturated ∆RM Dspc culture in a 5 mL overlay of BHI containing 0.75% agar, in the presence of 5 mM CaCl2. Infection plates were incubated at 30° for 24 hrs. Single plaques were resuspended in BHI, then propagated three times on ∆RM Dspc, a single plaque was isolated each time to ensure phage purity. High titer phage lysates were obtained by preparing top agar infections of ∆RM Dspc with plaques at near-confluent density, then soaking the agar with SM buffer (100mM NaCl, 10mM MgSO4, 50mM Tris-HCl pH 7.5). Plasmid construction and preparation All genetic constructs for expression in L. seeligeri were cloned into the following three compatible shuttle vectors, each of which contains an origin of transfer sequence for mobilization by transfer genes of the IncP-type plasmid RP4. These transfer genes are integrated into the genome of the E. coli conjugative donor strains SM10 λpir and S-17 λpir (30).All plasmids used in this study, along with details of their construction, can be found in Table S2. pPL2e – single-copy plasmid conferring erythromycin resistance that integrates into the tRNAArg locus in the L. seeligeri chromosome (31). pAM8 – E. coli – Listeria shuttle vector conferring chloramphenicol resistance (32). pAM326 - E. coli – Listeria shuttle vector conferring kanamycin resistance (constructed in this study).

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To express crRNAs containing engineered spacers, a minimal type VI CRISPR array containing the native promoter and a single repeat-spacer-repeat unit with BsaI entry sites was cloned into BamHI/SalI-digested pPL2e to generate pAM305. To clone new spacers, pAM305 was digested with BsaI, and ligated to spacer inserts consisting of annealed oligos with cohesive overhangs compatible with the sticky ends generated by BsaI-cleavage of pAM305. All plasmid targeting assays described in this study use the pAM8-derived plasmid pAM54 (32), in which a protospacer matching the endogenous type VI spc4 was cloned into the 3’ untranslated region of a chloramphenicol resistance cassette. The negative control for plasmid targeting assays is pAM8, which contains the chloramphenicol cassette without a protospacer. Putative anti-CRISPR constructs were assembled by cloning into HindIII/EagI-digested pAM326. E. coli – L. seeligeri conjugation All genetic constructs for expression in L. seeligeri were introduced by conjugation with the E. coli donor strains SM10 λpir, S-17 λpir (30), or for allelic exchange (see below), β2163 ∆dapA (33). Donor cultures were grown overnight in LB medium supplemented with the appropriate antibiotic (25 µg/mL chloramphenicol for pPL2e-derived plasmids, 100 µg/mL ampicillin for pAM8-derived plasmids, or 50 µg/mL kanamycin for pAM326-derived plasmids) at 37°C. Recipient cultures were grown overnight in BHI medium supplemented with the appropriate antibiotic (1 µg/mL erythromycin for pPL2e-derived plasmids, 10 µg/mL chloramphenicol for pAM8-derived plasmids, 50 µg/mL kanamycin for pAM326-derived plasmids) at 30°C. 100 µL each of donor and recipient culture were diluted into 10 mL of BHI medium, and concentrated onto a filter disc (Millipore-Sigma, HAWP04700) using vacuum filtration. Filter discs were laid onto BHI agar supplemented with 8 µg/mL oxacillin (which weakens the cell wall and enhances conjugation) and incubated at 37°C for 4 hr. Discs were removed, cells were resuspended in 2 mL BHI, and transconjugants were selected on medium containing 50 µg/mL nalidixic acid (which kills donor E. coli but not recipient L. seeligeri) in addition to the appropriate antibiotic for plasmid selection. Transconjugants were isolated after 2-3 days incubation at 30°C. Gene deletions in L. seeligeri Allelic exchange plasmids were generated by cloning 1kb homology arms flanking the genomic region to be deleted into the suicide vector pAM215 (11), which does not replicate in Listeria, and contains a chloramphenicol resistance cassette and lacZ from Geobacillus stearothermophilus. These plasmids were then transformed into the E. coli donor strain β2163 ∆dapA(33), which is auxotrophic for diaminopimelic acid (DAP), selecting on LB medium supplemented with the appropriate antibiotic and 1.2 mM DAP. Conjugation was carried out as described above, except all steps were carried out in the presence of 1.2 mM DAP. Transconjugants were selected on media lacking DAP and containing 50 µg/mL nalidixic acid, to ensure complete killing of donor E. coli, as well as 10 µg/mL chloramphenicol to select for integration of the pAM215-derived plasmid. Chloramphenicol-resistant colonies were patched on BHI supplemented with 100 µg/mL 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (X-gal) and confirmed lacZ+ by

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checking for blue colony color. Plasmid integrants were passaged 3-4 times in BHI at 30° in the absence of antibiotic selection, to permit loss of the integrated plasmid. Cultures were screened for plasmid excision by dilution and plating on BHI + X-gal. White colonies were checked for chloramphenicol sensitivity, then chromosomal DNA was prepared from each, and tested for the desired deletion by PCR using primers flanking the deletion site. Deletions were finally confirmed by Sanger sequencing. Bacterial genome sequencing, genome assembly, and ϕLS46 identification The ϕLS46 genome was sequenced by whole-genome sequencing and assembly of its parent lysogen, L. seeligeri LS46. Chromosomal DNA was prepared from LS46 by lysozyme digestion of the cell wall, followed by cell lysis with 1% sarkosyl, then phenol-chloroform extraction and ethanol precipitation. 1 ng of chromosomal DNA was used to make an NGS library using the Illumina Nextera XT DNA Library Preparation Kit according to the manufacturer’s instructions. Library quality was confirmed by analysis on Agilent TapeStation, then 2x150bp paired-end sequencing was carried out on the Illumina NextSeq platform. Raw reads were quality-trimmed using Sickle (https://github.com/najoshi/sickle) using a quality cutoff of 30 and length cutoff of 45. Trimmed reads were assembled using SPAdes (http://cab.spbu.ru/software/spades/) with the default parameters, which resulted in 140 assembled contigs with an N50 of 2841899. These contigs were mapped onto the completed reference genome of L. seeligeri SLCC3954 using Medusa (http://combo.dbe.unifi.it/medusa/) with the default parameters, which resulted in 105 scaffold assemblies. In our draft genome assembly, one scaffold (Scaffold 1) represents a 2.8 Mbp assembly, Scaffold 7 contains 46 Kbp, and each of the remaining 103 scaffolds contains between 100-1300 bp. To identify putative prophages in the assembled genome, we used Phaster (http://phaster.ca), which predicted a single prophage element, occupying the entirety of Scaffold 7. We confirmed that this prophage (ϕLS46) was the one isolated by mitomycin C induction of LS46 using PCR of the ∆RM Dspc-passaged phage stock with ϕLS46-specific primers. Construction of gene deletions in ϕLS46 Gene deletions in ϕLS46 were constructed in two ways. One group of deletions was obtained by selection of spontaneous escapers of Cas9 targeting of the anti-CRISPR locus in ϕLS46. A Cas9 spacer targeting the anti-CRISPR region (gp4) was cloned into the vector pAM307, which carries Cas9 from Streptococcus pyogenes along with a repeat-spacer-repeat construct with BsaI entry sites. This plasmid (pAM379) was introduced into ∆RM Dspc, which was then infected with ten-fold serial dilutions of ϕLS46 in a plaque assay on BHI top agar. Cas9-targeting reduced the efficiency of ϕLS46 plaquing by several orders of magnitude, but spontaneous Cas9-resistant escaper plaques were isolated and checked for deletions by PCR using primers flanking the anti-CRISPR locus. The deletions were then precisely mapped by Sanger sequencing. To generate an in-frame deletion of the acrVIA1 gene, we first assembled a homology repair template (pAM386) containing 1kb homology arms flanking an in-frame deletion of acrVIA1. In the deletion construct, the first six and last six codons of acrVIA1 remain, both to avoid Rho-dependent termination of untranslated RNA, as well as to preserve the Shine-Dalgarno sequence for the gp3 gene predicted to be present in the last six codons of acrVIA1. The repair template plasmid was introduced into ∆RM Dspc, this strain was

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infected with ϕLS46 in BHI top agar (allowing recombinants to be generated), and a phage stock was harvested. A Cas9 spacer targeting acrVIA1 was cloned into pAM307 to generate pAM377 and introduced into ∆RM Dspc. The ϕLS46 stock passaged on ∆RM Dspc carrying the pAM386 repair template was used to infect ∆RM Dspc carrying pAM377, and Cas9-resistant escaper mutants were isolated. Two mutant phage isolates were Sanger sequenced across the acrVIA1 gene, and found to contain the precise deletion.

