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WO2025129073A2 - Acriiia1, a protein–rna anti-crispr complex that targets core cas and accessory nucleases, and uses thereof - Google Patents

Acriiia1, a protein–rna anti-crispr complex that targets core cas and accessory nucleases, and uses thereof Download PDF

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WO2025129073A2
WO2025129073A2 PCT/US2024/060134 US2024060134W WO2025129073A2 WO 2025129073 A2 WO2025129073 A2 WO 2025129073A2 US 2024060134 W US2024060134 W US 2024060134W WO 2025129073 A2 WO2025129073 A2 WO 2025129073A2
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promoter
cell
polypeptide
acriiiai
polynucleotide
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WO2025129073A3 (en
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Asma Hatoum
Barbaros ASLAN
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University of Illinois at Urbana Champaign
University of Illinois System
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University of Illinois System
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • ACRIIIA1 A PROTEIN-RNA ANTI-CRISPR COMPLEX THAT TARGETS CORE CAS AND ACCESSORY NUCLEASES, AND USES THEREOF
  • CRISPR-Cas systems protect bacteria and archaea from their viruses, and anti- CRISPRs (Acrs) are small virus-encoded proteins that inhibit CRISPR-Cas immunity. Over 80 distinct families of Acrs have been described to date; however only three of these subvert Type III CRISPR-Cas immunity.
  • Type III systems employ a complex network of CRISPR-associated (Cas) and accessory nucleases to degrade viral nucleic acids. Methods are needed in the art to reduce or eliminate Type III CRISPR-Cas immunity.
  • An aspect provides polynucleotide encoding an AcrIIIAI polypeptide, wherein the Acrl I IA1 polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
  • the polynucleotide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2.
  • a polynucleotide comprising a heterologous promoter sequence operably linked to a polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
  • the promoter can be a constitutive promoter, a repressible promoter, or an inducible promoter.
  • a constitutive promoter can be an SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter.
  • An inducible promoter can be a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
  • Yet another aspect provides a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises a heterologous polynucleotide and a polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
  • a heterologous polynucleotide can be or encodes an affinity tag, an epitope sequence tag, a detectable marker polypeptide, an amino acid spacer, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, an amino acid linker, or a cleavable linker.
  • Yet another aspect comprises a vector comprising any of the polynucleotides described herein.
  • Another aspect provides an isolated AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
  • an isolated fusion protein comprising (1 ) one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and (2) one or more heterologous polypeptides.
  • the heterologous polypeptide can be an affinity tag, an epitope sequence tag, a detectable marker polypeptide, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand an amino acid spacer, an amino acid linker, or a cleavable linker.
  • Even another aspect provides a delivery vehicle comprising any of the AcrIIIAI polynucleotides, vectors, AcrIIIAI polypeptides, or isolated fusion proteins described herein.
  • the delivery vehicle can be a liposome or nanoparticle.
  • CRISPR-Cas expression system comprising :
  • a second polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide complex to a target polynucleotide in a cell, wherein the second polynucleotide is operably linked to a promoter;
  • a recombinant cell comprising:
  • the inducible promoter is a TRE3G inducible promoter, a tetracycline- regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
  • the expression of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, and/or the polynucleotide encoding one or more crRNA molecules can be under control of the same promoter.
  • An aspect provides a method of inhibiting a Casio polypeptide or Casio polypeptide complex in a cell, the method comprising introducing:
  • an AcrIIIAI polypeptide into the cell wherein the AcrIIIAI polypeptide is heterologous to the cell, and the AcrIIIAI polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and/or
  • an AcrIIIAI polynucleotide into the cell wherein the AcrIIIAI polynucleotide is heterologous to the cell, and the AcrIIIAI polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2, thereby inhibiting the Casio polypeptide or Cas 10 polypeptide complex in the cell.
  • the cell can naturally express the Casio polypeptide or Casio polypeptide complex.
  • the cell can be genetically engineered to express a heterologous Casio polypeptide or Casio polypeptide complex.
  • the polynucleotide encoding the AcrIIIAI polypeptide can be operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter.
  • the cell can be contacted with an agent or condition that induces expression of the AcrIIIAI polypeptide in the cell.
  • the cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell.
  • the method can occur ex vivo or in vitro.
  • Another aspect provides a method of reducing activity of a CRISPR-Cas10 system in a cell comprising delivering any of the AcrIIIAI polynucleotides, the vectors, or the AcrIIIAI polypeptides to the cell.
  • Even another aspect provides a method for regulation of a CRISPR-Cas system, comprising delivering the CRISPR-Cas expression systems described herein to a target cell.
  • the cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell.
  • An aspect provides a recombinant phage comprising a genetically modified genome comprising a heterologous AcrIIIAI polynucleotide.
  • the AcrIIIAI polypeptide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
  • Another aspect provides a method of improving administration of a whole phage therapeutic comprising genetically engineering phage making up the whole phage therapeutic to express a recombinant AcrIIIAI polypeptide and administering the whole phage therapeutic to a subject in need thereof.
  • the subject can have a bacterial infection caused by Staphylococcus sp. Therefore, provided herein are methods and compositions comprising AcrIIIAI , the first Type lll-A specific anti-CRISPR protein.
  • AcrIIIAI can bind to the Cas effector complex and can attenuate its DNase activity and second messenger production. Additionally, AcrIIIAI associates with fragmented t(m)RNAs (acrRNAs) that undergo extensive degradation during phage infection.
  • AcrIIIAI can directly antagonize core Cas machinery while acrRNAs absorb collateral damage dealt by accessory nucleases. This clever strategy hinders indiscriminate cleavage on multiple fronts and provides robust protection against Type III CRISPR-Cas immunity.
  • FIG. 1 panels A-D show a subset of S. epidermidis myophages overcome Type lll-A CRISPR-Cas immunity.
  • A Illustration of the phage-host system used for Type lll-A CRISPR-Cas functional assays in panels C and D.
  • CRISPR-associated cas and csm genes encoded in the chromosome (polygons) and plasmid-encoded repeat and spacer (boxes) are shown. Filled and open triangles indicate cleavage events by Cas and cellular nucleases, respectively.
  • C Phage challenge plate assays. Averages of triplicate measurements ( ⁇ S. D.) of plaque-forming units per milliliter (pfu/ml) are shown on top, and representative plate images from at least two independent trials are shown on bottom. Dotted line, limit of detection. EV, empty vector; RC, reverse-complement.
  • FIG. 2 panels A-G show phage twillingate gp17 ⁇ acrIIIAI) is necessary and sufficient for anti-CRISPR activity.
  • A Illustration of candidate acr genes within ‘gap 3’ of Twillingate. Arrows below genes show locations of CRISPR targeting spacers tested in phage challenge assays in panels B and E.
  • B Phage challenge plate assays with WT Twillingate.
  • C PCR products generated from the gap 3 region of ten ‘escaper’ phages (E1 -E10).
  • D Illustration showing deleted segments (dashed lines) in selected escapers.
  • FIG. 3 panels A-H show AcrIIIAI co-purifies with fragmented t(m)RNAs.
  • A Schematic of the purification process for AcrIIIAI .
  • IMAC immobilized metal affinity chromatography
  • SEC size exclusion chromatography
  • SUMO small ubiquitin-related modifier.
  • B Top: Size exclusion chromatogram for AcrIIIAI and protein markers.
  • BSA Bovine serum albumin.
  • mAU milli-absorbance units at 280 nm (solid line) and 254 nm (dashed line). Dotted vertical line indicates column void volume.
  • Bottom SDS-PAGE analysis of proteins from AcrIIIAI fractions. FT is the input into the column.
  • M prestained protein ladder.
  • H Read coverage profile of the tmRNA (Grey bars). TLD, tRNA-like domain; MLR, mRNA-like region. For F and H, numbers of reads that start and stop at each position are overlaid across coverage profiles. Asterisks indicate regions that sustain significant cleavage. For E-H, representatives from three independent trials are shown.
  • FIG. 4 panels A-F show AcrIIIAI binds to Cas10-Csm via interactions with Csm2.
  • A SDS-PAGE analysis of Cas10-Csrn Csm2H6N .
  • B Native PAGE analysis of Cas10- Csm Csm2H6N alone and combined with increasing amounts of AcrIIIAI (1 :1 , 1 :5, 1 :10, and 1 :50 molar ratios).
  • C Size exclusion chromatograms for AcrIIIAI and Csm2 fractionated individually and combined in indicated ratios. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dashed line).
  • FIG. 5 panels A-H show AcrIIIAI attenuates cOA production and DNA cleavage by Cas10-Csm.
  • A Urea-PAGE analysis of cOAs produced by Cas10-Csm Csm2H6N following pre-incubation with AcrIIIAI at indicated ratios. A10, ladder composed of ten adenine nucleotides.
  • B Relative amounts of cOAs produced in three trials of the experiment in panel A.
  • C Agarose gel showing cleavage of (
  • FIG. 10 panels A-F accompany FIG. 3 and show nuclease activity and function of AcrIIIAI vs. AcrIIIAI H63A ’ H64A .
  • A AcrIIIAI amino acid sequence and other salient features, pl, isoelectric point; MW, molecular weight; Da, Daltons. The underlined histidine residues were mutated in this study (SEQ ID NO:1 ).
  • B Nuclease assay in which AcrIIIAI flow-through (FT), which contains both RNA-free and RNA-bound forms, was combined with an in vitro transcribed tmRNA substrate from S. epidermidis in the presence/absence of indicated metals.
  • FT AcrIIIAI flow-through
  • FIG. 11 panels A-B accompany FIG. 4 and show AcrIIIAI does not bind to Csm5.
  • A Size exclusion chromatograms of AcrIIIAI and Csm5 when passed through an S75 sizing column individually and combined in a 4:1 ratio. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dotted line). Dashed vertical line indicates void volume of the column.
  • B SDS-PAGE analysis of proteins contained within indicated fractions. Each gel image corresponds to the chromatogram on the left. kDa, kilodaltons; M, PageRulerTM Plus Prestained Protein Ladder.
  • FIG. 12 panels A-C accompany FIG. 4 and show Cas10-Csm structure and AlphaFold 3 models with AcrIIIAI.
  • A Cas10-Csm “short apo” complex with subunit stoichiometry Cas10i:Csm22:Csm33:Csm4i:Csm5i:crRNAi determined by cryo-electron microscopy (PDB ID 7V02; Smith et al, Structure, 2022).
  • PDB ID 7V02 cryo-electron microscopy
  • A bound to four copies of AcrIIIAI .
  • C The same model in panel B colored according to pLDDT (predicted local distance difference test), a per-atom confidence estimate.
  • FIG. 14 panels A-D accompany FIG. 5 and show AcrIIIAI does not block target RNA cleavage.
  • A Urea-PAGE analysis of a radiolabeled 43 nucleotide target RNA that was subjected to degradation by Cas10-Csm and/or RNA-bound AcrIIIAI after various time points (0.5, 1.0, 2.0, and 4.0 minutes).
  • B Quantification of fractions of RNA cleaved in three trials of the experiment in panel A.
  • C Urea-PAGE analysis of a radiolabeled 43 nucleotide target RNA that was subjected to degradation by Cas10-Csm and/or RNA-free AcrIIIAI after various time points (0.5, 1.0, 2.0, and 4.0 minutes).
  • FIG. 15 panels A-C accompany FIG. 5 and show AcrIIIAI does not cleave cOAs or complex-associated crRNAs.
  • A Urea-PAGE analysis of cOAs subjected to degradation by AcrIIIAI . A10, ladder composed of ten adenine nucleotides.
  • B Fractions of cOAs remaining in three trials of the experiment in panel A. Average measurements ( ⁇ S.D) are shown.
  • C Urea-PAGE analysis of radiolabeled crRNAs and/or acrlllA1-RNAs extracted from reaction mixtures containing Cas10-Csm and/or AcrIIIAI following a 4 h incubation. Positions of crRNAs are labelled to the right.
  • DL Decade Ladder
  • nt nucleotide.
  • FIG. 16 accompanies FIG. 7 and shows AcrIIIAI functions in the absence of RNase R.
  • Indicated pcrispr-spc plasmids (or the empty vector (EV) control) were introduced into the strains prior to phage challenge.
  • Representative plate images (bottom) and averages of triplicate measurements ( ⁇ S. D.) of plaque-forming units per milliliter (pfu/ml) (top) are shown. Dotted line indicates the limit of detection in this assay. Data is representative of two independent trials.
  • FIG. 17 panels A-H accompany FIG. 7 show AlphaFold 3 models of RNase R bound to various RNAs.
  • A Crystal structure of E. coli RNase R (residues 87-725) (PDB ID 5XGU; Chu etal, Nucleic Acids Research, 2017). The RNA channel and active site Mg 2+ (sphere) are indicated.
  • B AlphaFold 3 models of S. epidermidis RNase R (residues 1- 719) bound to Mg 2+ (sphere).
  • the protein structure is colored according to pLDDT (predicted local distance difference test), a per-atom confidence estimate.
  • the pTM predicted template modeling
  • ipTM interface predicted template modeling
  • C A plot showing the pTM and ipTM scores for AlphaFold 3 models of RNase R-Mg 2+ in complex with 20 representative S. epidermidis tRNAs and tmRNA, both full-length and fragmented (i.e. the first 40 nts of their 5’ends). Dashed lines indicate confidence thresholds: Models with pTM > 0.5 likely represent true structures. Complexes with ipTM ⁇ 0.6 are failed predictions, while ipTM values above 0.8 are high- confidence representations. Complexes with ipTM scores between 0.6 and 0.8 are borderline. See Table 4 for source data.
  • RNAs (D-H) AlphaFold 3 models of S. epidermidis RNase R (residues 1 -719) bound to Mg 2+ in complex with indicated RNAs.
  • the protein structures are colored according to pLDDT as in Panel B, and RNAs are colored Magenta. The pTM and ipTM scores are also indicated.
  • CRISPRs clustered regularly-interspaced short palindromic repeats
  • Cas CRISPR-associated proteins
  • CRISPR-Cas systems are tremendously diverse, and have been grouped into two classes, six types, and many subtypes based upon their effector complex architectures, cas gene content, and mechanistic details (4).
  • CRISPR-Cas systems In response to the selective pressure imposed by CRISPR-Cas systems, the viruses of bacteria and archaea have evolved a variety of strategies to overcome immunity. Prime among these are anti-CRISPR proteins (Acrs). Acrs are a diverse group of small proteins ( ⁇ 80-150 amino acids) that typically work by binding Cas effector complexes and blocking their functions (5). Over 80 families of Acrs targeting five of the six CRISPR-Cas Types have been described thus far (6); however, only three of these have the capacity to overcome Type III immunity (7-10). Type III CRISPR-Cas systems are found in bacteria and archaea and are regarded as the most complex (4, 11 ).
  • Types lll-A and lll-B have historically garnered the most attention. These systems employ multi-subunit effector complexes and a variety of accessory/cellular nucleases to detect and destroy invading RNA and DNA.
  • members of the core Cas effector complex Upon crRNA binding to a complementary transcript, members of the core Cas effector complex perform at least three catalytic activities: Cas7/Csm3/Cmr4 shred target RNA (12-16), Casio degrades single-stranded DNA indiscriminately (15, 17- 20), and Casi o also produces cyclic-oligoadenylates (cOAs), second-messenger molecules that bind and stimulate accessory nucleases often bearing CRISPR-associated Rossman Fold (CARF) domains (21-25). These include Csm6/Csx1 , which are not a part of the core effector complex, but are encoded within or proximal to the CRISPR locus (26). Beyond these Cas accessory enzymes, Type III systems co-opt diverse cellular ‘housekeeping’ nucleases to facilitate crRNA processing and clear phage nucleic acids (27-30).
  • AcrlllB2 binds the Type lll-B effector complex and diminishes all three of its catalytic activities by reducing the disassociation rate of cleaved target RNA.
  • plasmid-encoded AcrlllB2 is effective when targeting both early- and late- expressed viral genes (9), a single genomic copy of AcrlllB2 protects viruses only when late genes are targeted (10).
  • Acrlll-1 does not bind/inhibit effector complexes, but rather it degrades cyclic tetra-adenylates (cOA4), and thus interferes with the downstream activation of CARF-containing accessory nucleases that rely upon cOA4 to propagate the immune response (8).
  • cOA4 cyclic tetra-adenylates
  • the effectiveness of Acrlll-1 during early- versus late- gene targeting has not been explored; however, since the cOA-responsive accessory nucleases are essential particularly when late-expressed genes are targeted (31 ), it is likely that Acrlll-1 is also more effective when the target is located in late-expressed genes. To date, Type lll-A specific Acrs have not been described.
  • Acrl I IA1 the first Type lll-A anti-CRISPR protein and methods of its use.
  • AcrIIIAI in a subset of phages that infect S. epidermidis RP62a. Genetic analyses revealed that native expression of acrIIIAI completely overcomes CRISPR immunity whether early- or late- expressed phage genes are targeted. Biochemical analyses showed that AcrIIIAI co-purifies with fragmented t(m)RNAs (herein referred to as acrIIIAI -RNAs), and both RNA-bound and RNA-free fractions associate with the effector complex and attenuate its DNase activity and cOA production.
  • acrIIIAI -RNAs fragmented t(m)RNAs
  • acrIIIAI -RNAs co-purify with effector complexes in the native host during phage infection, and acrIIIAI -RNAs specifically interact with RNase R, a cellular ‘housekeeping’ nuclease that promotes robust immunity (30) in a purified system.
  • RNase R a cellular ‘housekeeping’ nuclease that promotes robust immunity (30) in a purified system.
  • Polynucleotides including recombinant polynucleotides contain less than an entire microbial genome and can be single- or double-stranded nucleic acids.
  • a polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof.
  • a polynucleotide can comprise, for example, a gene, open reading frame, non-coding region, or regulatory element.
  • a gene is any polynucleotide molecule that encodes a polypeptide, protein, or fragments thereof, optionally including one or more regulatory elements preceding (5' noncoding sequences) and following (3' non-coding sequences) the coding sequence. In one aspect, a gene does not include regulatory elements preceding and following the coding sequence.
  • a native or wild-type gene refers to a gene as found in nature, optionally with its own regulatory elements preceding and following the coding sequence.
  • a chimeric or recombinant gene refers to any gene that is not a native or wild-type gene, optionally comprising regulatory elements preceding and following the coding sequence, wherein the coding sequences and/or the regulatory elements, in whole or in part, are not found together in nature.
  • a vector is a polynucleotide that can be used to introduce polynucleotides or expression cassettes into one or more host cells.
  • a vector can be an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the polynucleotide sequence.
  • An expression vector can have a promoter sequence operably linked to a polynucleotide sequence (e.g., transgene) to drive expression in a host cell, and in some aspects, also comprises a transcription terminator sequence.
  • Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs).
  • a plasmid vector can be a circular double-stranded DNA construct used as a cloning and/or expression vector. Plasmid vectors can be linear or closed circular plasmids.
  • a vector is a phagemid.
  • a phagemid can be provided as a component of a bacteriophage. Any suitable vector can be used to deliver polynucleotides or expression cassettes to a population of host cells.
  • expression vectors can comprise a recombinant polynucleotide encoding a recombinant AcrIIIAI polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced.
  • the various nucleic acid and control sequences described herein are joined together (i.e. , operably linked) to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the recombinant AcrIIIAI polypeptide at such sites.
  • an expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome).
  • a vector can be integrated into the genome of the host cell and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.
  • Csm2 polypeptide sequences include:
  • Csm4 polypeptides examples include:
  • Csm5 polypeptides examples include:
  • Csm6 polypeptides examples include:
  • Cas6 polypeptides examples include:
  • a Casio system can comprise polynucleotides encoding Casi o and one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5.
  • a Casi o system can further comprise one or more crRNAs.
  • a Cas10 system can comprise polynucleotides encoding Cas10; one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5; Csm6; Cas6; and combinations thereof.
  • the polynucleotides can be present in one or more vectors that can be delivered to cells.
  • Casio systems can have three nuclease activities.
  • the first is a sequence specific RNA cleavage performed by the Cas7.
  • the targeted RNA is positioned along the crRNA in the RNP and digested by the multiple copies of Cas7 present in the complex. As a result the RNA molecule is cleaved at fixed, 6-nt long intervals. Complementarity between crRNA and targeted RNA does not have to be strict, and the presence of mismatches does not abolish nuclease activity.
  • the second nuclease activity is non-specific ssDNA cleavage.
  • the RNA polymerase opens the DNA double helix to start transcription exposing the antisense making it accessible for the HD domain of Casi o of the RNP complex.
  • the DNase is activated by complementarity between crRNA and targeted RNA.
  • the safety switch for this activity relies on complementarity between the 5'crRNA handle and the 3' protospacer region of targeted RNA. Such complementarity inhibits cleavage, and thus prevents the host’s CRISPR array from being targeted.
  • the third nuclease activity is non-specific RNA degradation. Similarly to non-specific ssDNA cleavage, this activity also depends on Casi o.
  • the non-specific RNA degradation is triggered by the binding of the RNP complex with targeted RNA with simultaneous non-complementarity between crRNA handle and targeted RNA.
  • Systems described herein can comprise one or more polynucleotide sequences that encode a CRISPR RNA (crRNA) that can specifically target a polynucleotide of interest.
  • the one or more crRNAs e.g., 1 , 2, 3, 4, 5, 6, 7, or more crRNAs
  • the one or more crRNAs can be operatively linked to a promoter.
  • CRISPR repeats The structure of a naturally occurring CRISPR locus includes a number of short repeating sequences (“repeats”). Repeats can occur in clusters and can be regularly spaced by unique intervening sequences called “spacers.” CRISPR repeats can vary in length and are partially palindromic. Repeats can be arranged in clusters of repeated units. Spacers are located between two repeats and can have a unique sequence of about 20- 72 bp in length. Repeat/spacer arrays can be transcribed as a long precursor that is cleaved within repeat sequences and processed into smaller crRNAs by Casio. crRNAs retain spacer sequences that specify the targets of CRISPR interference.
  • crRNAs function as complementary guides in Cas/crRNA ribonucleoprotein complexes that cleave the nucleic acids carrying a cognate sequence (“the protospacer” or “spacer”).
  • the spacer- derived sequence of the crRNA can anneal to its DNA target (i.e. , the spacer).
  • targeting can be prevented by excessive base pairing between the repeat-derived crRNA and the corresponding DNA sequence.
  • the crRNA comprises sufficient mismatching (e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, or more mismatches) between the crRNA tag region and corresponding DNA sequence adjacent to the spacer.
  • a crRNA comprises a segment that is the same as or complementary to a DNA target sequence (a spacer) in the targeted cell.
  • a crRNA can be operably linked to a promoter.
  • a crRNA that is complexed with Cas10 during cleavage of a DNA target sequence can be about 25, 30, 35 or more nucleotides in length.
  • a crRNA has at its 5' terminus an eight nucleotide sequence derived from an upstream repeat sequence, followed by a variable length of nucleotides derived from the spacer, and in some cases additional nucleotides derived from downstream sequences.
  • Type III CRISPR systems can generally recognize their target despite mismatches (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more) mismatches within the target sequence as described above.
  • mismatches e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more
  • the eight nucleotide sequence can be well conserved.
  • An example is ACGAGAAC.
  • the recombinant cells can comprise a recombinant AcrIIIAI polynucleotides, wherein the recombinant Acrl I IA1 polynucleotides are heterologous to the cell.
  • a recombinant AcrIIIAI polynucleotide encodes a polypeptide that has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1.
  • An AcrIIIAI polynucleotide can have greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:2.
  • Recombinant cells can also comprise a recombinant AcrIIIAI polypeptide, wherein the recombinant AcrIIIAI polypeptide is heterologous to the cell and the recombinant AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1.
  • Recombinant cells can further comprise a polynucleotide encoding a gene or gene cluster encoding a Cas10 polypeptide system, wherein the gene or gene cluster is operably linked to a promoter; or a Casi o polypeptide system.
  • Recombinant cells can naturally express a Casi o polypeptide system or can be genetically engineered to express a gene or gene cluster encoding a Casio polypeptide system.
  • Recombinant cells can further comprise a polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide system to a target polynucleotide.
  • the polynucleotide encoding one or more crRNA molecules can be present on part of the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system. Alternatively, the polynucleotide encoding one or more crRNA molecules can be separate from the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system.
  • Expression of a recombinant AcrIIIAI polynucleotide, a polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system, and/or a polynucleotide encoding one or more crRNA molecules can be each independently under the control of a constitutive promoter, a repressible promoter, or an inducible promoter.
  • the expression of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide system, and/or the polynucleotide encoding one or more crRNA molecules can be under control of the same promoter.
  • a constitutive promoter can be, e.g., a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, a SFFV promoter, or any other suitable promoter.
  • An inducible promoter can be a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, a UAS inducible promoter, or any other suitable promoter.
  • AcrIIIAI polypeptides in the recombinant cells can modulate the activity of a Casio polypeptide system (either naturally occurring or recombinantly added to a cell).
  • a recombinant AcrIIIAI polypeptide can be used to prevent or interfere with the activity of the Casio polypeptide system as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay or a Cas10-Csm DNA cleavage assay.
  • a suitable assay e.g., a Cas10-Csm cOA production assay or a Cas10-Csm DNA cleavage assay.
  • AcrIIIAI polypeptides can reduce the biological activity of a Casio polypeptide system by about 20, 30, 40, 50, 60, 70, 80, 90% or more as measured using a suitable assay.
  • a recombinant cell can be a eukaryotic cell, a mammalian cell, a human cell, or a prokaryotic cell.
  • a Casio polypeptide system can derived from, e.g., Pyrococcus furiosis, Thermococcus onnurineus, Sulfolobus solfataricus, Roseifluxus sp. RS-1, Psuedothermotoga lettingae, Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., Methanopyrus kandleri, or Thermus thermophilus or any other or any organism harboring a Type III CRISPR-Cas system.
  • systems that can comprise a polynucleotide encoding an AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the first polynucleotide is operably linked to a promoter.
  • Another system can comprise a polynucleotide encoding one or more crRNA molecules that are capable of guiding a Casio polypeptide complex to a target polynucleotide or gene in a cell, wherein the polynucleotide is operably linked to a promoter; and a polynucleotide encoding an AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID N0:1 , wherein the polynucleotide is operably linked to a promoter.