In vitro RNA cleavage assays 10 µM synthetic RNA substrates (listed in Table S1) were labeled with ATP [ɣ-32P] for 30 min at 37° with 1ul NEB T4 Polynucleotide Kinase, then purified using GE MicroSpin G-50 columns. In a 10 µL reaction, 1 nM purified L. seeligeri Cas13-His6:crRNA complex was combined with 10 nM synthetic target RNA, in buffer containing 10mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 1 mM β-mercaptoethanol, and 5% glycerol, at room temperature for the indicated time. Reactions were quenched by addition of an equal volume of loading dye (95% formamide, 14 mM EDTA, 0.025% SDS, 0.04% bromophenol blue, 0.04% xylene cyanol), then denatured by boiling 5 min, then crash cooled on ice for 1 min before loading on denaturing TBE-Urea PAGE gels with 15% acrylamide. Reactions were exposed to phosphoscreen 1 hour and imaged with Beckman Coulter FLA7000IP Typhoon storage phosphorimager.

Co-immunoprecipitation ∆CRISPR strains of L. seeligeri harboring pAM364 (Cas13-his6 cloned into a pPL2e backbone) and pAM395 (Ptet-AcrVIA1-3xFlag cloned into a pAM326 backbone) along with empty vector controls, were cultured in 50 mL BHI supplemented with 50 µg/mL kanamycin and 100 ng/mL aTc at 30°C until the OD600 reached 0.7. 30 mL culture samples were harvested, pelleted by centrifugation at 8,000 rpm for 2min, and frozen at -80°C. Pellets were resuspended in 0.5 mL ice-cold lysis buffer (50 mM HEPES pH 7.0, 200 mM NaCl, 5 mM MgCl2, 5% glycerol, 1 mg/mL lysozyme, supplemented with Roche cOmplete EDTA-free protease inhibitor cocktail. Samples were incubated at 37°C for 5 min, then placed on ice and lysed by sonication. Insoluble material was pelleted by centrifugation at 15,000 rpm for 1 hr at 4°C. A “load” sample was harvested, then the remaining soluble fraction was applied to 30 µL of pre-equilibrated ANTI-FLAG M2 Affinity Gel (Millipore-Sigma #A2220) for 4 hr at 4°C. The resin was pelleted by centrifugation at 2,000 rpm for 1 min, then the “unbound” sample was harvested. The resin was washed three times by centrifugation and resuspension in 1 mL wash buffer (20 mM HEPES pH 7.0, 200 mM NaCl, 5 mM MgCl2, 5% glycerol). All wash buffer was then removed, and the resin was resuspended in 40 µL 2x Laemmli SDS-PAGE loading buffer lacking β-mercaptoethanol, and boiled for 5 min. The resin was pelleted and supernatant was harvested as the “IP” sample. 5% β-mercaptoethanol was added to all samples before separation on 4-20% acrylamide SDS-PAGE gels. For immunoblot analysis, proteins were transferred to a methanol-activated PVDF membrane, blocked with 5% nonfat milk, and probed with anti-His6 (Genscript #A00186), anti-Flag (Sigma #F3165) and anti-σABacillus subtilis (34) primary antibodies, then with horseradish peroxidase-conjugated anti-mouse (Bio-Rad #170-5047) or anti-rabbit (Bio-Rad #170-

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5046) secondary antibodies. Proteins were detected using Western Lightning Plus-ECL chemiluminescence reagent (Perkin-Elmer).

Electrophoretic mobility shift assay Synthetic RNA substrates were radiolabeled as described for RNA cleavage assays. In vitro RNP assembly was performed for 30 min in a 10 µL reaction at room temperature in the presence of 5mM HEPES pH 7, 10 mM NaCl, 1 mM BME, 5 mM MgCl2, 1 µg/mL bovine serum albumin, 10 µg/mL salmon sperm DNA, and 5% glycerol. Labeled RNA substrates were added at a final concentration of 10 nM, dCas13a (R445A, H450A, R1016A, H1021A) at 500 nM, and AcrVIA1 at 1800 nM. Reactions were placed on ice 1 min, then 10 µL of non-denaturing loading dye (25% glycerol, 0.05% xylene cyanol, 0.05% bromophenol blue, 50 mM HEPES pH 7.0) was added, and samples were electrophoretically separated by 10% acrylamide native PAGE at 4°C. Gels were exposed and imaged as described for RNA cleavage assays.

RNA sequencing and analysis L. seeligeri ∆RM Dspc was infected with ϕLS46 at OD600 of 0.5, MOI of 0.1 in BHImedium containing 5 mM CaCl2 at 30°C. At each time point, 1.5 mL of culture washarvested, pelleted by centrifugation at 8,000 rpm for 2 min, and frozen at -80°C. Toharvest RNA, samples were resuspended in 90 µL of RNase-free phosphate-bufferedsaline containing 2 mg/mL lysozyme, and incubated at 37°C for 3 min. 10 µL of 10%sarkosyl was immediately added to lyse the cells. 300 µL of TRI Reagent (ZymoResearch Direct-Zol RNA Miniprep Plus Kit) was added to each sample, then RNA wasprepared according to the manufacturer’s instructions, eluting in 50 µL RNase-free water.Ribosomal RNA was removed from 1 µg of purified RNA using the NEBNext rRNADepletion Kit (Bacteria) according to the manufacturer’s instructions. After rRNAremoval, samples were concentrated using the Zymo Research RNA Clean andConcentrator-5 Kit according to the manufacturer’s instructions, eluting RNA in 6 µLRNase-free water. Libraries were prepared for deep sequencing using the IlluminaTruSeq Stranded mRNA Library Preparation Kit, skipping mRNA purification andbeginning at the RNA fragmentation step. Quality control of libraries was carried out onan Agilent TapeStation. Paired-end (2x75bp) sequencing was performed on the NextSeqplatform. Raw paired-end reads were mapped to the ϕLS46 genome using Bowtie2 withparameters “very-sensitive” and I = 40. Using a custom script, the coverage at eachposition on the ϕLS46 genome was calculated by tallying a count for each of thepositions covered by each mapped read. Read counts at each genomic position werenormalized to the total number of reads in each library.

Protein Expression and Purification The L. seeligeri type VI CRISPR array alongside Cas13a-His6 or dCas13a-His6 (R445A, H450A, R1016A, H1021A) were cloned into pAM8 as described in Table S1, and conjugated into L. seeligeri ∆spc ∆cas13a. For expression, these strains were cultured at 30 °C in BHI supplemented with 10 µg/mL chloramphenicol for ~24 hr. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5% glycerol, 20 mM imidazole, 7 mM b-mercaptoethanol). The harvested cells were then lysed by an EmulsiFlex-C3 homogenizer (Avestin) and centrifuged at

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20,000 rpm for 30 min in a JA-20 fixed angle rotor (Avanti J-E series centrifuge, Beckman Coulter). The supernatant was applied to 5 mL HisPur™ Cobalt Resin (Thermo Fisher Scientific). The protein was eluted with lysis buffer supplemented with 500 mM imidazole after washing the column with 10 column volumes of lysis buffer. The elution fractions were further dialyzed against buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7mM b-mercaptoethanol), and applied on a 1 mL HiTrap SP Fast flow column (GE Healthcare). Proteins were eluted by a linear gradient from 100 mM to 1 M NaCl in 20 column volumes, and then concentrated in 50 kDa molecular mass cut-off concentrators (Amicon) before further purification over a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT). AcrVIA1 was cloned into a pRSF-Duet-1 vector (Novagen), in which the acrVIA1 gene was attached with N-terminal His6-SUMO tag following an ubiquitin-like protease (ULP1). The vector was transformed into Escherichia coli BL21 (DE3) strain and expressed by induction with 0.25 mM isopropyl-b-D-1-thiogalactopyranoside (GoldBio) at 16 °C for 20 hr. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 7 mM b-mercaptoethanol). The harvested cells were then lysed by an EmulsiFlex-C3 homogenizer (Avestin) and centrifuged at 20,000 rpm for 30 min in a JA-20 fixed angle rotor (Avanti J-E series centrifuge, Beckman Coulter). The supernatant was applied to 5 mL HisTrap Fast flow column (GE Healthcare). The protein was eluted with lysis buffer supplemented with 500 mM imidazole after washing the column with 10 column volumes of lysis buffer and 2 column volumes of lysis buffer supplemented with 40 mM imidazole. The elution fractions were further dialyzed against buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7mM b-mercaptoethanol), and applied on a 5 mL HiTrap Q Fast flow column (GE Healthcare). Proteins were eluted by a linear gradient from 100 mM to 1 M NaCl in 20 column volumes, and then concentrated in 10 kDa molecular mass cut-off concentrators (Amicon) before further purification over a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT). Leptotrichia buccalis Cas13 purification was conducted as previously described(32), and the same samples were used in this study.