  • Yet another system can comprise a first polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex or system, wherein the first polynucleotide is operably linked to a promoter; a second polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide system to a target polynucleotide or gene in a cell, wherein the second polynucleotide is operably linked to a promoter; and a third polynucleotide encoding an AcrIIIAI polypeptide, wherein the Acrl I IA1 polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the third polynucleotide is operably linked to a promoter.
  • Each of the polynucleotides of these systems can be operably linked to the same promoter or different promoters.
  • Each of the polynucleotides can be present in a one vector or all polynucleotides can be present in the same vector.
  • polynucleotides encoding a CRISPR Casi o system and/or AcrIIIAI polypeptides can be transiently present or expressed in a cell.
  • Polynucleotides encoding CRISPR Casi o systems and/or AcrIIIAI polypeptides can be stably present in a cell, and can be integrated into a chromosome.
  • a vector can be maintained in a cell using a selectable marker, which can also be encoded by the vector, such as an antibiotic resistance gene, or a gene that subjects the cells comprising the vector to a nutritional selection.
  • Vectors can be introduced into cells using any suitable technique and delivery system, such as electroporation, lipid-based transfection systems, plasmid transformation systems, e.g., using competent cells, phage or viral transduction, micro-injection, including direct injection of a vector or crRNA.
  • Casio and Csm proteins and/or AcrIIIAI polypeptides can be directly injected into a cell configured to express a suitable crRNA.
  • a polynucleotide encoding an AcrIIIAI polypeptide can be operably linked to an inducible promoter, or to a promoter that is sensitive to another stimulus. Therefore, the expression of an AcrIIIAI polypeptide can be controlled by switching expression on and off using the inducible or controllable promoter.
  • the promoter operably linked to the polynucleotide encoding a Acrl I IA1 polypeptide can be activated so that AcrIIIAI is expressed. Once expressed the AcrIIIAI polypeptide can reduce or eliminate the activity of the Casi o system in the cell.
  • the promoter the promoter operably linked to the polynucleotide encoding a AcrIIIAI polypeptide can be deactivated so that AcrIIIAI is not expressed.
  • the Casi o system can then return to operation.
  • any of the polynucleotides, polypeptides, fusion proteins, or vectors described herein can be present with a delivery vehicle.
  • a polynucleotide, polypeptide, or vector can be combined with or encapsulated within a delivery vehicle such a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform.
  • a delivery vehicle such as a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome
  • nucleic acid molecule, polypeptide, vector, or combinations thereof into a host cell can be used to introduce the nucleic acid molecule, polypeptide, or vector into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like).
  • a target cell e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like.
  • Methods for introduction of a nucleic acid molecule, polypeptide, vector, or combinations thereof include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery.
  • PKI polyethyleneimine
  • a polypeptide described herein is provided as a nucleic acid molecule (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the polypeptide.
  • a polypeptide as described herein can be introduced into a cell (provided to the cell) by any convenient method, e.g., injection directly into a cell, nucleofection; via a protein transduction domain (PTD) conjugated to one or more components of the Casi o system.
  • PTD protein transduction domain
  • a nucleic acid molecule, polypeptide, vector, or combination thereof can be delivered to a cell in a particle or associated with a nanoparticle or particle.
  • a delivery nano particle or particle can comprise a lipid, a lipidoid (see, U.S. Pat. Publ. 20110293703), or a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, a cationic lipid.
  • Sugar-based particles for example GalNAc (see WO2014118272 and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961 ) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell.
  • lipid nanoparticles LNPs
  • SNATM Spherical Nucleic Acid constructs and other nanoparticles (e.g., gold nanoparticles) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell.
  • SNATM Spherical Nucleic Acid
  • a nanoparticle can be any particle having a diameter of less than 1000 nm.
  • nanoparticles suitable for use in delivering a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell can have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm.
  • Nanoparticles suitable for use in delivery can comprise, e.g., solid nanoparticles (e.g., metal such as silver, gold, iron, titanium, non-metal, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof), metal, dielectric, and semiconductor nanoparticles, hybrid structures (e.g., core-shell nanoparticles).
  • Semi-solid and soft nanoparticles are also suitable for use in delivery e.g., liposomes, exosomes. Liposomes can be made from several different types of lipids, e.g., phospholipids.
  • a stable nucleic-acid-lipid particle (SNALP) can be used for delivery.
  • a SNALP formulation can contain the lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl]-1 ,2-dimyristyloxy- propylamine (PEG-C-DMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40: 10:48 molar percent ratio.
  • PEG-C-DMA lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl]-1 ,2-dimyristyloxy- propylamine
  • DLinDMA 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane
  • DSPC ,2-distearoyl-sn-glycero-3-phosphocholine
  • a SNALP liposome can be prepared by formulating D-Lin-DMA and PEG-C- DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C- DMA.
  • a resulting SNALP liposomes can be about 80-100 nm in size.
  • a nucleic acid molecule, polypeptide, vector, or combination thereof can be delivered encapsulated in PLGA microspheres (see, e.g., US Pat. Publ. 20130252281 ; US Pat. Publ. 20130245107; US Pat. Publ. 20130244279).
  • Supercharged proteins can also be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof.
  • Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation.
  • Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.
  • CPPs Cell Penetrating Peptides
  • CPPs can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof.
  • CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
  • the method can comprising introducing an AcrIIIAI polypeptide into the cell, wherein the AcrIIIAI polypeptide is heterologous to the cell, and the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1 .
  • the methods can cause a reduction of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more of the biological activity of the CRISPR-Cas10 system in the cell as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay, or a Cas10-Csm DNA cleavage assay.
  • a cell can naturally express the Casi o polypeptide or Casi o polypeptide system.
  • a cell can be genetically engineered to express a heterologous Casi o polypeptide or Casi o polypeptide system.
  • an AcrIIIAI polypeptide, an AcrIIIAI polynucleotide, or vector capable of expressing an AcrIIIAI polypeptide, as described above, can be delivered to a cell.
  • the delivery of the polypeptide, polynucleotide, or vector to the cell can cause a reduction of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more of the biological activity of the CRISPR-Cas10 system in the cell as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay, or a Cas10-Csm DNA cleavage assay.
  • a suitable assay e.g., a Cas10-Csm cOA production assay, or a Cas10-Csm DNA cleavage assay.
  • Whole phage therapy is the administration of bacteriophage (i.e., phage), which can infect and kill bacteria, to a patient as an anti-bacterial therapy.
  • phage bacteriophage
  • Intact whole phage or a mixture of many different phages to target bacteria can be used in phage therapy.
  • specific phage that are effective against particular strains of infectious bacteria are selected and administered e.g., orally, topically, or intravenously, to a subject.
  • Whole phage therapy can be improved by Introducing a polynucleotide encoding AcrIIIAI into a therapeutic phage genome to improve the phage’s ability to kill bacteria when delivered to a patient during whole phage therapy.
  • the efficacy of a whole phage therapy can be improved by about 10, 20, 30, 40, 50, 60, 70, 80, 90% or more as compared to a system that does not include AcrIIIAI as described herein.
  • Another aspect provides methods of treatment of a bacterial infection (e.g., Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Clostridium, Salmonella, Enterococcus, and others) comprising administering a whole phage therapeutic comprising genetically engineering phage making up the whole phage therapeutic to express a recombinant AcrIIIAI polypeptide and administering the whole phage therapeutic to a subject in need thereof.
  • a subject can be a mammal such as a human, a non-human mammal, or a non-human primate.
  • a therapeutic or pharmaceutical composition can include at least one of the polypeptides, polynucleotides, phage, or vectors described herein in a pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” refers to at least one component of a pharmaceutical preparation that is normally used for administration of active ingredients.
  • a pharmaceutically acceptable carrier can contain any pharmaceutical excipient used in the art and any form of vehicle for administration.
  • the compositions can be, for example, injectable solutions, aqueous suspensions or solutions, non-aqueous suspensions or solutions, solid and liquid oral formulations, salves, gels, ointments, intradermal patches, creams, lotions, tablets, capsules, sustained release formulations, and the like.
  • Additional excipients can include, for example, colorants, tastemasking agents, solubility aids, suspension agents, compressing agents, enteric coatings, sustained release aids, and the like. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
  • the pharmaceutical composition can further comprise an additional active ingredient(s), such as an antibiotic.
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97, 1997).
  • An effective amount of a pharmaceutical composition as described herein will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the methods in use.
  • An effective amount can comprise one or more administrations (1 , 2, 3, 4, 5 or more) of a composition depending on the aspect. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment.
  • a viral vector e.g., an AAV viral vector
  • AAV viral vector can be administered to a subject at a dose ranging from about 10 13 to about 10 16 viral vector particles.
  • Polypeptide compositions can be delivered at about 0.001 , 0.01 , 0.1 , 0.5, 1.0, 5.0 mg/kg, or any other suitable amount.
  • a vector such as a viral vector, a polynucleotide, a polypeptide, or a pharmaceutical composition as described herein can be administered to a subject intravenously, intrathecally, intracistema-magna, intracerebrally, intraventricularly, intranasally, intratracheally, intra- aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracistemally, intranervally, intrapleurally, topically, intralymphatically, intracistemally or intranerve.
  • treating include administering polypeptides, polynucleotides, vectors, and/or pharmaceutical compositions described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.
  • a composition can be administered to an individual for a prophylactic or therapeutic purpose, and thus can be provided as a pharmaceutical preparation by mixing a polynucleotide, polypeptide, phage, or vector described herein with a pharmaceutically acceptable carrier.
  • prophylactic or therapeutic purpose purposes include but are not necessarily limited to inhibiting the growth of, reducing the amount of and/or killing undesirable and/or pathogenic microorganisms, or to reduce the pathogenicity and/or antibiotic resistance of such microorganisms.
  • the pharmaceutical composition can be administered using any suitable route and type of formulation, and the dosing of the formulation can be determined by those skilled in the art given the benefit of the present disclosure.
  • the compositions and methods of the invention relate to reducing pathogenic bacteria.
  • the compositions and methods are adapted for veterinary purposes.
  • compositions and methods of this disclosure are adapted for use in eliminating pathogenic bacteria from a non-living surface, such as a the surface of a device, or the surface of an area used for any procedure wherein the reduction of pathogenic bacteria is important, including but not necessarily limited to surfaces used for food preparation or medical purposes.
  • compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art.
  • the terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.
  • the term “and/or” includes any and all combinations of one or more of the associated listed items.
  • the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise.
  • the term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).
  • compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
  • S. epidermidis RP62a 32
  • Acrispr known as LAM 104 in (33)
  • Acrispr-cas LM1680 in (34)
  • S. epidermidis strains and S. aureus RN4220 were grown in Brain Heart Infusion (BHI, BD Diagnostics) and Tryptic Soy Broth (TSB, BD Diagnostics), respectively.
  • E. coli DH5a was grown in Luria Bertani (LB) broth (VWR), and E. coli Rosetta 2 (BL21 (DE3)) was grown in Terrific Broth (TB, VWR).
  • antibiotics 10 pg/ml chloramphenicol (to select for pC194-based plasmids), 10 pg/ml erythromycin (to select for pTet-based plasmids), 15 pg/ml anhydrotetracycline (to activate the inducible promoter), 15 pg/ml neomycin (to select for S. epidermidis), 30 pg/ml chloramphenicol (to select for E. coli Rosetta2 plasmids), and 50 pg/ml kanamycin (to select for pET28b-10HisSmt3-based plasmids). All bacterial strains were grown at 37 °C, except for protein induction at 17 °C. Liquid cultures were propagated with constant agitation in an orbital shaker set to 180-200 rpm.
  • Phage propagation and enumeration S. epidermidis phage PhiIBB (Genbank: NC_041928.1 ) was a generous gift from Luis Melo (36). PhiIBB, Terranova (Genbank: MH542234.1 ), Twillingate (Genbank: MH321491.1 ), and Quidividi (Genbank: MH321490.1 ) (37) were propagated on S. epidermidis RP62a Acrispr-cas. Concentrated phage stocks were prepared by picking 1 -5 purified phage plaques and resuspending them into 500 pl of TSB by vortexing for 30 sec.
  • top agar layer was allowed to solidify ⁇ 10 min at room temperature and then incubated overnight at 37 °C. The following day, the entire top agar layer was harvested with a sterile cell scraper and resuspended in 20 mL fresh TSB. Phages were released into suspension by vortexing for 5 min. Suspensions were then pelleted via centrifugation at 10,000 x g for 10 min, and the supernatant was passed through a 0.45 pm bottle filter to obtain high concentrations of each phage. Phage concentrations were determined using the double-agar overlay method as described in (38).
  • the phage concentration (i.e., titer) in plaque-forming units per ml (pfu/ml) was determined by the following formula: [(number of plaques counted on the most diluted spot) I (dilution factor)]*200.
  • Phage stocks were stored at 4 °C. Phage identities were routinely confirmed/authenticated via PCR amplification and sequencing of genomic regions unique to each phage.
  • Phage challenge assays For all phage challenge plate assays, concentrated phage stocks were diluted in 10-fold increments (10°-10 -7 ), and dilutions were spotted atop lawns of cells. Following overnight incubation, plaques were enumerated using the protocol described in the section above titled “phage propagation and enumeration”. For assays using the anhydrotetracycline- (aTc-) inducible system, plates and top agar were supplemented with 15 pg/ml aTc.