In Vitro Assembly of AcrVIA1-Cas13acrRNA Complex To assemble the AcrVIA1-Cas13acrRNA complex, the purified Cas13acrRNA was mixed with AcrVIA1 at the molar ratio of 1:4 and incubated on ice for 30 min. The reconstructed complex was then purified by gel filtration chromatography over a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT). The peak fractions containing AcrVIA1-Cas13acrRNA complex were concentrated in 50 kDa molecular mass cut-off concentrators (Amicon) and added with 5 mM MgCl2 before grid preparation.

Cryo-EM Sample Preparation and Data Acquisition 3.0 µl of ~0.8 mg/ml purified Cas13acrRNA and AcrVIA1-Cas13acrRNA complex were applied onto glow-discharged UltrAuFoil 300 mesh R1.2/1.3 grids (Quantifoil), respectively. The grids were glow-discharged for 60 s at 0.37 mBar, 15 mA in a Pelco

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easiGlow air system (TED PELLA). Grids were blotted with Vitrobot™ filter paper (Electron Microscopy Sciences) for 2s at ~100% humidity and flash frozen in liquid ethane using an FEI Vitrobot Mark IV. Images were collected on FEI Titan Krios electron microscope operated at an acceleration voltage of 300 kV with a Gatan K3 Summit detector with a 1.08 Å pixel size at Memorial Sloan Kettering Cancer Center. The defocus range was set from -1.0 µm to 2.5 µm. Movies were recorded in super-resolution mode at an exposure rate of 19.9 e-/ pixel /s with a total exposure time of 3 s, for an accumulated electron exposure of 49.3 e-/ Å2. Intermediate frames were recorded every 0.075 s for a total number of 40 frames.

Image Processing For Cas13acrRNA dataset, motion correction was performed with MotionCor2 (35). Contrast transfer function parameters were estimated by Ctffind4 (36). All other steps of image processing were performed by RELION 3 (37). Templates for automated particle selection were generated from 2D-averages of ~2,000 manually picked particles. Automated particle selection resulted in 4,085,566 particles from 3,249 images. After two rounds of 2D classification, a total of 1,263,761 particles were selected for 3D classification using the initial model generated by RELION as reference. Particles corresponding to the best class with the highest-resolution features were selected and subjected to the second round of 3D classification. One of 3D classes with good secondary structural features and the corresponding 443,629 particles were polished using RELION particle polishing, yielding an electron microscopy map with a resolution of 3.2 Å after 3D auto-refinement. A preferred orientation of the sample was observed during the cryo-EM reconstruction. Due to the observed directional anisotropy of the reconstruction, the corresponding 443,629 particles were further subjected to another round of 2D classification and some classes with similar orientations were discarded to reduce the impact of the preferred views on the resolution of final reconstruction. 190,543 particles were used for the final reconstruction with a resolution of 3.2 Å after 3D auto-refinement (Fig. S6). The directional resolution volumes were evaluated by the 3D FSC tool (38). The dataset for AcrVIA1-Cas13acrRNA complex was processed by the same procedure as above. Briefly, 4,372,366 particles were autopicked from 3,806 images, 717,725 particles were selected for the final 3D reconstruction after two rounds of 2D and 3D classification, resulting in a AcrVIA1-Cas13acrRNA complex map with an overall resolution of 3.0 Å (Fig. S6). All resolutions were estimated using RELION ‘post-processing’ by applying a soft mask around the protein density and the Fourier shell correlation (FSC) = 0.143 criterion. Local resolution estimates were calculated from two half data maps using cryoSPARC(39). Further details related to data processing and refinement are summarized in table S1.

Atomic Model Building and Refinement For the AcrVIA1-Cas13acrRNA complex, the initial models of Cas13acrRNA and AcrVIA1 were manually built in COOT based on the bulky side chains to register the sequence(40). All models were refined against summed maps using phenix.real_space_refine by applying geometric and secondary structure restraints. For Cas13acrRNA complex, the model of Cas13crRNA in AcrVIA1-Cas13acrRNA complex was

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docked into the cryo-EM density map using UCSF Chimera (41) and then manually rebuilt in COOT (40). All models were refined against summed maps using phenix.real_space_refine (42) by applying geometric and secondary structure restraints. All figures were prepared by PyMol (http://www.pymol.org) or Chimera (41). The statistics for data collection and model refinement are shown in table S1.

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Fig. S1: Acr screen in listeriophages. (A) Diagram of L. seeligeri SLCC3954 genetic elements modified during this study. (B) Detection of phages in lysates of L. seeligeri (11 phages isolated) or L. monocytogenes (4 phages isolated) strains after treatment with mitomycin C. Ten-fold dilutions of lysate were spotted on lawns of L. seeligeri ∆RM Dspc. (C) Confirmation of lysogen formation by the phages isolated in (B). Putative lysogens were treated with mitomycin C to induce and detect integrated prophages. Ten-fold dilutions of induced culture filtrates from each lysogen were spotted on lawns of L. seeligeri ∆RM ∆spc. For ϕLS6 and ϕLS48, lysogens were less stable and spontaneous plaques were detectable during growth of the uninduced lysogen. (D) Transfer of a conjugative plasmid with or without the spc4 target of the L. seeligeri SLCC3954 type VI-A CRISPR-Cas system into different strains: wild-

A

B C

L. seeligeriSLCC3954

(WT)

cas13aType VI-A CRISPR-Cas system

M S N NType I RM system A

M S NType I RM system B

spacers(spc)

ϕLS3

ϕLS4

ϕLS6

ϕLS10

ϕLS14

ϕLS46

ϕLS48

ϕLS51

ϕLS57

ϕLS59

ϕLS62

ϕEGDe

U153

A118

ϕ10403S

Induced from L. seeligeri strains

Induced from L. monocytogenes strains

5 4 3 2 1

x

spacerinsertion cassette

(ΩspcX)

ϕLS48

ϕLS46

ϕLS6

ϕLS57

ϕLS4

ϕ10403S

ϕEGDe

U153

ϕLS62

Induction of lysogens D

WT(ϕLS4)

WT(ϕLS48)

WT(ϕLS6)

WT(U153)

WT(ϕEGDe)

WT(ϕLS46)

WT(ϕLS57)

WT

∆spc∆cas13a

no target spc4 targetconjugative plasmid

WT(ϕ10403S)

ϕLS51

WT(ϕLS51)

RR R R R

R R

E

late lytic early lytic lgϕLS46

acrVIA1 acrIIA6

1 2 3 4

late lytic early lytic lgϕRR4

acrIIA1acrIIA2

1 2 3 4 5 6

anti-CRISPR region

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type (WT), Dspc Dcas13a or WT harboring the ϕLS46,ϕLS57, ϕ10403S, ϕLS4, ϕLS48, ϕLS6, U153, or ϕEGDe prophages. Ten-fold dilutions of transconjugants were plated on selective media. (E) Comparison of the ϕRR4 andϕLS46 genomes, with acr regions highlighted in blue, and genes containing homology to known anti-CRISPRs indicated. The ϕRR4 andϕLS46 genomes are organized into four regions that can be classified according to gene function: these include an early lytic region containing predicted replicases and recombinases involved in phage circularization and genome replication; a late lytic region containing phage structural genes; and a lysogeny region containing transcriptional regulators and a predicted site-specific integrase. In ϕRR4, the fourth region contains two predicted homologs of the Cas9 inhibitors AcrIIA1 and AcrIIA2, and our previous experiments showed that this region is not essential for phage viability but provides inhibitory activity against Cas9 from Streptococcus pyogenes. BLAST searches for each of the ORFs in the analogous operon in ϕLS46 revealed no sequence-similarity for genes of known function, but a more sensitive HHpred search revealed similarity between the gp3 protein and AcrIIA6 from Streptococcus phage DT1. We therefore hypothesized that this operon may be an anti-CRISPR region in the ϕLS46 genome.

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Fig. S2: ϕLS46 gp3 encodes an AcrIIA6 homolog that represses acr transcription. (A) Diagram of ϕLS46 acr operon containing four genes controlled by Pacr promoter.The plasmid pPacr-lacZ contains Pacr fused to a lacZ reporter to measure transcriptionfrom the promoter. Pacr transcription was measured in the presence and absence of pgp3,which expresses the gp3 gene. (B) Autorepression of Pacr transcription by Gp3. β-galactosidase activity was measured in the absence of lacZ reporter (-), pPacr-lacZ, orpPacr-lacZ in the presence of pgp3. Data from 3 biological replicates are shown.

-

pPac

r-lacZ

pPac

r-lacZ

+ pgp3

0

20

40

60

80

100

beta

gal

acto

sida

se a

ctiv

ity (M

U)

ϕLS46Pacr

lacZ

A BacrVIA1

acrIIA6

gp1 gp2 gp3 gp4

pPacr-lacZPacr

gp1

pgp3Ptet

gp3

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Fig. S3: AcrVIA1 inhibits type VI-A CRISPR-Cas targeting of plasmids and phages.