  • aTc- anhydrotetracycline-
  • phage challenge assays in liquid culture, overnight cultures were diluted 1 :100 in fresh BHI broth, and phages were added to reach multiplicities of infection (i.e., phage:cell ratios) of 0.1 , 0.5, or 1.0. Phage-host mixtures (200 pl) were then aliquoted into triplicate wells of a 96-well plate. Cells were grown at 37 °C with agitation for 15 hours in a SpectraMax M2e microplate/cuvette reader (Molecular Devices), and OD600 (optical density at 600 nm) measurements were taken every 15 min. For all phage challenge assays, graphs show an average of triplicate measurements (+S.D.) as a representative of at least two independent trials.
  • pAH011 40
  • pcrispr-cas 41
  • pcrispr-cas-spcl 42
  • pC194 43
  • pTarget 15
  • pE194 44
  • All assembled plasmids were first introduced into S.
  • aureus RN4220 via electroporation (described in the section below), and inserted sequences were confirmed by PCR amplification and Sanger sequencing (performed by Eurofins MWG Operon). At least two transformants were confirmed by sequencing and at least one of each construct was purified using the EZNA Plasmid Mini Kit (Omega Bio-tek). Where indicated, plasmids were introduced into S. epidermidis RP62a Acrispr or Acrispr- cas via electroporation.
  • Electroporation into Staphylococci Electrocom petent cells were prepared as described in (45). Briefly, fresh media was used to dilute 10 mL of overnight culture until OD600 of 0.5. Diluted cultures were incubated at 37 °C for 30 min, then placed on ice for 10 min. All subsequent steps were performed on ice. Cells were pelleted at 4000 x g for 10 min, and then washed with an equal volume ice-cold water two times. Cell pellets were washed twice more with 10% glycerol (ice-cold) using 1/20- and 1/25- the volume of culture, respectively.
  • Phage hybridization and genotyping Fresh overnight culture of S. epidermidis RP62a Acrispr-cas was diluted 1 :100 in BHI (2 ml) supplemented with 5 mM CaCL. Cells were grown to an OD600 of 0.5, and then infected with two phages: Twillingate at an MOI of 0.05 and PhiIBB at a MOI of 8. The bacteria : phage mixtures were incubated in a 37 °C water bath for 5 minutes (without agitation) to allow phage absorption. Then, infected cells were collected via centrifugation at 17,000 x g for 5 min.
  • the pelleted cells were washed with 1 mL of fresh BHI and centrifuged again at 17,000 x g for 5 min to remove unabsorbed phages. The final pellet was resuspended in 150 mL fresh BHI. 10-fold dilutions (out to 1 O’ 5 ) of the cell suspension were spotted atop a semisolid layer of 0.5 x HIA agar containing 5 mM CaCL and a 1 :100 dilution of overnight culture of S. epidermidis RP62a Acrispr bearing pcrispr-spc ⁇ f>-polA. Spots were air-dried, and plates were incubated overnight at 37 °C.
  • putative hybrid phages ten well-isolated plaques, now referred to as putative hybrid phages, were picked from plates and placed into separate tubes containing 0.5 mL fresh BHI. The tubes were vortexed, centrifuged, and filtered as described in “Phage propagation and enumeration” to separate the phages from the agar. Ten-fold dilutions (10°-10’ 7 ) of the supernatant were plated atop a semisolid HIA agar layer containing 5 mM CaCL and a 1 :100 dilution of overnight S. epidermidis RP62a Acrispr cells bearing pcrispr-spc ⁇ f>-polA.
  • Genomic DNA was extracted from the high-titer lysates by combining 300 mL with an equal volume of phenokCHC isoamyl alcohol, vortexing for 15 sec, and subjecting to centrifugation at 10,000 x g for 2 min. The upper aqueous phase was removed into a fresh tube and DNA was ethanol precipitated and resuspended into nuclease-free water.
  • Phage DNA sequencing and assembly Phage DNA sequencing and assembly. Library preparation and sequencing were performed at the SeqCenter (Pittsburgh, PA) using the Illumina Library Prep Tagmentation kit. Sequencing was performed on an Illumina NextSeq 2000. Adapters and indexes were removed by bcl2fastq v. 1.8.4 (Illumina) and FastQC v. 0.11.9 was used to confirm data quality. Sequencing reads were assembled using SPAdes v. 3.15.5 (46) in isolate mode with the kmer values of 21 , 33, 55, 77 and 99. The resulting assembly graph was inspected using Bandage v. 0.8.1 (47) and the highest coverage contig with the proper length representing the phage genome was extracted as a fasta file.
  • Phage genome sequence alignments with MAUVE Genome sequences of wildtype and/or the newly-assembled hybrid phage (TwillBB-H8) were first were re-opened such that position 1 in all phages occurs at homologous loci. Re-opened genomes were then saved as fasta files and multiple genome alignments were generated using MAUVE by downloading the software and following the instructions on the website (darlinglab.org/mauve/mauve.html)
  • pET28b-10His-Smt3-plasmids Constructing pET28b-10His-Smt3- plasmids.
  • pET28b-10His-Smt3-acr///A7 pET28b-10His-Smt3-ac/7//A7/-/63A,/-/64A, and pET28b-10His-Smt3-cs/7?2 were each constructed using a two-piece Gibson assembly with primers and templates listed in Table 2.
  • the PCR products derived from the plasmid backbone were treated with 1 ,000 Units of Dpnl (NEB) for 1 h at 37 °C, followed by heat inactivation at 80 °C for 20 min.
  • Dpnl Dpnl
  • Transformation of E. coli For preparation of chemically-competent E. coli, overnight cultures were diluted in LB (1 : 100) and incubated with agitation at 37 °C until the OD600 reached ⁇ 0.5. The culture was placed on ice for 10 min, and subjected to centrifugation at 4000 x g for 5 min. The resulting pellet was resuspended in transformation and storage (TSS) buffer (85% LB medium, 10% (w/v) PEG MW 8000, 5% (v/v) DMSO, 50 mM MgCL) at 1/10 the starting culture volume. Cells were distributed in 50 ml aliquots and stored at -80 °C.
  • TSS transformation and storage
  • acrlllA1 -RNAs were end-labeled with T4 Polynucleotide Kinase in a reaction containing y-[ 32 P]-ATP (PerkinElmer) for 1 h at 37 °C. Then, radiolabeled acrlllA1 -RNAs were subjected to ethanol precipitation to remove excess y-[ 32 P]-ATP. Purified acrlllA1 -RNAs were mixed 1 :1 with 95% formamide loading dye and resolved on 12% Urea PAGE gels. The sizes were estimated with a DecadeTM Marker RNA ladder (Thermo Fisher Scientific). Gels were exposed to storage phosphor screens and visualized using Amersham Typhoon biomolecular imager (Cytiva).
  • RNA-seq analysis of acrlllA1-RNAs Extracted acrlllA1 -RNAs (100-300 ng) were submitted to the Roy J Carver Biotechnology Center at the University of Illinois at Urbana- Champaign for RNA-seq. The small RNAs were treated with Antarctic Phosphatase and Polynucleotide Kinase, and libraries were constructed with the Next Small RNA Sample Prep kit (NEB) according to the manufacturer’s instructions. The library pool was quantitated by qPCR and sequenced on a MiSeq flowcell for 151 cycles from each end of the fragments using a MiSeq 300-cycle sequencing kit version 2.
  • RNA-seq reads were first mapped to the entire E. coli Rosetta 2 (BL21 (DE3)) genome (NZ_CP083274.1 ). Subsequent to the identification of coverage peaks, reads were aligned to individual tRNA and tmRNA sequences, which were extracted from the GenBank file and reverse complemented if necessary to match the strand orientation. Coverage profiles were calculated as a function of position using Bowtie2 (v. 2.4.4) and Samtools (v. 1.13) (49).
  • each tRNA raw coverage profile previously generated was first normalized to one, absolute value of its derivative (with respect to position) calculated, and mapped onto its corresponding alignment parsed from the cmalign output file. Only unique tRNA sequences (differing at most by two nucleotides) which had a peak raw coverage above 2000 were used in the analysis. The tRNA type, isotype and loop positions were also independently verified using tRNAscan-SE (54).
  • the S. epidermidis tmRNA substrate was synthesized using in vitro transcription. Briefly, the transcription template was amplified from S. epidermidis RP62a using primer pairs L328/L329 (Table 2). The PCR product was purified using the EZNA Cycle Pure Kit (Omega Bio-tek), and 0.5-1.0 pg of purified PCR fragment was used as template. The tmRNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). The reaction was carried out at 37 °C for 2 h.
  • the mixture was homogenized by inverting the tube gently several times and then sonicated on ice (three 30 sec pulses with 1 min rest in between). Insoluble materials were removed via centrifugation at 10,000 x g and 13,500 x g for 20 min, respectively. Lysates were then filtered through a 0.2 pm PES membrane. Filtered lysates were passed through pre equilibrated Ni-NTA columns to capture Cas10-Csm complexes.
  • Ni-NTA columns were packed in 5 ml gravity columns (G-Biosciences) using 1 ml Ni-NTA agarose resin slurry (Thermo Fisher Scientific) and equilibrated with 10 ml of 1x resuspension buffer (300 mM NaCI, 50 mM NaH2PO4, pH 8.0).
  • Ni-NTA columns with bound Cas10-Csm complexes were washed with 10 ml Wash Buffer 1 (resuspension buffer supplemented with 20 mM imidazole) and then 10 ml Wash Buffer 2 (resuspension buffer supplemented with 20 mM imidazole and 10% glycerol).
  • Cas10-Csm complexes were eluted with five 0.5 ml fractions of elution buffer (resuspension buffer supplemented with 250 mM imidazole and 10% glycerol). Samples from each fraction were resolved in 15% SDS PAGE gels and stained with 0.1 % Coomassie G-250. Protein sizes were estimated with PageRulerTM Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific). Gels were imaged using an Azure 400 imager (Azure Biosystems). Protein samples were stored in elution buffer at -80 °C.
  • Cas10-Csrn Csm2H6N or Cas10-Csrn ACsm2 Csm4H6N complexes (30 pmol) were combined with increasing amounts of AcrIIIAI FT (flow-through containing both RNA-free and RNA-bound fractions) (30, 150, 300, and 1500 pmol) and allowed to chill on ice for ⁇ 5 min.
  • Cas10-Csm complexes (30 pmol) and AcrIIIAI (1500 pmols) were also loaded individually on the same gel as controls, alongside the Native Mark Protein Standard (Thermo Fisher Scientific).
  • Proteins were resolved in a 6% native polyacrylamide gel (29:1 acrylamide/bisacrylamide) with 0.75 mm thickness.
  • Tris-Glycine Buffer 25 mM Tris, 250 mM glycine, pH 8.5
  • Gels were run for 80 min at 100 V in a Mini-PROTEAN Tetra Cell (Bio-Rad) submerged in an ice water bath.
  • Gels were stained with 0.1 % Coomassie G-250 for 10-20 min, and then submerged in destaining solution (50% methanol and 10% acetic acid) for 1 -2 h.
  • Destained gels were imaged with an Azure 400 imager (Azure Biosystems).
  • the Coomassie Blue Destaining Solution was completely removed from the gel by soaking gels in dH2O (with agitation) for at least three hours, while changing out the water every hour. Gels were then soaked in TAE buffer (40 mM Tris Base, 20 mM acetic acid, 1 mM EDTA) containing 5% ethidium bromide with agitation for 30-45 min. Gels were then imaged with an Azure 400 imager (Azure Biosystems) under UV light.
  • TAE buffer 40 mM Tris Base, 20 mM acetic acid, 1 mM EDTA
  • Protein sizes were estimated with PageRulerTM Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific). Gels were imaged using an Azure 400 imager (Azure Biosystems). For each pairwise interaction, at least two independent trials were performed.
  • Cas10-Csm RNA cleavage assays The Cas10-Csm RNA cleavage assay was adapted from (24). Briefly, a target RNA that bears complementarity to the crRNA encoded by the first spacer in the S. epidermidis RP62a CRISPR-Cas locus (ssRNA-01 , Table 3), was radiolabeled on its 5’ end with T4 polynucleotide kinase and y-[ 32 P]-ATP and purified over a G25 column (IBI Scientific). Next, Cas10-Csm Csm2H6N complexes (20 pmols) and AcrIIIAI (200 pmols) were pre-incubated alone or combined for 30 min at room temperature.
  • ssRNA-01 a target RNA that bears complementarity to the crRNA encoded by the first spacer in the S. epidermidis RP62a CRISPR-Cas locus
  • nuclease buffer 25 mM Tris-HCI pH 7.5, 10 mM MgCl2, and 2 mM DTT
  • radiolabeled ssRNA-01 were added to each tube.
  • Aliquots (10 pl) were withdrawn from reaction mixes at various time points (0.5, 1 , 2, and 4 min) and quenched by adding an equal volume of 95% formamide loading buffer.
  • Reactions were resolved on a 15% UREA PAGE gel at 55 W for 2 h. Gels were exposed overnight to a storage phosphor screen and visualized using an Amersham Typhoon biomolecular imager. ImageQuant software was used for densitometric analysis.
  • Fractions of substrates cleaved were determined using the following equation: density of uncut substrates for each timepoint were divided by the density of uncut substrate in the lane where no protein was added. Three independent trials were performed, and averages (+S.D.) were calculated.
  • Cas10-Csm cOA production assays The cOA production assay was carried out as described in (24) with minor modifications. Briefly, Cas10-Csrn Csm2H6N (20 pmols) and AcrIIIAI (20, 100, or 200 pmols) were combined and pre-incubated at room temperature for 1 h. As controls, Cas10-Csm (20 pmols) and AcrIIIAI (200 pmols) were incubated alone for the same amount of time.
  • cOA production was initiated in these reactions by adding TNG Buffer (50 mM Tris HCI pH 8.0, 150 mM NH 4 CI, 5% (v/v) glycerol, 500 pM ATP and 10 mM Mg 2+ ), 5 pCi a-[ 32 P]-ATP, and unlabeled ssRNA-01 (Table 3). Reactions were incubated at 37 °C for 10 min and quenched by adding an equal volume of 95% formamide loading buffer. Produced cOAs were resolved on a 15% UREA PAGE gel at 55 W for 1 .25 h alongside A10, an oligonucleotide marker which consists of ten consecutive adenine residues.
  • TNG Buffer 50 mM Tris HCI pH 8.0, 150 mM NH 4 CI, 5% (v/v) glycerol, 500 pM ATP and 10 mM Mg 2+
  • Radiolabeled cOAs were first produced by Cas10-Csm complexes according to the protocol described above. cOAs were then extracted from reaction mixtures by adding an equal volume of phenol: CHCh: isoamyl alcohol (25:24:1 ), vortexing for 15 sec, and subjecting to centrifugation at 10,000 x g for 2 min. The upper aqueous phase was removed into a fresh tube and extracted as above with an equal volume of CHCh. The aqueous phase was removed into a fresh tube and purified over a G25 column (IBI Scientific).
  • nuclease buffer 25 mM Tris-HCI pH 7.5, 10 mM MgCh, and 2 mM DTT
  • AcrIIIAI 200 pmols
  • Reactions were allowed to proceed for 1 h at 37 °C and then quenched with an equal volume of 95% formamide loading buffer. Reactions were resolved on a 15% UREA PAGE gel at 55 W for 1.25 h alongside A10, an oligonucleotide marker which consists of ten consecutive adenine residues.
  • ImageQuant software was used for densitometric analysis.
  • Cas10-Csm DNA cleavage assay For DNA cleavage assays, Cas10- Csrn Csm2H6N Csm3D32A (30 pmols) was pre-incubated with AcrIIIAI (30, 150, or 300 pmols) in nuclease buffer (20 mM Tris pH 8.5, 40 mM KOI, 5 mM MgCh, 1 mM DTT) at 37 °C for 5 min. As controls, Cas10-Csrn Csm2H6N Csm3D32A (30 pmols) and AcrIIIAI (300 pmols) were pre-incubated alone under the same conditions.
  • a target RNA that bears complementarity to the crRNA encoded by the first spacer in the S. epidermidis RP62a CRISPR-Cas locus (ssRNA-01 , Table 3) was added to a final concentration of 200 nM.
  • a reaction mixture containing the Cas10-Csm complex without ssRNA-01 was included as a control.
  • )X174 DNA (0.05 mg) was added to each tube. Reaction mixtures were incubated at 37 °C for 4 h and then quenched by the addition of quench buffer (2:2:1 vol/vol/vol Proteinase K:water:0.5 M EDTA, pH 8.0).
  • AcrIIIAI crRNA cleavage assay To test for AcrIIIAI -mediated cleavage of complex-associated crRNAs, reactions were set up exactly as above using the maximum amount of AcrIIIAI (300 pmols) in the absence of target RNA and (
  • One-step growth curve for Twillingate WT and E6 was adapted from (56) with minor modifications. Briefly, 50 ml of mid-log S. epidermidis RP62a Acrispr was combined with phage lysate to achieve an MOI of 0.1. Phages were allowed to adsorb to the host for 5 min at room temperature without agitation. Cells were pelleted by centrifugation at 8,000 x g for 10 min and washed with 50 ml of TSB twice. Following washes, cells were incubated with agitation at 37 °C, and 200 pl samples were taken at 10 min intervals and passed through 0.45 pm syringe filters.
  • Cells were pelleted at 5000 x g for 5 minutes at 4°C, and the pellets were washed with an equal volume of BHI broth, followed by another wash with PBS. Supernatants were discarded, and the pellets were stored at -80 °C.
  • cell pellets were resuspended in 200 pL of sterile water and transferred into microtubes. Resuspended pellets were then incubated with lysostaphin (100 pg/mL) and MgCl2 (5 mM) at 37 °C for 2 hours.
  • the Wizard® Genomic DNA Purification Kit (Promega) was used to extract the genomic DNA according to the manufacturer’s instructions.
  • DNA pellets were dissolved in 50-60 pL of prewarmed DNase-free water. DNA concentrations were measured using the NanoDropTM 2000 Spectrophotometer (Thermo Fisher Scientific), and the samples were stored at -20 °C until being sent for sequencing.
  • Phage DNA cleavage analysis via Nanopore sequencing Phage DNA cleavage analysis via Nanopore sequencing.
  • Library preparation and Nanopore sequencing were performed at the SeqCenter (Pittsburgh, PA).
  • the PCR-free Ligation Sequencing Kit (Oxford Nanopore Technologies, SQK- NBD1 14.24) was used. Sequence reads within the resultant fastq files were aligned to the corresponding phage genomes using Minimap2 aligner (v. 2.26-r1175) (57). Subsequent alignment files were sorted and indexed into bam format using Samtools program with the respective "sort” and "index” commands.
  • CCDF complementary cumulative distribution functions
  • RNAs were extracted from ⁇ 500 pmols of Cas10-Csm complex using 750 ml TRIzol Reagent (Invitrogen) as described in the section titled “Extraction and visualization of AcrIIIAI - associated nucleic acids (acrlllA1 -RNAs)”. Seven biological replicates were performed for untreated samples and samples treated with Twillingate WT. Three biological replicates were performed for Twillingate E6. Cas10-Csm associated RNAs were analyzed using DESeq2 according to the section below.
  • RNA extracted from Cas10-Csm complexes were submitted to the Roy J Carver Biotechnology Center at the University of Illinois at Urbana-Champaign for RNA-seq.
  • RNA- seq was performed exactly as described in the section above titled “RNA-seq analysis of acrlllA1 -RNAs”.
  • Raw Illumina reads in fastq format were aligned to S. epidermidis RP62a Acrispr-cas genome using Bowtie2 (v. 2.4.4), producing a Sequence Alignment Map (SAM) file.
  • SAM Sequence Alignment Map
  • Codon usage analysis ofS. epidermidis RP62a and Twillingate Raw frequency data for codons and corresponding amino acids in S. epidermidis RP62a and phage Twillingate were generated using the online application at bioinformatics.org/sms2/codon_usage.html.
  • the frequency data for synonymous codons those that code for the same amino acid — were first aggregated, and then absolute counts and the relative percentages of each codon within its respective amino acid group were calculated and exported into a table. Quantification and statistical analysis. Student’s Wests were performed to determine if observed drops in plaque counts (Fig. 2) or enzymatic activities (Fig. 5) were statistically significant. Tests were performed using Microsoft Excel, and a difference was deemed significant if the p-value was below 0.05. Details for numbers of replicates etc. for specific experiments can be found in the corresponding figure legends and methods.
  • strains bearing pcrispr-spc encoding a non-targeting spacer (EV) or spacers producing crRNAs that are identical to the target sequence are completely susceptible to phage infection, as evidenced by the appearance of billions of plaques (zones of bacterial death) when dilutions of phage are plated on lawns of each strain ( Figure 1 C).
  • strains expressing crRNAs complementary to the transcript of each targeted gene were completely protected against PhiIBB and Terranova, as evidenced by the absence of plaques.
  • these same targeting strains remained sensitive to Quidividi and Twillingate, suggesting that these phages have the capacity to resist immunity.
  • AcrIIIAI provides robust protection against immunity whether early- or late- expressed genes are targeted by the CRISPR system ( Figure 1 ). This inhibition is due, at least in part, to direct interactions with the Cas10-Csm complex — AcrIIIAI binds to Csm2 ( Figures 4 and 12) and causes diminished DNase activity and cOA production by Casi o ( Figure 5). Reduced cOA production, in turn, would result in diminished activation of the cOA- responsive accessory nuclease Csm6. Thus, AcrIIIAI on its own has the capacity to inhibit collateral damage to phage nucleic acids by both core Cas and accessory nucleases.
  • acrlllA1 -RNAs RNAs — t(m)RNA fragments herein referred to as acrlllA1 -RNAs ( Figure 3).
  • acrlllA1 -RNAs associate with the Cas10-Csm complex in the native host during Twillingate infection ( Figure 6), which establishes their physiological relevance.
  • t(m)RNAs are necessarily derived from the host since Twillingate is devoid of its own t(m)RNAs (37), and AcrIIIAI is very likely to be responsible for their fragmentation (Figure 10), although more experimentation is required to identify the precise active site.
  • RNase R is a nonspecific 3’-5’ exoribonuclease that has the capacity to cleave through structured RNAs (73, 74). It plays critical roles in cellular RNA metabolism, including rRNA quality control (75) and the degradation of nonstop mRNAs following rescue of stalled ribosomes (76).
  • RNase R bolsters Type III CRISPR-Cas immunity — it interacts with Csm5 within the Cas10-Csm complex and collaborates with PNPase (another 3’-5’ exonuclease) to catalyze crRNA maturation and targeted transcript degradation, particularly when targeted transcripts are expressed at low levels (30).
  • PNPase another 3’-5’ exonuclease
  • acrlllA1 -RNAs might constitute excess nonspecific substrates that absorb collateral damage dealt by RNase R at the site of phage infection.
  • RNase R is subjected to tight regulation in the cell through its interactions with tmRNA and associated protein SmpB, which confine RNase R to the ribosomes and reduce RNase R’s stability (reviewed in Bechhofer and Lieber, 2019).
  • acrIIIAI - RNAs might have an impact on other accessory nucleases that are co-opted for immunity, including PNPase and RNase J2 (29, 30).
  • PNPase and RNase J2 are members of the RNA degradosome, a large protein complex that catalyzes bulk RNA decay (78) and plays a critical role in quality control of tRNAs (79).
  • acrIIIAI -RNAs might impact degradosome-associated nucleases through competitive inhibition or some other mechanism yet to be identified.
  • CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science. 315, 1709-1712.
  • Arginine-rich motifs present multiple interfaces for specific binding by RNA. RNA 11, 1848-1857.
  • HTSlib C library for reading / writing high-throughput sequencing data. Giga 1-6.
  • a type lll-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity. Elife 8, e45393.
  • RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex Cell 139, 945-956.
  • a Type lll-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding. Nucleic Acids Res. 46, 10319-10330.
  • RNA length is measured by a ruler mechanism anchored at the precursor processing site.
  • RNA degradosome promotes tRNA quality control through clearance of hypomodified tRNA. Proc. Natl. Acad. Sci. 116, 1394-1403.
  • Anti-CRISPRs go viral : The infection biology of CRISPR-Cas inhibitors. Cell Host Microbe 29, 704-714.
  • Type III CRISPR - Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543-548.
  • Plasmid Copy Number Control Isolation and Characterization of High-Copy-Number Mutants of Plasmid pE194. J. Bacteriol. 137, 635-643.

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Abstract

Provided herein are methods and compositions comprising AcrIIIA1, the first Type III-A specific anti-CRISPR protein. AcrIIIA1 can bind to the Cas10 effector complex and can attenuate its DNase activity and second messenger production. Additionally, AcrIIIA1 associates with fragmented t(m)RNAs (acrRNAs) that undergo extensive degradation during phage infection. AcrIIIA1 can directly antagonize core Cas10 machinery while acrRNAs absorb collateral damage dealt by accessory nucleases. These compositions and methods can, inter alia, provide robust protection against Type III CRISPR-Cas immunity.

Description

ACRIIIA1, A PROTEIN-RNA ANTI-CRISPR COMPLEX THAT TARGETS CORE CAS AND ACCESSORY NUCLEASES, AND USES THEREOF
Government Support
This invention was made with government support under 2054755 awarded by the National Science Foundation. The government has certain rights in the invention.
Priority
This application claims the benefit of U.S. Ser. No. 63/610,603, filed on December 15, 2023, which is incorporated herein by reference in its entirety.
Background
CRISPR-Cas systems protect bacteria and archaea from their viruses, and anti- CRISPRs (Acrs) are small virus-encoded proteins that inhibit CRISPR-Cas immunity. Over 80 distinct families of Acrs have been described to date; however only three of these subvert Type III CRISPR-Cas immunity. Type III systems employ a complex network of CRISPR-associated (Cas) and accessory nucleases to degrade viral nucleic acids. Methods are needed in the art to reduce or eliminate Type III CRISPR-Cas immunity.
Summary
An aspect provides polynucleotide encoding an AcrIIIAI polypeptide, wherein the Acrl I IA1 polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1. The polynucleotide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2.
Another aspect provides a polynucleotide comprising a heterologous promoter sequence operably linked to a polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1. The promoter can be a constitutive promoter, a repressible promoter, or an inducible promoter. A constitutive promoter can be an SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter. An inducible promoter can be a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
Yet another aspect provides a polynucleotide encoding a fusion protein, wherein the polynucleotide comprises a heterologous polynucleotide and a polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1. A heterologous polynucleotide can be or encodes an affinity tag, an epitope sequence tag, a detectable marker polypeptide, an amino acid spacer, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, an amino acid linker, or a cleavable linker.