A

B

C∆RM ∆spc

ϕLS59

ϕLS46

100 10-1 10-2 10-3 10-410-510-610-7

D

phage dilution

∆RM Ωspc59

100 10-1 10-2 10-3 10-410-510-610-7phage dilution

late lytic early lytic lg

ϕLS46

E1

A1

E2

1 2 3 4

L2 L4L1

L5 L6 L7 L8

ΩspcA1

ΩspcE1

ΩspcE2

ΩspcL1

ΩspcL2

ΩspcL4

ΩspcL5

ΩspcL6

ΩspcL7

ΩspcL8

100 10-1 10-2 10-3 10-410-510-610-7phage dilution

ΦLS46ΦLS46 ∆acrVIA1

∆spc

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

ΦLS46ΦLS46 ∆acrVIA1

no target spc2 targetconjugative plasmid

∆RM ∆spc

∆RM Ωspc2

no target spc4 targetconjugative plasmid

∆RM ∆spc

∆RM Ωspc4

14

(A) Transfer of a conjugative plasmid with or without the spc2 or spc4 target of the L.seeligeri SLCC3954 type VI-A CRISPR-Cas system into strains ∆RM Dspc, ∆RM Wspc2or ∆RM Wspc4. Ten-fold dilutions of transconjugants were plated on selective media. (B)Detection of phage propagation after spotting ten-fold dilutions of the phages ϕLS46 orϕLS59, on lawns of L. seeligeri ∆RM Dspc or ∆RM Wspc59. (C) Schematic of theϕLS46 genome showing the four main transcription units (acr in blue; lysogeny cassettein red; early- and late-expressed lytic genes in green and purple, respectively). Thelocation of the targets of the spacers used in this study are shown in grey. Top and bottomlocations refer to the DNA strand that is transcribed to produce a target transcript that iscomplementary to the crRNA derived from each spacer. (C) Same as (B) but spottingphages ϕLS46 or ϕLS46 DacrVIA1 on lawns of bacteria expressing crRNAs from thespacers shown in (C).

15

Fig. S4: Purification and functional test of Cas13a-His6 and AcrVIA1-3xFLAG (A) SDS-PAGE of Cas13a-His6 after expression and purification from L. seeligeri. M, protein size marker; E, elution. (B) Same as (A) but for AcrVIA1 purified from E. coli. (C) Transfer of a conjugative plasmid with or without the spc4 target of the L. seeligeri SLCC3954 type VI-A CRISPR-Cas system into L. seeligeri strains expressing Cas13a-His6 and harboring either an empty vector or p(AcrVIA1-3xFLAG). Three ten-fold dilutions of transconjugants were plated on selective media.

A B

C

M E

AcrVIA1

25015010075503725201510

25015010075

5037

25201510

kDa

Cas13a

M EkDa

no target spc4 targetconjugative plasmid

Cas13a-His6(empty vector)

Cas13a-His6p(AcrVIA1-3xFLAG)

16

Fig. S5: AcrVIA1 directly interacts with Cas13acrRNA on complex formation. Gel filtration profiles of AcrVIA1 (upper panel), Cas13acrRNA (middle panel), and AcrVIA1 bound to Cas13acrRNA (bottom panel). Lower insert shows SDS-PAGE profile of AcrVIA1 with Cas13acrRNA after gel filtration.

Cas13a

AcrVIA1

UV 280UV 260

AcrVIA1

Cas13acrRNA

AcrVIA1+Cas13acrRNA

10 15 tret (min)

17

Fig. S6: Cryo-EM Reconstruction of Cas13acrRNA and AcrVIA1-Cas13acrRNA complexes. (A) Flow chart of image processing for Cas13acrRNA binary complex. (B and E) FourierShell Correlation (FSC) curve of Cas13acrRNA (B) and AcrVIA1-Cas13acrRNA complex (E)and between two half maps that were calculated from two half datasets, and between thecryo-EM map and corresponding model. (C and F) Euler angle distribution of cryo-EMparticles for calculating the final EM map of Cas13acrRNA (C) and AcrVIA1-Cas13acrRNA

complex (F). The position of each sphere relative to the density map (gray in the center)corresponds to its angular assignment, with the radius of sphere proportional to the

Resolution (A)

2.8 3.0 3.5 4.0 5.0

180

90

Dire

ctio

nal F

ourie

r She

ll C

orre

latio

n

Perc

enta

ge o

f per

Ang

le F

SC (%

)

Spatial Frequency (A-1)

0.0

0.2

0.4

0.6

0.8

1.0

0

20

40

60

80

0.1 0.2 0.3 0.4

0.143 FSC 3.0 A

Histogram of Directional FSC

Global FSC+1 S.D. from Mean of Directional FSC_

Sphericity= 0.974

Resolution (A)

2.8 3.2 4.0 5.0 6.0

180

90

Dire

ctio

nal F

ourie

r She

ll C

orre

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Perc

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SC (%

)

Spatial Frequency (A-1)

0.0

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0

20

40

60

80

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0.143 FSC 3.2 A

Histogram of Directional FSC

Global FSC+1 S.D. from Mean of Directional FSC_

Sphericity= 0.964

3.2 A

3D refinement and2D classification

particle polishing

190,543 particles

(22.7%)(35.0%)(7.0%)

(27.4%) (3.1%) (4.8%)

3D classification

1,263,761particles

(2 rounds)

3,249 movies

Autopick

4,085,566 particles

(2 rounds)2D classification

Cas13acrRNA binary complex

G

F

E

D

C

B

A

18

number of particles in that orientation. Preferred orientations of both samples were observed during the cryo-EM reconstruction. (D and G) Final 3D reconstructed map of Cas13acrRNA (D) and AcrVIA1-Cas13acrRNA complex (G) colored according to local resolution.

19

Fig. S7: crRNA alignement in Cas13acrRNA (A) Cryo-EM structure of Cas13acrRNA. The insert shows detailed interactions between Cas13a and the cleavage site of pre-crRNA. The repeat and spacer regions within crRNA are shown in black and red, respectively. (B) Structure of crRNA in Cas13acrRNA. A stacking alignment is observed for segments 8 to 12 and 13 to 19. (C) Transfer of a conjugative plasmid with or without the spc4 target of the L. seeligeri type VI-A CRISPR-Cas system into Dspc Dcas13a harboring plasmids expressing wild-type or mutant (R1048A, K1049A) Cas13a. (D) Transfer of a conjugative plasmid with or

20

without the spc4 target of the L. seeligeri type VI-A CRISPR-Cas system into wild-type L. seeligeri harboring pgp2 plasmids expressing wild-type or mutant different mutantversions of AcrVIA1.

21

Fig. S8: EM densities for representative interfaces between AcrVIA1 and Cas13acrRNA. (A) EM densities for interface shown in Fig. 3F. (B) EM densities for interface shown in Fig. 3G. (C and D) EM densities for interface shown in Fig. 3H. The maps are contoured at 5σ.

Y213

Y197

AcrVIA1

LinkerR881

V892

AcrVIA1

Linker

C18

C13

R90

S68

F130

S135

P91

G22

U20N259 K261

N43

Y39

S93

I97

DC

BA

22

Fig. S9: Structural comparison of Cas13acrRNA and AcrVIA1-Cas13acrRNA complex. (A) Structure comparison following superposition of Cas13acrRNA and AcrVIA1-Cas13acrRNA complex. Vector length correlates with the domain movement scale. AcrVIA1 and crRNA originate from the AcrVIA1-Cas13acrRNA complex. (B) Superposition of crRNA in Cas13acrRNA (in grey) and AcrVIA1-Cas13acrRNA complex (in red). (C) Detailed presentation of binding mode of 3’ end of crRNA in Cas13acrRNA (in grey) and AcrVIA1-Cas13acrRNA complex (in red).