Even another aspect comprises a vector comprising any of the polynucleotides described herein.
Another aspect provides an isolated AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
Yet another aspect provides an isolated fusion protein comprising (1 ) one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and (2) one or more heterologous polypeptides. The heterologous polypeptide can be an affinity tag, an epitope sequence tag, a detectable marker polypeptide, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand an amino acid spacer, an amino acid linker, or a cleavable linker.
Even another aspect provides a delivery vehicle comprising any of the AcrIIIAI polynucleotides, vectors, AcrIIIAI polypeptides, or isolated fusion proteins described herein. The delivery vehicle can be a liposome or nanoparticle.
Another aspect provides CRISPR-Cas expression system comprising :
(a) a first polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex, wherein the first polynucleotide is operably linked to a promoter;
(b) a second polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide complex to a target polynucleotide in a cell, wherein the second polynucleotide is operably linked to a promoter; and
(c) a third polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the third polynucleotide is operably linked to a promoter. The third polynucleotide can be operably linked to an inducible promoter, repressible promoter, or constitutive promoter. The gene or gene cluster encoding a Cas10 polypeptide complex can comprise CRISP R-associated genes encoding Casio, Csm2, Csm3, Csm4, Csm5, Csm6, and/or Cas6.
Also provided herein is a recombinant cell comprising:
(a) a recombinant AcrIIIAI polypeptide, wherein the recombinant AcrIIIAI polypeptide is heterologous to the cell and the recombinant AcrIIIAI polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 ; or
(b) a recombinant AcrIIIAI polynucleotide, wherein the recombinant AcrIIIAI polynucleotide is heterologous to the cell and the recombinant AcrIIIAI polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2.
The recombinant cell can further comprise a polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex, wherein the gene or gene cluster is operably linked to a promoter; or a Casio polypeptide complex. The recombinant cell can naturally express a Cas10 polypeptide complex. The recombinant call can further comprise a polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide complex to a target polynucleotide. The AcrIIIAI polypeptide can modulate an activity of a Casi o polypeptide complex. The cell can be a eukaryotic cell, a mammalian cell, a human cell, or a prokaryotic cell. The polynucleotide encoding one or more crRNA molecules can present on part of the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex.
The Casi o polypeptide complex can be derived from Pyrococcus furiosis, Thermococcus onnurineus, Sulfolobus solfataricus, Roseifluxus sp. RS-1, Psuedothermotoga lettingae, Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., Methanopyrus kandleri, Thermus thermophilus, or any organism harboring a Type III CRISPR-Cas system. Expression of the recombinant AcrIIIAI polynucleotide, the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex, and/or the polynucleotide encoding one or more crRNA molecules is independently under control of a constitutive promoter, a repressible promoter, or an inducible promoter. The constitutive promoter can be a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter. The inducible promoter is a TRE3G inducible promoter, a tetracycline- regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter. The expression of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, and/or the polynucleotide encoding one or more crRNA molecules can be under control of the same promoter.
An aspect provides a method of inhibiting a Casio polypeptide or Casio polypeptide complex in a cell, the method comprising introducing:
(i) an AcrIIIAI polypeptide into the cell, wherein the AcrIIIAI polypeptide is heterologous to the cell, and the AcrIIIAI polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and/or
(ii) an AcrIIIAI polynucleotide into the cell, wherein the AcrIIIAI polynucleotide is heterologous to the cell, and the AcrIIIAI polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2, thereby inhibiting the Casio polypeptide or Cas 10 polypeptide complex in the cell. The cell can naturally express the Casio polypeptide or Casio polypeptide complex. The cell can be genetically engineered to express a heterologous Casio polypeptide or Casio polypeptide complex. The polynucleotide encoding the AcrIIIAI polypeptide can be operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter. The cell can be contacted with an agent or condition that induces expression of the AcrIIIAI polypeptide in the cell. The cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell. The method can occur ex vivo or in vitro.
Another aspect provides a method of reducing activity of a CRISPR-Cas10 system in a cell comprising delivering any of the AcrIIIAI polynucleotides, the vectors, or the AcrIIIAI polypeptides to the cell.
Even another aspect provides a method for regulation of a CRISPR-Cas system, comprising delivering the CRISPR-Cas expression systems described herein to a target cell. The cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell.
An aspect provides a recombinant phage comprising a genetically modified genome comprising a heterologous AcrIIIAI polynucleotide. The AcrIIIAI polypeptide can have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
Another aspect provides a method of improving administration of a whole phage therapeutic comprising genetically engineering phage making up the whole phage therapeutic to express a recombinant AcrIIIAI polypeptide and administering the whole phage therapeutic to a subject in need thereof. The subject can have a bacterial infection caused by Staphylococcus sp. Therefore, provided herein are methods and compositions comprising AcrIIIAI , the first Type lll-A specific anti-CRISPR protein. AcrIIIAI can bind to the Cas effector complex and can attenuate its DNase activity and second messenger production. Additionally, AcrIIIAI associates with fragmented t(m)RNAs (acrRNAs) that undergo extensive degradation during phage infection. AcrIIIAI can directly antagonize core Cas machinery while acrRNAs absorb collateral damage dealt by accessory nucleases. This clever strategy hinders indiscriminate cleavage on multiple fronts and provides robust protection against Type III CRISPR-Cas immunity.
Brief Description of the Figures
FIG. 1 panels A-D show a subset of S. epidermidis myophages overcome Type lll-A CRISPR-Cas immunity. (A) Illustration of the phage-host system used for Type lll-A CRISPR-Cas functional assays in panels C and D. CRISPR-associated cas and csm genes encoded in the chromosome (polygons) and plasmid-encoded repeat and spacer (boxes) are shown. Filled and open triangles indicate cleavage events by Cas and cellular nucleases, respectively. Diamonds, cOAs; R, RNase R; P, PNPase; J, RNase J1/2; RNAP, RNA polymerase. (B) Alignment of phage genomes generated using MAUVE (darlinglab.org/mauve/mauve.html). Arrows indicate genetic loci targeted by CRISPR spacers in functional assays. Histograms indicate homologous regions in the nucleotide sequences of the different phages. (C) Phage challenge plate assays. Averages of triplicate measurements (±S. D.) of plaque-forming units per milliliter (pfu/ml) are shown on top, and representative plate images from at least two independent trials are shown on bottom. Dotted line, limit of detection. EV, empty vector; RC, reverse-complement.
(D) Phage challenge assays in liquid culture. Averages of triplicate measurements (±S. D.) are shown. MOI, multiplicity of infection; OD600, optical density at 600 nm.
FIG. 2 panels A-G show phage twillingate gp17 {acrIIIAI) is necessary and sufficient for anti-CRISPR activity. (A) Illustration of candidate acr genes within ‘gap 3’ of Twillingate. Arrows below genes show locations of CRISPR targeting spacers tested in phage challenge assays in panels B and E. (B) Phage challenge plate assays with WT Twillingate. (C) PCR products generated from the gap 3 region of ten ‘escaper’ phages (E1 -E10). (D) Illustration showing deleted segments (dashed lines) in selected escapers.
(E) Phage challenge plate assays with Twillingate E6. (F) Illustration of the inducible system used to screen candidate genes for anti-CRISPR activity. (G) Phage challenge plate assays with ISP using the inducible system in panel F. aTc, anhydrotetracycline. For all phage challenge plate assays, averages of triplicate measurements (±S. D.) of plaque- forming units per milliliter (pfu/ml) are shown on top, and representative plate images from at least two independent trials are shown on bottom. Dotted line, limit of detection. Asterisks indicate significant reductions in pfu/ml when compared to the empty-vector (EV) control in two-tailed t-tests (*, p<0.05; **, p<0.005).
FIG. 3 panels A-H show AcrIIIAI co-purifies with fragmented t(m)RNAs. (A) Schematic of the purification process for AcrIIIAI . IMAC, immobilized metal affinity chromatography; SEC, size exclusion chromatography; SUMO, small ubiquitin-related modifier. (B) Top: Size exclusion chromatogram for AcrIIIAI and protein markers. BSA, Bovine serum albumin. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dashed line). Dotted vertical line indicates column void volume. Bottom: SDS-PAGE analysis of proteins from AcrIIIAI fractions. FT is the input into the column. M, prestained protein ladder. (C) Radiolabeled nucleic acids extracted from AcrIIIAI fractions. Dashed line separates adjacent lanes in the same gel with different exposures. (D) Nuclease digestion of AcrIIIAI -bound nucleic acids. For C and D, representatives of at least two independent trials are shown. DL, Decade RNA ladder. (E) RNA-seq analysis of AcrIIIAI - associated RNAs showing Illumina reads mapped to the genome of the overexpression strain. (F) Read coverage profile of a representative tRNA (grey bars). Vertical grey shading delimits positions of loops in the tRNA structure. D, D-loop; A, anticodon loop, V, variable loop, T, T C loop. (G) Heatmap showing changes in normalized raw coverage across tRNAs that co-purify with AcrIIIAI . (H) Read coverage profile of the tmRNA (Grey bars). TLD, tRNA-like domain; MLR, mRNA-like region. For F and H, numbers of reads that start and stop at each position are overlaid across coverage profiles. Asterisks indicate regions that sustain significant cleavage. For E-H, representatives from three independent trials are shown.
FIG. 4 panels A-F show AcrIIIAI binds to Cas10-Csm via interactions with Csm2. (A) SDS-PAGE analysis of Cas10-CsrnCsm2H6N. (B) Native PAGE analysis of Cas10- CsmCsm2H6N alone and combined with increasing amounts of AcrIIIAI (1 :1 , 1 :5, 1 :10, and 1 :50 molar ratios). (C) Size exclusion chromatograms for AcrIIIAI and Csm2 fractionated individually and combined in indicated ratios. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dashed line). Dotted vertical line indicates void volume of the column. (D) SDS-PAGE analyses of proteins within fractions corresponding to chromatograms on the left. (E) SDS-PAGE analysis of Cas10-CsrnACsm2 Csm4H6N. (F) Native PAGE analysis of Cas10-CsmACsm2 Csm4H6N combined with increasing amounts of AcrIIIAI (1 :1 , 1 :5, 1 :10, and 1 :50 molar ratios). kDa, kilodaltons; M, Protein Ladder. For B and F, representatives of three independent trials are shown. M, NativeMark unstained protein ladder.
FIG. 5 panels A-H show AcrIIIAI attenuates cOA production and DNA cleavage by Cas10-Csm. (A) Urea-PAGE analysis of cOAs produced by Cas10-CsmCsm2H6N following pre-incubation with AcrIIIAI at indicated ratios. A10, ladder composed of ten adenine nucleotides. (B) Relative amounts of cOAs produced in three trials of the experiment in panel A. (C) Agarose gel showing cleavage of (|)X174 DNA by Cas10- CsrnCsm2H6N Csm3D32A in the absence or presence of AcrIIIAI at indicated ratios. KL, 1 kb DNA ladder; kb, kilobase. (D) Relative fractions of DNA cleaved from three trials of the experiment shown in panel C. For B and D, average measurements (±S.D) are shown. Asterisks indicate significant reductions in two-tailed t-tests (*, p<0.05; **, p<0.005, ***, p<0.0005). (E) Illustration of experiment used to detect DNA cleavage in vivo. (F) Nanopore sequencing reads mapped to genomes of Twillingate (Twill) WT and E6 following infection of S. epidermidis Acrispr bearing pcrispr-spc plasmids. (G) Fractions of Nanopore reads with various lengths that map to phage genomes following infection of indicated strains. (H) Length distributions of Nanopore reads that map to phage (blue) and bacterial (black) genomes following the experiment in E.
FIG. 6 panels A-D show Cas10-Csm co-purifies with acrlllA1-RNAs following Twillingate infection. (A) Schematic of the experiment used to capture Cas10-Csm associated acrlllA1 -RNAs in cells following phage infection. (B) RNA-seq analysis of Cas10-Csm associated RNAs that map to S. epidermidis RP62a Acrispr-cas following infection with Twillingate WT (top) and E6 (bottom). Fold enrichment of each RNA species was calculated as a ratio of normalized reads from infected/uninfected cells. Peaks corresponding to tRNAs (oval) and the tmRNA (asterisk) are highlighted. (C) Volcano plots showing RNA species that are significantly enriched in Cas10-Csm following seven and three independent trials of infecting with Twillingate WT (left) and E6 (right), respectively. Dots, tRNAs; black dot and *, tmRNA. (D) Heatmap showing changes in normalized raw coverage across tRNAs that co-purify with Cas10-Csm following infection with Twillingate. D, D-loop; A, anticodon loop, V, variable loop, T, T C loop.