F69

17 18

20

21

22

2021

V94

Y39H128

5’

3’

2021

22NTD

Helical-1

HEPN-1

Helical-2

LinkerHEPN-2

crRNA

AcrVIA1

AcrVIA1

CBA

23

Fig. S10: Comparison of Listeria seeligeri Cas13a with Leptotrichia buccalis Cas13a. (A) AcrVIA1 does not inhibit Leptotrichia buccalis Cas13a. cis-RNA cleavage time course with purified L. buccalis Cas13a, AcrVIA1 and/or AcrVIA1-3xFLAG using radiolabeled spc2-target RNA substrates. Cas13a was present at 10nM, synthetic crRNA at 10nM, and AcrVIA1 or AcrVIA1-3xFLAG was added at 400, 100, 10, or 1 nM. The

3’

NTD

NTD

AcrVIA1AcrVIA1

AcrVIA1Helical-1

HEPN-1Helical-2LinkerHEPN-2

3’

3’

NTD

NTD

Helical-1

HEPN-1

Helical-2

Linker

HEPN-2 crRNA

ED

CB

L. buccalis Cas13a

AcrVIA1-3xFLAGAcrVIA1

spc2 target RNA ++ + ++ + ++

+++ +

+ + ++ + +

+A

24

products of degradation after 5, 10, and 20 minutes were analyzed by denaturing PAGE. (B) Structural comparison following superposition of L. seeligeri Cas13crRNA (in color) with L. buccalis Cas13acrRNA (in grey, PDB 5XWY). The insert presents the structural differences between NTD domains in the two species of Cas13a. (C) Structural comparison of crRNA in L. seeligeri Cas13acrRNA (in color) with L. buccalis Cas13acrRNA (in grey). (D) Structural comparison following superposition of L. seeligeri AcrVIA1-Cas13acrRNA (in color) with L. buccalis Cas13acrRNA (in grey). The insert presents the structural differences of the NTD domain of Cas13a in the two systems. The clashes between AcrVIA1 and NTD domain in L. buccalis Cas13acrRNA (in grey) are shown in red circle. (E) Structure comparison of crRNA in Cas13acrRNA (in color) with L. buccalis Cas13acrRNA (in grey). The missing interactions between AcrVIA1 and crRNA in L. buccalis Cas13acrRNA (in grey) are shown in green circle.

25

Fig. S11: AcrVIA1 enables full phage escape from type VIA CRISPR-Cas immunity. (A) Efficiency of plaquing (relative to the number of plaques formed in lawns of L. seeligeri (∆RM Dspc)) of phages ϕLS46 or ϕLS46 DacrVIA1 in lawns of bacteria expressing spcL1, spcL2, spcL4, spcL5, spcL6, spcL7 or spcL8. These spacers target transcripts produced by the late-expressed region of ϕLS46; shown in Figure S3C (B-H) Growth of L. seeligeri (∆RM WspcL1), (∆RM WspcL2), (∆RM WspcL4), (∆RM WspcL5), (∆RM WspcL6), (∆RM WspcL7) and (∆RM WspcL8), measured as OD600 over time, infected with ϕLS46 or ϕLS46 DacrVIA1 phages, or uninfected. The average curves of three biological replicates are reported, with +/- SEM values shown in lighter colors.

A

B C D

E F

1EO

PΦLS46ΦLS46 ΔacrVIA1

ΦLS46ΦLS46 ΔacrVIA1uninfected

Δspc spcL1 spcL2 spcL4 spcL5 spcL6 spcL7 spcL810-8

10-6

10-4

10-2

0 5 10 15 20

0.2

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time (hours)

OD

600

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600

0 5 10 15 20

0.2

2

time (hours)

OD

600

G H

spcL1 spcL2 spcL4

spcL5 spcL6 spcL7 spcL8

26

Fig. S12: RNA-seq duringϕLS46 infection. (A) Strand-specific read coverage of phage-mapped reads is plotted along the ϕLS46 genome, and normalized to total reads in each sample. Targeting location of the spacers used in this study is also shown. The average of two biological replicates is shown. (B) Zoom-in of read coverage for the acr region of ϕLS46, showing co-operonic transcription of the four genes at both early and late time points during the phage life cycle. The average of two biological replicates is shown.

E1

A1

E2L2 L4

L1L5 L6 L7 L8

-6-4-20

0246

log1

0 no

rmal

ized

read

s

acr late lytic early lytic lg

10 min

90 min

E1

A1

E2L2 L4

L1L5 L6 L7 L8

-6-4-20

0246

log1

0 no

rmal

ized

read

s

acr late lytic early lytic lg

30 min

E1

A1

E2L2 L4

L1L5 L6 L7 L8

-6-4-20

0246

log1

0 no

rmal

ized

read

s

acr late lytic early lytic lg

ϕLS46 genome acr operon

1gp1 acrVIA11gp1 gp3 gp40

2

4

6

log1

0 no

rmal

ized

read

s

0

2

4

6

log1

0 no

rmal

ized

read

s1gp1 acrVIA11gp1 gp3 gp4

0

2

4

6

log1

0 no

rmal

ized

read

s

1gp1 acrVIA11gp1 gp3 gp4

A B

27

Table S1. Cryo-EM Statistics for Data Collection and Model Refinement. Cas13acrRNA AcrVIA1-Cas13acrRNA Data collection and processing Microscope Titan Krios Titan Krios Detector Gatan K3 Gatan K3 Automation software SerialEM SerialEM Nominal magnification 22,500 22,500 Calibrated magnification 47,262 47,262 Voltage (kV) 300 kV 300 kV Electron exposure (e-/Å2) 49.3 49.3 Exposure rate ( e-/pixel/s) 19.9 19.9 Number of frames collected 40 40 Defocus range (µm) -1.0 to -2.5 -1.0 to -2.5 Pixel size (Å) 1.08 1.08 Micrographs (collected/used) 4,465/3,249 4,398/3,806 Total extracted particles (no.) 4,085,566 4,372,366 Refined particles (no.) 2,929,591 3,070,831 Final particles (no.) 190,543 717,725 Estimated accuracy of translations/rotations 0.749/2.479 0.513/2.160 Resolution (global) FSC 0.5 (unmasked/masked, Å) 4.2/3.6 3.6/3.2 FSC 0.143 (unmasked/masked, Å) 3.6/3.2 3.2/3.0 Resolution range (local, Å) 2.8~6.5 2.8~5.5 Resolution range due to anisotropy (Å) 3.2~3.5 2.8~3.0 Map sharpening B factor (Å2) -126.35 -100.0 Refinement Refinement package Phenix Phenix Model resolution (Å) 3.22 2.98 FSC threshold 0.5 0.5 Map Correlation Coefficient 0.77 0.79 Model composition Protein residues 1,007 1,257 Nonhydrogen atoms 9,429 11,404 R.m.s. deviations Bond lengths (Å) 0.004 0.009 Bond angles (°) 0.864 1.107 Validation MolProbity score 1.46 1.47 Clashscore 2.55 2.21 Poor rotamers (%) 0.21 0.51 Ramachandran plot Favored (%) 92.2 92.1 Allowed (%) 7.8 7.9 Disallowed (%) 0 0 C-β deviations (%) 0 0 CaBLAM outliers (%) 0.88 0.48

28

EM-Ringer Score 3.50 3.29 Average B-factors protein (Å2) 37.17 31.67 RNA (Å2) 37.81 30.47 PDB code 6VRC 6VRB EMDB code EMD-21367 EMD-21366

29

Table S2. Bacterial strains used in this study. Species Strain Alias Genotype Origin L. seeligeri LS1 wild-type wild type ATCC SLCC3954 L. seeligeri LS2 RR4 wild type (43) L. seeligeri LS3 FSL N1-067 wild type FSL, Cornell L. seeligeri LS4 FSL H6-011 wild type FSL, Cornell L. seeligeri LS5 FSL H6-169 wild type FSL, Cornell L. seeligeri LS6 ATCC 51334 wild type ATCC L. seeligeri LS7 ATCC 51335 wild type ATCC L. seeligeri LS8 FSL S4-0171 wild type FSL, Cornell L. seeligeri LS9 FSL B8-0099 wild type FSL, Cornell L. seeligeri LS10 FSL C7-0024 wild type FSL, Cornell L. seeligeri LS11 FSL S10-0305 wild type FSL, Cornell L. seeligeri LS12 FSL B8-0287 wild type FSL, Cornell L. seeligeri LS13 FSL C7-0049 wild type FSL, Cornell L. seeligeri LS14 FSL H6-0007 wild type FSL, Cornell L. seeligeri LS15 FSL C7-0030 wild type FSL, Cornell L. seeligeri LS16 FSL S4-0116 wild type FSL, Cornell L. seeligeri LS17 FSL S10-0823 wild type FSL, Cornell L. seeligeri LS18 FSL B8-0050 wild type FSL, Cornell L. seeligeri LS19 FSL C7-0481 wild type FSL, Cornell L. seeligeri LS20 FSL T4-0118 wild type FSL, Cornell L. seeligeri LS21 FSL S4-0037 wild type FSL, Cornell L. seeligeri LS22 FSL C7-0115 wild type FSL, Cornell L. seeligeri LS23 FSL S4-0039 wild type FSL, Cornell L. seeligeri LS24 FSL S10-1784 wild type FSL, Cornell L. seeligeri LS25 FSL B8-0253 wild type FSL, Cornell L. seeligeri LS26 FSL C7-0156 wild type FSL, Cornell L. seeligeri LS27 FSL S10-0300 wild type FSL, Cornell L. seeligeri LS28 FSL C7-0082 wild type FSL, Cornell L. seeligeri LS29 FSL L5-0045 wild type FSL, Cornell L. seeligeri LS30 FSL S10-0030 wild type FSL, Cornell L. seeligeri LS31 FSL F6-1136 wild type FSL, Cornell L. seeligeri LS32 FSL L5-0086 wild type FSL, Cornell L. seeligeri LS33 FSL S10-1611 wild type FSL, Cornell L. seeligeri LS34 FSL R9-8498 wild type FSL, Cornell L. seeligeri LS35 FSL C7-0251 wild type FSL, Cornell L. seeligeri LS36 FSL M6-0039 wild type FSL, Cornell L. seeligeri LS37 FSL S10-2970 wild type FSL, Cornell

30

L. seeligeri LS38 FSL C7-0134 wild type FSL, Cornell L. seeligeri LS39 FSL C7-0462 wild type FSL, Cornell L. seeligeri LS40 FSL S10-1769 wild type FSL, Cornell L. seeligeri LS41 FSL H6-0027 wild type FSL, Cornell L. seeligeri LS42 FSL R8-6874 wild type FSL, Cornell L. seeligeri LS43 FSL S4-0544 wild type FSL, Cornell L. seeligeri LS44 FSL S4-0616 wild type FSL, Cornell L. seeligeri LS45 FSL S4-0939 wild type FSL, Cornell L. seeligeri LS46 FSL C7-0218 wild type FSL, Cornell L. seeligeri LS47 FSL C7-0499 wild type FSL, Cornell L. seeligeri LS48 FSL S6-0001 wild type FSL, Cornell L. seeligeri LS49 FSL L5-0058 wild type FSL, Cornell L. seeligeri LS50 FSL A5-0405 wild type FSL, Cornell L. seeligeri LS51 FSL C7-0081 wild type FSL, Cornell L. seeligeri LS52 FSL L5-0018 wild type FSL, Cornell L. seeligeri LS53 FSL R2-0626 wild type FSL, Cornell L. seeligeri LS54 FSL R8-7055 wild type FSL, Cornell L. seeligeri LS55 FSL C7-0498 wild type FSL, Cornell L. seeligeri LS56 FSL R9-4405 wild type FSL, Cornell L. seeligeri LS57 FSL S10-0788 wild type FSL, Cornell L. seeligeri LS58 FSL C7-0167 wild type FSL, Cornell L. seeligeri LS59 FSL M6-0259 wild type FSL, Cornell L. seeligeri LS60 FSL S10-0889 wild type FSL, Cornell L. seeligeri LS61 FSL B8-0378 wild type FSL, Cornell L. seeligeri LS62 N/A wild type FSL, Cornell L. seeligeri LS1 N/A ∆spc ∆cas13a (32) L. seeligeri LS1 N/A ∆RM, ∆spc This Study L. seeligeri LS1 94°::Ptet-spc4 (11)

L. seeligeri LS1 ∆spc ∆cas13a, 94°::Ptet-spc4 (11)

E. coli Rosetta2 N/A

F- ompT hsdSB(rB- mB-) gal dcm (DE3) pRARE2 (CamR) Millipore-Sigma

E. coli SM10

thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu Km λpir (30)

E. coli b2163 dapA::erm-pir116 RP4-2-Tc::Mu Km (33)

*FSL, Food Science Lab

31

Table S3. Phages used in this study. Phage Host Genotype Origin ϕLS3 L. seeligeri wild type This Study, induced from L. seeligeri LS3 ϕLS4 L. seeligeri wild type This Study, induced from L. seeligeri LS4 ϕLS6 L. seeligeri wild type This Study, induced from L. seeligeri LS6 ϕLS10 L. seeligeri wild type This Study, induced from L. seeligeri LS10 ϕLS14 L. seeligeri wild type This Study, induced from L. seeligeri LS14 ϕLS46 L. seeligeri wild type This Study, induced from L. seeligeri LS46 ϕLS48 L. seeligeri wild type This Study, induced from L. seeligeri LS48 ϕLS51 L. seeligeri wild type This Study, induced from L. seeligeri LS51 ϕLS57 L. seeligeri wild type This Study, induced from L. seeligeri LS57 ϕLS59 L. seeligeri wild type This Study, induced from L. seeligeri LS59 ϕLS62 L. seeligeri wild type This Study, induced from L. seeligeri LS62 ϕEGDe L. seeligeri wild type This Study, induced from L. monocytogenes EGDe ϕ10403S L. seeligeri wild type This Study, induced from L. monocytogenes 10403S U153 L. seeligeri wild type (33) A118 L. seeligeri wild type (33) ϕLS46 L. seeligeri ∆gp1-4 This Study, escaper of pAM379 targeting

ϕLS46 L. seeligeri ∆acrVIA1 This Study, pAM386-recombinant escaper of pAM377 targeting

32

Table S4. Plasmids used in this study.

Plasmid Description Source Construction Notes

pAM8 E. coli-Listeria shuttle vector (cat) Meeske, et al. 2018 Mol Cell N/A

pAM52 spc2 cat target in pAM8 Meeske, et al. 2018 Mol Cell N/A

pAM54 spc4 cat target in pAM8 Meeske, et al. 2018 Mol Cell N/A

pAM326 E. coli-Listeria shuttle vector (kan) This Study

Gibson assembly of kanR (oAM1055/1056), pSK1 ori (oAM1057/1058), oriT (oAM1059/1060)

pPL2e Listeria integrating vector (erm) Lauer, et al. 2002 J Bacteriol. N/A

pAM215 lacZ suicide vector (cat) Meeske, et al. 2019 Nature N/A

pAM305 Type VI RSR in pPL2e with BsaI sites for spacer cloning This Study

Gibson assembly of BamHI/SalI digested pPL2e and BsaI RSR synthetic fragment (oAM462/451)

pAM375 pgp1-4 This Study

Gibson assembly of HindIII/EagI digested pAM326 and gp1-4 region (oAM1176/1177)

pAM388 pgp1 This Study

Gibson assembly of HindIII/EagI digested pAM326, Ptet (oAM1107/572) and gp1 (oAM1178/1179)

pAM383 pgp2 This Study

Gibson assembly of HindIII/EagI digested pAM326, Ptet (oAM1107/572) and gp2 (oAM1180/1181)

pAM384 pgp3 This Study

Gibson assembly of HindIII/EagI digested pAM326, Ptet (oAM1107/572) and gp3 (oAM1182/1183)

pAM385 pgp4 This Study

Gibson assembly of HindIII/EagI digested pAM326, Ptet (oAM1107/572) and gp4 (oAM1184/1185)

pAM449 spc2 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM464/465

pAM450 spc4 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM770/771

pAM422 spcA1 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1270/1271

pAM380 spcE1 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1192/1193

pAM381 spcE2 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1194/1195

pAM382 spcL1 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1196/1197

pAM434 spcL2 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1370/1371

pAM436 spcL4 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1374/1375

pAM437 spcL5 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1376/1377

pAM438 spcL6 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1378/1379

33

pAM439 spcL7 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1382/1383

pAM440 spcL8 targeting ϕLS46 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM1384/1385

pAM442 Array of 3 spc (spcA1, spcE1, spcE2) targeting ϕLS46 in pAM305 This Study

Gibson assembly of BamHI/SalI digested pPL2e and 3spc synthetic fragment

pAM365 spc59 targeting ϕLS59 in pAM305 This Study Ligation of BsaI-digested pAM305 and annealed oAM559/560

pAM386 repair template for acrVIA1 deletion This Study

Gibson assembly of HindIII/EagI digested pAM326 and ∆gp2 upstream (oAM1176/1206) and ∆gp2 downstream (oAM1207/1177)

pAM307

type II CRISPR system (S. pyogenes) in pAM8 with SapI sites for spacer cloning

Meeske, et al. 2019 Nature N/A

pAM377 Cas9 spc targeting gp4 of ϕLS46 in pAM307 This Study

Ligation of SapI-digested pAM307 and annealed oAM1186/1187

pAM379 Cas9 spc targeting gp2 of ϕLS46 in pAM307 This Study

Ligation of SapI-digested pAM307 and annealed oAM1190/1191

pAM364 CRISPR array + Cas13-His6 in pPL2e This Study

Gibson assembly of BamHI/SalI digested pPL2e and CRISPR locus (oAM462/1111)

pAM395 AcrVIA1-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 and AcrVIA1-3xflag synthetic fragment

pAM414 pPacr-lacZ in pAM8 This Study

Gibson assembly of BamHI/EagI digested pAM8, Pacr (oAM1256/1257) and lacZ (oAM304/702)

pAM417 CRISPR array + Cas13-His6 in pAM8 (for purification) This Study

Gibson assembly of BamHI/EagI digested pAM8 and CRISPR locus (oAM631/1260)

pAM419 CRISPR array + dCas13-His6 in pAM8 (for purification) This Study

Gibson assembly of BamHI/EagI digested pAM8 and CRISPR locus (oAM631/1260, amplified from dCas13 strain)

pAM452 AcrVIA1(Y39A, S40A, N43A, S93A, Q96A)-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 and mutant Acr allele synthetic fragment

pAM453 AcrVIA1(∆E131-E134)-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 and mutant Acr allele synthetic fragment

pAM454 AcrVIA1(I2A, Y4A)-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 and mutant Acr allele synthetic fragment

pAM455 AcrVIA1(S68A, F69A)-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 and mutant Acr allele synthetic fragment

pAM456 AcrVIA1(∆N173-N232)-3xFlag in pAM326 This Study

Gibson assembly of HindIII/EagI digested pAM326 mutant Acr allele synthetic fragment

34

Table S5. Oligonucleotide primers used in this study.