FIG. 7 panels A-J show RNase R captures and cleaves acrlllA1-RNAs. (A-C) Native PAGE analyses of indicated nucleases alone and combined with RNA-bound AcrIIIAI in a 1 :20 molar ratio (A, B) or 1 :10, 1 :20, and 1 :40 molar ratios (C). R, RNase R; PNP, PNPase; J2, RNase J2. (D-F) Native gels from panels A-C soaked in ethidium bromide and imaged under UV light. (G) Size exclusion chromatograms for AcrIIIAI and RNase R fractionated on an S200 column individually and combined in indicated ratios. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dashed line). Dotted vertical line indicates void volume of the column. (H) SDS-PAGE analyses of proteins within fractions corresponding to chromatograms on the left. (I and J) Urea-PAGE analysis of radiolabeled acrlllA1 -RNAs (I) or a linear 43 nucleotide target RNA (J) that were subjected to degradation by RNase R for various times (0.5, 1 , 5, and 10 minutes). Dotted line separates non-adjacent wells in the same gel. All data are representatives of two independent trials. kDa, kilodaltons; M, Protein Ladder.
FIG. 8 shows a proposed model for AcrIIIAI function. The interference stage of Type lll-A CRISPR-Cas immunity is shown in the absence (A) and presence (B) of AcrIIIAI .
FIG. 9 panels A-E accompany FIG. 1 and show phage hybridization identifies six candidate Acrs. (A) Nucleotide alignment of Twillingate and PhilBB. The ten ‘gap’ regions are highlighted. (B) Schematic of the hybridization protocol used to generate Twillingate-PhiIBB recombinants. (C) Plot summarizing the genotypes of each gap region in eight selected recombinants. (D) Nucleotide alignment of PhilBB and Twill-BB H8, the hybrid that harbors the shortest region derived from Twillingate. (E) Illustration of the genomic region in Twill-BB H8 that contains Twillingate-derived sequence. For A and D, histograms were generated using the MAUVE open source software (darlinglab.org/mauve/mauve.html).
FIG. 10 panels A-F accompany FIG. 3 and show nuclease activity and function of AcrIIIAI vs. AcrIIIAI H63A’ H64A. (A) AcrIIIAI amino acid sequence and other salient features, pl, isoelectric point; MW, molecular weight; Da, Daltons. The underlined histidine residues were mutated in this study (SEQ ID NO:1 ). (B) Nuclease assay in which AcrIIIAI flow-through (FT), which contains both RNA-free and RNA-bound forms, was combined with an in vitro transcribed tmRNA substrate from S. epidermidis in the presence/absence of indicated metals. (C) Elution profiles of AcrIIIAI (top) and AcrIIIAI H63A’ H64A (bottom) following fractionation on an S75 size exclusion column. Void volume is 8.5 ml. mAU, milli- absorbance units. (D and E) Nuclease assays in which fractions of AcrIIIAI (left) and AcrIIIAI H63A’ H64A (right) were combined with radiolabeled substrates — tmRNA from S. epidermidis (D) or a linear RNA substrate (E). Dotted line separates non-contiguous lanes in the same gel. A representative of at least two independent trials is shown. L, Decade Ladder; nt, nucleotide. (F) Phage challenge plate assays with ISP using the inducible system in Figure 2F. aTc, anhydrotetracycline. Averages of triplicate measurements (±S. D.) of plaque-forming units per milliliter (pfu/ml) are shown on top, and representative plate images are shown on bottom. Two independent trials were performed. Dotted line, limit of detection. # indicates plaque counts represent an estimate as they are too small to count.
FIG. 11 panels A-B accompany FIG. 4 and show AcrIIIAI does not bind to Csm5. (A) Size exclusion chromatograms of AcrIIIAI and Csm5 when passed through an S75 sizing column individually and combined in a 4:1 ratio. mAU, milli-absorbance units at 280 nm (solid line) and 254 nm (dotted line). Dashed vertical line indicates void volume of the column. (B) SDS-PAGE analysis of proteins contained within indicated fractions. Each gel image corresponds to the chromatogram on the left. kDa, kilodaltons; M, PageRuler™ Plus Prestained Protein Ladder.
FIG. 12 panels A-C accompany FIG. 4 and show Cas10-Csm structure and AlphaFold 3 models with AcrIIIAI. (A) Cas10-Csm “short apo” complex with subunit stoichiometry Cas10i:Csm22:Csm33:Csm4i:Csm5i:crRNAi determined by cryo-electron microscopy (PDB ID 7V02; Smith et al, Structure, 2022). (B) AlphaFold 3 model of Cas10- Csm with subunit stoichiometry as in (A) bound to four copies of AcrIIIAI . (C) The same model in panel B colored according to pLDDT (predicted local distance difference test), a per-atom confidence estimate.
FIG. 13 accompanies FIG. 5 and shows CRISPR function in the presence of catalytic mutants. Phage challenge plate assays with ISP using the inducible system shown in FIG. 2F.
Representative plate images (bottom) and averages of triplicate measurements (± S. D.) of plaque-forming units per milliliter (pfu/ml) (top) are shown. Data shown is a representative of two independent trials. EV, empty vector; aTc, anhydrotetracycline. Casi o HD refers to the H14A, D15A mutant; Casi o palm refers to the G584-587A mutant. Dotted line indicates the limit of detection in this assay.
FIG. 14 panels A-D accompany FIG. 5 and show AcrIIIAI does not block target RNA cleavage. (A) Urea-PAGE analysis of a radiolabeled 43 nucleotide target RNA that was subjected to degradation by Cas10-Csm and/or RNA-bound AcrIIIAI after various time points (0.5, 1.0, 2.0, and 4.0 minutes). (B) Quantification of fractions of RNA cleaved in three trials of the experiment in panel A. (C) Urea-PAGE analysis of a radiolabeled 43 nucleotide target RNA that was subjected to degradation by Cas10-Csm and/or RNA-free AcrIIIAI after various time points (0.5, 1.0, 2.0, and 4.0 minutes). (D) Quantification of fractions of RNA cleaved in three trials of the experiment in panel C. For A and C, asterisks indicate positions at which the target RNA was cleaved by Cas10- Csm; DL, Decade Ladder, nt, nucleotide. For B and D, average measurements (± S.D) are shown.
FIG. 15 panels A-C accompany FIG. 5 and show AcrIIIAI does not cleave cOAs or complex-associated crRNAs. (A) Urea-PAGE analysis of cOAs subjected to degradation by AcrIIIAI . A10, ladder composed of ten adenine nucleotides. (B) Fractions of cOAs remaining in three trials of the experiment in panel A. Average measurements (± S.D) are shown. (C) Urea-PAGE analysis of radiolabeled crRNAs and/or acrlllA1-RNAs extracted from reaction mixtures containing Cas10-Csm and/or AcrIIIAI following a 4 h incubation. Positions of crRNAs are labelled to the right. DL, Decade Ladder; nt, nucleotide.
FIG. 16 accompanies FIG. 7 and shows AcrIIIAI functions in the absence of RNase R. Phage challenge plate assays with Twillingate WT and E6 against S. epidermidis RP62a and a mutant harboring an in-frame deletion of rnr, the gene that encodes RNase R. Indicated pcrispr-spc plasmids (or the empty vector (EV) control) were introduced into the strains prior to phage challenge. Representative plate images (bottom) and averages of triplicate measurements (± S. D.) of plaque-forming units per milliliter (pfu/ml) (top) are shown. Dotted line indicates the limit of detection in this assay. Data is representative of two independent trials.
FIG. 17 panels A-H accompany FIG. 7 show AlphaFold 3 models of RNase R bound to various RNAs. (A) Crystal structure of E. coli RNase R (residues 87-725) (PDB ID 5XGU; Chu etal, Nucleic Acids Research, 2017). The RNA channel and active site Mg2+ (sphere) are indicated. (B) AlphaFold 3 models of S. epidermidis RNase R (residues 1- 719) bound to Mg2+ (sphere). The protein structure is colored according to pLDDT (predicted local distance difference test), a per-atom confidence estimate. The pTM (predicted template modeling) and ipTM (interface predicted template modeling) scores are also indicated. The C-terminal 72 amino acids constitute a long disordered region and were removed for clarity. (C) A plot showing the pTM and ipTM scores for AlphaFold 3 models of RNase R-Mg2+ in complex with 20 representative S. epidermidis tRNAs and tmRNA, both full-length and fragmented (i.e. the first 40 nts of their 5’ends). Dashed lines indicate confidence thresholds: Models with pTM > 0.5 likely represent true structures. Complexes with ipTM < 0.6 are failed predictions, while ipTM values above 0.8 are high- confidence representations. Complexes with ipTM scores between 0.6 and 0.8 are borderline. See Table 4 for source data. (D-H) AlphaFold 3 models of S. epidermidis RNase R (residues 1 -719) bound to Mg2+ in complex with indicated RNAs. The protein structures are colored according to pLDDT as in Panel B, and RNAs are colored Magenta. The pTM and ipTM scores are also indicated.
Detailed Description Introduction
The co-evolutionary arms race between bacteria and their viruses (phages) has given rise to a vast array of immune systems and counter-defense mechanisms that phages have evolved to overcome them. Over 40% of bacteria and nearly all archaea employ clustered regularly-interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins to protect against their viruses (1 ). CRISPR-Cas systems maintain memories of past infections by capturing short stretches (~30-40 nucleotides) of viral DNA and integrating them into the CRISPR locus as ‘spacers’ in between DNA repeats with similar lengths (2, 3). Spacer sequences are transcribed and processed into small CRISPR RNAs (crRNAs), which combine with one or more Cas protein(s) to form an effector complex. Effector complexes surveil the interior of the cell and degrade foreign nucleic acids bearing ‘protospacer’ sequences that are complementary to crRNAs. CRISPR-Cas systems are tremendously diverse, and have been grouped into two classes, six types, and many subtypes based upon their effector complex architectures, cas gene content, and mechanistic details (4).
In response to the selective pressure imposed by CRISPR-Cas systems, the viruses of bacteria and archaea have evolved a variety of strategies to overcome immunity. Prime among these are anti-CRISPR proteins (Acrs). Acrs are a diverse group of small proteins (~80-150 amino acids) that typically work by binding Cas effector complexes and blocking their functions (5). Over 80 families of Acrs targeting five of the six CRISPR-Cas Types have been described thus far (6); however, only three of these have the capacity to overcome Type III immunity (7-10). Type III CRISPR-Cas systems are found in bacteria and archaea and are regarded as the most complex (4, 11 ). Of the six subtypes currently identified (A-F), Types lll-A and lll-B have historically garnered the most attention. These systems employ multi-subunit effector complexes and a variety of accessory/cellular nucleases to detect and destroy invading RNA and DNA. Upon crRNA binding to a complementary transcript, members of the core Cas effector complex perform at least three catalytic activities: Cas7/Csm3/Cmr4 shred target RNA (12-16), Casio degrades single-stranded DNA indiscriminately (15, 17- 20), and Casi o also produces cyclic-oligoadenylates (cOAs), second-messenger molecules that bind and stimulate accessory nucleases often bearing CRISPR-associated Rossman Fold (CARF) domains (21-25). These include Csm6/Csx1 , which are not a part of the core effector complex, but are encoded within or proximal to the CRISPR locus (26). Beyond these Cas accessory enzymes, Type III systems co-opt diverse cellular ‘housekeeping’ nucleases to facilitate crRNA processing and clear phage nucleic acids (27-30).
The elaborate network of accessory nucleases in the Type III immune response presents unique challenges for Type III Acrs and implies that targeting the effector complex alone might not be sufficient to completely protect against immunity. Indeed, the first Type III anti-CRISPR described, Acrl 11 B 1 binds the Cas effector complex and inhibits its function, but it rescues viruses only when late-expressed genes are targeted by the CRISPR system (7). Late gene targeting results in a delayed immune response that gives viruses a window of opportunity to begin replicating undetected. This ‘head start’ in viral replication, coupled with the inhibitory activity of Acrl I IB1 , allows viruses to complete their life cycle and escape immunity. In addition, AcrlllB2 has been reported (9, 10). AcrlllB2 binds the Type lll-B effector complex and diminishes all three of its catalytic activities by reducing the disassociation rate of cleaved target RNA. Although plasmid-encoded AcrlllB2 is effective when targeting both early- and late- expressed viral genes (9), a single genomic copy of AcrlllB2 protects viruses only when late genes are targeted (10). In contrast to these Type lll-B specific Acrs, Acrlll-1 does not bind/inhibit effector complexes, but rather it degrades cyclic tetra-adenylates (cOA4), and thus interferes with the downstream activation of CARF-containing accessory nucleases that rely upon cOA4 to propagate the immune response (8). The effectiveness of Acrlll-1 during early- versus late- gene targeting has not been explored; however, since the cOA-responsive accessory nucleases are essential particularly when late-expressed genes are targeted (31 ), it is likely that Acrlll-1 is also more effective when the target is located in late-expressed genes. To date, Type lll-A specific Acrs have not been described.
Provided herein is Acrl I IA1 , the first Type lll-A anti-CRISPR protein and methods of its use. We identified AcrIIIAI in a subset of phages that infect S. epidermidis RP62a. Genetic analyses revealed that native expression of acrIIIAI completely overcomes CRISPR immunity whether early- or late- expressed phage genes are targeted. Biochemical analyses showed that AcrIIIAI co-purifies with fragmented t(m)RNAs (herein referred to as acrIIIAI -RNAs), and both RNA-bound and RNA-free fractions associate with the effector complex and attenuate its DNase activity and cOA production. Moreover, acrIIIAI -RNAs co-purify with effector complexes in the native host during phage infection, and acrIIIAI -RNAs specifically interact with RNase R, a cellular ‘housekeeping’ nuclease that promotes robust immunity (30) in a purified system. Altogether, these results support a model in which AcrIIIAI uses a unique multi-layered strategy to mitigate indiscriminate nucleic acid cleavage on multiple fronts in order to provide robust protection against Type III CRISPR immunity.