Primer Sequence

oAM224 GGCGTGAAAATCAACGACCC

oAM225 TTTGCTTCAATGTCGCCAGC

oAM304 ATGAATGTGTTATCCTCAATTTGTT

oAM451 GTACCGGGCCCCCCCTCGAGGTTTTGTGATGCATGATTTGTTCTG

oAM462 CGGCCGCTCTAGAACTAGTGAGTGCCAAGTAACTGTGC

oAM464 AAACTTAGTCAACCCCTCGCTGCATTTTCACATT

oAM465 TTACAATGTGAAAATGCAGCGAGGGGTTGACTAA

oAM559 TTACAAAAAGAAGCTAAAGAAGTAAAAGAAGAAG

oAM560 AAACCTTCTTCTTTTACTTCTTTAGCTTCTTTTT

oAM572 GTTTAACTCACTCTATCAATGATAGAGAGCTTATTTTAATTAT

oAM631 GCAAGACGTAGCCCAGCGCGTCAGTGCCAAGTAACTGTGC

oAM770 AAACCATATTTCCAAACTCCACTTTGACTACACC

oAM702 GCGACCACACCCGTCCTGTGCTAAACCTTCCCGGCTTC

oAM771 TTACGGTGTAGTCAAAGTGGAGTTTGGAAATATG

oAM1055 CCCAGCGAACCATTTGAGG

oAM1056 TTATGCATCCCTTAACTTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCC

oAM1057 TACTGGATGAATTGTTTTAAGTTAAGGGATGCATAAACTGCATC

oAM1058 ATCCATGGCCTGGATCCATCAAGCTTAAAATTAGTATAATTATAGCACGAGCTCTGAT

oAM1059 GCTTGATGGATCCAGGCCATGGATGGCGGCCGCCTCTCGCCTGTCCCCTC

oAM1060 ACCTCAAATGGTTCGCTGGGTTAATCGCTTGCCCTCATCTGT

oAM1107 CTATAATTATACTAATTTTACATCACGGAAAAAGGTTATGC

oAM1111 TACCGGGCCCCCCCTCGAGGTTAATGATGATGGTGGTGATGCTTCATCGTTAATAGCGTTCTTACTAG

oAM1176 CGTGCTATAATTATACTAATTTTATATATTCGTTGACTACATTTTTCTACTATAATAGAAG

oAM1177 TGAGGGGACAGGCGAGAGGCTATTACATTACAGCTAGTGATAAGTATGTACAG

oAM1178 CATTGATAGAGTGAGTTAAACACTTTACAAGTTTAACATATTATGTTAATATATAAATATAGC

oAM1179 TGAGGGGACAGGCGAGAGGCGCCCATTTATTATTTGTTATATTTGTTGTAAAAATTTAC

oAM1180 ATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTAC

oAM1181 GAGGGGACAGGCGAGAGGCTTAATTTAGCTCCTCTTTTAAAATTTGTTTGC

oAM1182 CATTGATAGAGTGAGTTAAACTAAAAGAGGAGCTAAATTAAATGACAAATTTAATC

oAM1183 GAGGGGACAGGCGAGAGGCATTTATATAAAAAGTTTAAATTTCTGCATTAAATTCTTGG

oAM1184 CATTGATAGAGTGAGTTAAACTTTAGGAGGAATTAAAATGAATAAATTTGCAT

oAM1185 TGAGGGGACAGGCGAGAGGCCTGATGTATTATATTAATCCTTGCTCTTTTTTATC

oAM1186 AAACCAAGGTAAATTTGAAGTACAGATTCAAAAA

oAM1187 AACTTTTTGAATCTGTACTTCAAATTTACCTTGG

oAM1190 AAACTCATTTCTTTTGATTTCTAAAAATATAGTA

oAM1191 AACTACTATATTTTTAGAAATCAAAAGAAATGAG

35

oAM1192 TTACAACAAATATGGAAAGTAATTTATTTAAATT

oAM1193 AAACAATTTAAATAAATTACTTTCCATATTTGTT

oAM1194 TTACTTTATTCGATAAAGACAGCACGAATAAAAA

oAM1195 AAACTTTTTATTCGTGCTGTCTTTATCGAATAAA

oAM1196 TTACCAACTATCGAAATTGATTGGAAAATAAATA

oAM1197 AAACTATTTATTTTCCAATCAATTTCGATAGTTG

oAM1206 CTCCTCTTTTAAAATTTGTTTGTAGATCATCGTTTTTTCCTCCTATAGTC

oAM1207 CGATGATCTACAAACAAATTTTAAAAGAGGAGCTAAATTAAATG

oAM1256 AAGACGTAGCCCAGCGCGTCTATATTCGTTGACTACATTTTTCTACTATAATAGAAG

oAM1257 CAAATTGAGGATAACACATTCATCGTTTTTTCCTCCTATAGTCGGTTATC

oAM1260 GCGACCACACCCGTCCTGTGTTAATGATGATGGTGGTGATGCTTCATCGTTAATAGCGTTCTTACTAG

oAM1270 TTACGATGATTAAAATGATGACTGAAAAACAAAA

oAM1271 AAACTTTTGTTTTTCAGTCATCATTTTAATCATC

oAM1370 TTACTACAAACAACACGTATAAAAACAAAAAATT

oAM1371 AAACAATTTTTTGTTTTTATACGTGTTGTTTGTA

oAM1374 TTACTAATAGAAGAGGTTGTAAAAGATTGTAAAG

oAM1375 AAACCTTTACAATCTTTTACAACCTCTTCTATTA

oAM1376 TTACTTTATTACAATATATTCCGCAAACATTCAA

oAM1377 AAACTTGAATGTTTGCGGAATATATTGTAATAAA

oAM1378 TTACTTGCAAAAAGAAATTACAAAGGACTTTCATT

oAM1379 AAACAATGAAAGTCCTTTGTAATTTCTTTTTGCAA

oAM1382 TTACGAATCTGGACATTTAATTGATTTTGCAAAA

oAM1383 AAACTTTTGCAAAATCAATTAAATGTCCAGATTC

oAM1384 TTACTAAAGCAATAGCGAAATACATTGAAGAAAA

oAM1385 AAACTTTTCTTCAATGTATTTCGCTATTGCTTTA

oAM1415 GTTTGCCTAAAAATGCGCTTAAATCAGC

oAM1416 CGTCGTGCAATGCTAATCAAGATTGC

36

Table S6. RNA oligonucleotides used in this study RNA oligo Sequence

non-target GGCACACCCGCAGGGAGGAGCCAAAGCACGUCCAUCAUUCCGUUGCCACAGCAGAAGCCC

spc2 target GGCACACCCGCAGGGAAAUGUGAAAAUGCAGCGAGGGGUUGACUAACACAGCAGAAGCCC

37

Table S7. Synthetic gene fragments used in this study. Description Sequence

Type VI RSR in with BsaI sites for spacer cloning

ccgggccccccctcgaggTTTTGTGATGCATGATTTGTTCTGTATTATCTTGCATTTCATTTTCATAAACTAACTTGCCCCCGTTTTTATCCCTAGAAATTAGTACTTTTTTTCTATCAACCTCTACTTTAGTAATTCTCATAGTTTTCACCTCAATGATTTTTTTCTCTCTTCTATTGTACATATAATCACAAAAAAATAAAACACCTAAATGATGGATAAGCGTTTTTATACTTATCCAcatTAGACGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACTGAGACCAGTCTCGGAAGCTCAAAGGTCTCAGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACCCTACTTAATAATAGTAATTAAAACAACCAATGTAAAGGATATAATCAATATATTTAAAGTTTGCACGAGAATGCAATCATTTTATTCATAAATATCATATCATTTATAAGCTCTATTTTCCATTTTCTAAGGCTAATAAATAAAACTGCTGTACCTATGGATCTAAGGAAGACTTATGCACACAGTACAGCAACTTTTCAGCATGATTTGTGTTAAAAACATTTAATTTATTGTAGCAATTTCTTGAGAATCTATAAACCATTTTCCGGTTTCAATTTTAACCAACTCCAAATCTAACAATACTTGATTTTCGGTTTTCTTTTGATTGTCGACTTGAAAAGCAGACCAGGCTCGCACAGTTACTTGGCACTcactagttctagaGCggcc

Array of 3 spc (spcA1, spcE1, spcE2) targeting ϕLS46

TTTTGTGATGCATGATTTGTTCTGTATTATCTTGCATTTCATTTTCATAAACTAACTTGCCCCCGTTTTTATCCCTAGAAATTAGTACTTTTTTTCTATCAACCTCTACTTTAGTAATTCTCATAGTTTTCACCTCAATGATTTTTTTCTCTCTTCTATTGTACATATAATCACAAAAAAATAAAACACCTAAATGATGGATAAGCGTTTTTATACTTATCCAcatTAGACGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACGATGATTAAAATGATGACTGAAAAACAAAAGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACTTTATTCGATAAAGACAGCACGAATAAAAAGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACAACAAATATGGAAAGTAATTTATTTAAATTGTTTTAGTCCTCTTTCATATAGAGGTAGTCTCTTACCCTACTTAATAATAGTAATTAAAACAACCAATGTAAAGGATATAATCAATATATTTAAAGTTTGCACGAGAATGCAATCATTTTATTCATAAATATCATATCATTTATAAGCTCTATTTTCCATTTTCTAAGGCTAATAAATAAAACTGCTGTACCTATGGATCTAAGGAAGACTTATGCACACAGTACAGCAACTTTTCAGCATGATTTGTGTTAAAAACATTTAATTTATTGTAGCAATTTCTTGAGAATCTATAAACCATTTTCCGGTTTCAATTTTAACCAACTCCAAATCTAACAATACTTGATTTTCGGTTTTCTTTTGATTGTCGACTTGAAAAGCAGACCAGGCTCGCACAGTTACTTGGCACT

AcrVIA1-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTACTATATAAAAGATTTAAAAGTGAAAGGAAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATATACTCAAGAGAAAATTAT

38

TCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTATAGTTTTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAATCTGTACTTCAAATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTGAAGCGGAAGAATCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAAACAAGAAAAGTCAAAAATACAATCAAATTCAAGGAAAAATACAAGAAATTAATAAGGAAATAGCTACACTTTATGAATCGTCAGAGGATTATATATTTACTGAATATGTTAGTAATTTATATAGAGAGTCTGCAAAGTTGGAGCAACACAGCAAACAAATTTTAAAAGAGGAGCTAAATGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

AcrVIA1(Y39A, S40A, N43A, S93A, Q96A)-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTACTATATAAAAGATTTAAAAGTGAAAGGAAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATAGCCGCAAGAGAAGCATATTCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTATAGTTTTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAAGCTGTACTTGCTATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTGAAGCGGAAGAATCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAAACAAGAAAAGTCAAAAATACAATCAAATTCAAGGAAAAATACAAGAAATTAATAAGGAAATAGCTACACTTTATGAATCGTCAGAGGATTATATATTTACTGAATATGTTAGTAATTTATATAGAGAGTCTGCAAAGTTGGAGCAACACAGCAAACAAATTTTAAAAGAGGAGCTAAATGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

AcrVIA1(∆E131-E134)-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGT

39

AAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTACTATATAAAAGATTTAAAAGTGAAAGGAAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATATACTCAAGAGAAAATTATTCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTATAGTTTTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAATCTGTACTTCAAATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTTCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAAACAAGAAAAGTCAAAAATACAATCAAATTCAAGGAAAAATACAAGAAATTAATAAGGAAATAGCTACACTTTATGAATCGTCAGAGGATTATATATTTACTGAATATGTTAGTAATTTATATAGAGAGTCTGCAAAGTTGGAGCAACACAGCAAACAAATTTTAAAAGAGGAGCTAAATGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

AcrVIA1(I2A, Y4A)-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGGCCTACGCTATAAAAGATTTAAAAGTGAAAGGAAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATATACTCAAGAGAAAATTATTCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTATAGTTTTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAATCTGTACTTCAAATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTGAAGCGGAAGAATCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAAACAAGAAAAGTCAAAAATACAATCAAATTCAAGGAAAAATACAAGAAATTAATAAGGAAATAGCTACACTTTATGAATCGTCAGAGGATTATATATTTACTGAA

40

TATGTTAGTAATTTATATAGAGAGTCTGCAAAGTTGGAGCAACACAGCAAACAAATTTTAAAAGAGGAGCTAAATGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

AcrVIA1(S68A, F69A)-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTACTATATAAAAGATTTAAAAGTGAAAGGAAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATATACTCAAGAGAAAATTATTCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTACGCTGCTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAATCTGTACTTCAAATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTGAAGCGGAAGAATCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAAACAAGAAAAGTCAAAAATACAATCAAATTCAAGGAAAAATACAAGAAATTAATAAGGAAATAGCTACACTTTATGAATCGTCAGAGGATTATATATTTACTGAATATGTTAGTAATTTATATAGAGAGTCTGCAAAGTTGGAGCAACACAGCAAACAAATTTTAAAAGAGGAGCTAAATGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

AcrVIA1(∆N173-N232)-3xFlag

ctcgtgctataattatactaattttACATCACGGAAAAAGGTTATGCTGCTTTTAAGACCCACTTTCACATTTAAGTTGTTTTTCTAATCCGCATATGATCAATTCAAGGCCGAATAAGAAGGCTGGCTCTGCACCTTGGTGATCAAATAATTCGATAGCTTGTCGTAATAATGGCGGCATACTATCAGTAGTAGGTGTTTCCCTTTCTTCTTTAGCGACTTGATGCTCTTGATCTTCCAATACGCAACCTAAAGTAAAATGCCCCACAGCGCTGAGTGCATATAATGCATTCTCTAGTGAAAAACCTTGTTGGCATAAAAAGGCTAATTGATTTTCGAGAGTTTCATACTGTTTTTCTGTAGGCCGTGTACCTAAATGTACTTTTGCTCCATCGCGATGACTTAGTAAAGCACATCTAAAACTTTTAGCGTTATTACGTAAAAAATCTTGCCAGCTTTCCCCTTCTAAAGGGCAAAAGTGAGTATGGTGCCTATCTAACATCTCAATGGCTAAGGCGTCGAGCAAAGCCCGCTTATTTTTTACATGCCAATACAATGTAGGCTGCTCTACACCTAGCTTCTGGGCGAGTTTACGGGTTGTTAAACCTTCGATTCCGACCTCATTAAGCAGCTCTAATGCGCTGTTAATCACTTTACTTTTATCTAATCTAGACATCATTAATTCCTCCTTTTTGTTGACATTATATCATTGATAGAGTTATTTGTCAAACTAGTTTTTTATTTGGATCCCCTCGAGTTCATGAAAAACTAAAAAAAATATTGACACTCTATCATTGATAGAGCATAATTAAAATAAGCTCTCTATCATTGATAGAGTGAGTTAAACCTATAGGAGGAAAAAACGATGATCTACTATATAAAAGATTTAAAAGTGAAAGG

41

AAAAATATTTGAAAATCTAATGAACAAAGAGGCTGTAGAAGGATTAATTACTTTTTTAAAGAAAGCGGAATTTGAGATATACTCAAGAGAAAATTATTCAAAATACAACAAATGGTTTGAAATGTGGAAAAGCCCAACTTCGAGCCTTGTGTTTTGGAAAAATTATAGTTTTCGCTGTCATCTTCTTTTTGTCATAGAAAAAGATGGTGAATGCCTTGGAATTCCTGCATCTGTTTTTGAATCTGTACTTCAAATTTACCTTGCGGATCCGTTCGCTCCCGATACGAAAGAACTTTTTGTTGAGGTTTGTAATTTATATGAATGTTTAGCGGATGTCACTGTCGTAGAACATTTTGAAGCGGAAGAATCAGCGTGGCATAAATTAACCCATAATGAGACCGAAGTATCAAAAAGAGTCTATAGTAAAGATGATGACGAACTTCTTAAATATATTCCAGAATTTCTTGACACCATAGCGACAGACTACAAGGATCATGATGGTGATTATAAAGATCACGACATCGATTACAAAGATGATGACGATAAATAAGCctctcgcctgtcccctcagttcagtaatttc

42

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