Polynucleotides
Polynucleotides including recombinant polynucleotides contain less than an entire microbial genome and can be single- or double-stranded nucleic acids. A polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof. A polynucleotide can comprise, for example, a gene, open reading frame, non-coding region, or regulatory element.
A gene is any polynucleotide molecule that encodes a polypeptide, protein, or fragments thereof, optionally including one or more regulatory elements preceding (5' noncoding sequences) and following (3' non-coding sequences) the coding sequence. In one aspect, a gene does not include regulatory elements preceding and following the coding sequence. A native or wild-type gene refers to a gene as found in nature, optionally with its own regulatory elements preceding and following the coding sequence. A chimeric or recombinant gene refers to any gene that is not a native or wild-type gene, optionally comprising regulatory elements preceding and following the coding sequence, wherein the coding sequences and/or the regulatory elements, in whole or in part, are not found together in nature. Thus, a chimeric gene or recombinant gene comprise regulatory elements and coding sequences that are derived from different sources, or regulatory elements and coding sequences that are derived from the same source but arranged differently than is found in nature. A gene can encompass full-length gene sequences (e.g., as found in nature and/or a gene sequence encoding a full-length polypeptide or protein) and can also encompass partial gene sequences (e.g., a fragment of the gene sequence found in nature and/or a gene sequence encoding a protein or fragment of a polypeptide or protein). A gene can include modified gene sequences (e.g., modified as compared to the sequence found in nature). Thus, a gene is not limited to the natural or full-length gene sequence found in nature.
Polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides. For example, the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. A polynucleotide existing among hundreds to millions of other polynucleotide molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest are not to be considered a purified polynucleotide. Polynucleotides can encode the polypeptides described herein (e.g., SEQ ID NO:1 ).
Polynucleotides can comprise additional heterologous nucleotides that do not naturally occur contiguously with the polynucleotides. As used herein the term “heterologous” refers to a combination of elements that are not naturally occurring or that are obtained from different sources.
Polynucleotides can be isolated. An isolated polynucleotide is a naturally-occurring polynucleotide that is not immediately contiguous with one or both of the 5' and 3' flanking genomic sequences that it is naturally associated with. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid sequences naturally found immediately flanking the recombinant DNA molecule in a naturally-occurring genome is removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules. Polynucleotides can encode full-length polypeptides, polypeptide fragments, and variant or fusion polypeptides.
Degenerate polynucleotide sequences encoding polypeptides described herein, as well as homologous nucleotide sequences that are at least about 80, or about 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% identical to polynucleotides described herein (e.g., SEQ ID NO:2) and the complements thereof are also polynucleotides. Degenerate nucleotide sequences are polynucleotides that encode a polypeptide described herein or fragments thereof, but differ in nucleic acid sequence from the wild-type polynucleotide sequence, due to the degeneracy of the genetic code. Complementary DNA (cDNA) molecules, species homologs, and variants of polynucleotides that encode biologically functional polypeptides also are polynucleotides.
Polynucleotides can be obtained from nucleic acid sequences present in, for example, a bacteria like Streptococcus. Polynucleotides can also be synthesized in the laboratory, for example, using an automatic synthesizer. An amplification method such as PCR can be used to amplify polynucleotides from either genomic DNA or cDNA encoding the polypeptides.
Polynucleotides can comprise non-coding sequences or coding sequences for naturally occurring polypeptides or can encode altered sequences that do not occur in nature.
Unless otherwise indicated, the term polynucleotide or gene includes reference to the specified sequence as well as the complementary sequence thereof.
The expression products of genes or polynucleotides are often proteins, or polypeptides, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA. The process of gene expression is used by all known life forms, i.e., eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses, to generate the macromolecular machinery for life. Several steps in the gene expression process can be modulated, including the transcription, up-regulation, RNA splicing, translation, and post-translational modification of a protein.
Polynucleotides as set forth in SEQ ID NO:2 or functional fragments thereof are provided herein.
ATGACTGAAAAAATGATTGCAAAAAAACAAGGATGTGAATTCTTTGAATTCACAAAAA CAAAAACAGAAGGTATTGACACTTTTGTTACTGTTAAAGATATTAAAGGGTTTGCACC TTTTGGCACTCTTGACCGTGTGTTTTTTAGTCGTGAAACTATAGTACAAGAATTTGAC GGTACTATAACACATCACTTTGAAATCATTCAAAATGACGATTTCATTATTGATGAAG ATTCAGAAGACTTTGGAACCGTTAAAGGGTTCTAA SEQ ID NO:2.
In an aspect, a fragment of SEQ ID NO:2 is provided. For example, a fragment of about 7, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250 or more nucleotides can be used as, for example, a primer or probe. In another example, a fragment of about 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 25, 20,15, 10, 7 or less nucleotides can be used as, for example, a primer or probe. A primer or probe can comprise a detectable label or marker. In some aspects, isolated polynucleotides have at least 70%, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, and any number or range in between, identity to SEQ ID NO:2 or a functional fragment thereof. A polynucleotide can encode an Acrl I IA1 polypeptide, wherein the Acrl I IA1 polypeptide has greater than 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO:1 MTEKMIAKKQGCEFFEFTKTKTEGIDTFVTVKDIKGFAPFGTLDRVFFSRETIVQEFDGTI THHFEIIQNDDFIIDEDSEDFGTVKGF. In an aspect, a fragment of SEQ ID NO:1 is provided. For example, a fragment of about 7, 10, 15, 20, 25, 30, 40, 50, 75, 80 or more amino acids can be used as, for example, a clinical marker. In another example, a fragment of about 8075, 50, 40, 30, 25, 20,15, 10, 7 or less amino acids can be used as, for example, a clinical marker.
A polynucleotide can comprise a heterologous promoter sequence operably linked to a polynucleotide encoding one or more Acrl I IA1 polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO:1. A promoter can be a constitutive promoter, a repressible promoter, or an inducible promoter. A constitutive promoter can be, for example a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter. An inducible promoter can be, for example, a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
A polynucleotide can encode a fusion protein, wherein the polynucleotide comprises a heterologous polynucleotide and a polynucleotide encoding one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1 . The heterologous polynucleotide can be or can encode a heterologous protein such as a therapeutic protein, an affinity tag, an epitope sequence tag, a detectable marker polypeptide, an amino acid spacer, an amino acid linker, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, or a cleavable linker. An amino acid spacer is a sequence of amino acids that are not usually contiguously associated with an AcrIIIAI polypeptide of the invention in nature. An amino acid spacer can comprise about 1 ,5, 10, 20, 100, 1 ,000 or more amino acids.
In some aspects, a cleavage site or cleavable linker can be present in a polynucleotide as described herein. In some cases, a cleavable linker can be a selfcleaving linker (e.g., a 2A peptide or an intein). In some aspects a cleavable linker or cleavage site can be cleavable by one or more proteases. A cleavable linker or cleavage site can have any suitable length. In some cases, a cleavable linker or cleavage site is about 1 , 2, 5, 10, 15, or more amino acids in length.
In some aspects, a cleavable linker is cleavable by one or more host cell proteases (e.g., an extracellular protease such as a matrix metalloproteinase, or an endopeptidase- 2; an intracellular protease such as a cysteine protease or a seine protease; etc.). Any convenient cleavable linker can be used. In an aspect a cleavable linker or cleavage site can be cleaved by a protease such as chymotrypsin-like elastase family member 2A, anionic trypsin-2, chymotrypsin-C, chymotrypsinogen B, elastase 1 SGPTAAPA (SEQ ID NO:3), elastase 3, trypsin (SGPTGHGR SEQ ID NO:4; SGPTGMAR (SEQ ID NO:5), and chymotrypsin (SGPTASPL SEQ ID NO:6), (e.g., chymotrypsin B (SGPTTAPF SEQ ID NO:7)). Thus, in some cases, a cleavable linker of a fusion protein is cleavable by one or more proteases such as a trypsin, a chymotrypsin, and an elastase. In some cases, a cleavable linker of a subject secreted fusion protein is cleavable by one or more proteases selected from: chymotrypsin-like elastase family member 2A (cleavage site: Leu (L), Met (M) and Phe (F)), anionic trypsin-2 (cleavage site: Arg (R), Lys (K)), chymotrypsin-C (cleavage site: Leu (L), Tyr (Y), Phe (F), Met (M) Trp (W), Gin (Q), Asn (N)), chymotrypsinogen B (cleavage site: Tyr (Y), Trp (W), Phe (F), Leu (L)), elastase 1 (cleavage site: Ala (A)), and elastase 3 (cleavage site: Ala (A)).
Polypeptides
A polypeptide is a polymer where amide bonds covalently link three or more amino acids. A polypeptide can be post-translationally modified. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used in synthesis of the polypeptide, or combinations thereof. A polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used in synthesis of the polypeptide has less than about 30%, 20%, 10%, 5%, 1 % or less of other polypeptides, culture medium, chemical precursors, and/or other chemicals used in synthesis. Therefore, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% or more pure.
The term “polypeptides” can refer to one or more types of polypeptides or a set of polypeptides. “Polypeptides” can also refer to mixtures of two or more different types of polypeptides including, but not limited to, full-length proteins, truncated polypeptides, or polypeptide fragments. The term “polypeptides” or “polypeptide” can each mean “one or more polypeptides.”
In one aspect, a polypeptide or fragment thereof is non-naturally occurring. In an aspect, a polypeptide or fragment thereof comprises 5, 10, 20, 30, 40, 50, 75, 80, 85, or more amino acids of SEQ ID NO:1 or any other polypeptide described herein. In an aspect, the non-naturally occurring amino acids can provide a beneficial property such as increased solubility of the polypeptide or increased biological activity.
The terms “sequence identity” or “percent identity” are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first polypeptide or polynucleotide for optimal alignment with a second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions (i.e., overlapping positions)x100). In some aspects the length of a reference sequence aligned for comparison purposes is at least 50, 60, 70, or 80% of the length of the comparison sequence, and in some aspects is at least 90% or 100%. In an aspect, the two sequences are the same length.
Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a disclosed sequence and a claimed sequence can be at least 80%, at least 83%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence.
Polypeptides that are sufficiently similar to polypeptides described herein (e.g., SEQ ID NO:1 ) can be used herein. SEQ ID NO:1 MTEKMIAKKQGCEFFEF TKTKTEGIDTFVTVKDIKGFAPFGTLDRVFFSRETIVQEFDGTITHHFEIIQNDDFIIDEDSE DFGTVKGF.
A polypeptide variant differs by about, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more amino acid residues (e.g., amino acid additions, substitutions, or deletions) from a peptide shown SEQ ID NO: 1 or a fragment thereof. Where this comparison requires alignment, the sequences are aligned for maximum homology. The site of variation can occur anywhere in the polypeptide. In one aspect, a variant has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to the original polypeptide.
In some aspects, a polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the amino acid sequence put forth in SEQ ID NO:1 , or a fragment thereof. In some aspects, a polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to at least one portion of the amino acid sequence put forth in SEQ ID NO:1 , or a fragment thereof.
Variant polypeptides can generally be identified by modifying one of the polypeptide sequences described herein and evaluating the properties of the modified polypeptide to determine if it is a biological equivalent. A variant is a biological equivalent if it reacts substantially the same as a polypeptide described herein in an assay such as a Cas10- Csm cOA production assay, a Cas10-Csm DNA cleavage assay, or other suitable assay. In other words, a variant is a biological equivalent if it has 90-110% of the activity of the original polypeptide.
Variant polypeptides can have one or more conservative amino acid variations or other minor modifications and retain biological activity, i.e., are biologically functional equivalents to SEQ ID NO:1 , or a fragment thereof. Variant polypeptides can have labels, tags, additional amino acids, amino acids that can be used for purification, amino acids that can be used to increase solubility of the polypeptide, amino acids to improve other characteristics of the polypeptide, or other amino acids. In an aspect, the additional amino acids are not Streptococcus amino acids.
Methods of introducing a mutation into an amino acid sequence are well known to those skilled in the art. See, e.g., Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989). Mutations can also be introduced using commercially available kits such as “QuikChange™ Site-Directed Mutagenesis Kit” (Stratagene). The generation of a functionally active variant polypeptide by replacing an amino acid that does not influence the function of a polypeptide can be accomplished by one skilled in the art. A variant polypeptide can also be chemically synthesized.
Variant polypeptides can have conservative amino acid substitutions at one or more predicted nonessential amino acid residues. A conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1 ) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. In one aspect a polypeptide has about 1 , 2, 3, 4, 5, 10, 20 or fewer conservative amino acid substitutions.
A polypeptide can be a fusion protein, which can contain other amino acid sequences, such as amino acid linkers, amino acid spacers, signal sequences, TMR stop transfer sequences, transmembrane domains, as well as ligands useful in protein purification, such as glutathione-S-transferase, histidine tag (e.g., about 6, 7, 8, 9, 10, or more His residues), and staphylococcal protein A, or combinations thereof. In an aspect, a polypeptide comprises one or more epitope tags, such as FLAG (for example, DYKDDDDK; SEQ ID NO:8), HA (YPYDVPDYAC; SEQ ID NO:9), myc (EQKLISEEDLC; SEQ ID NO:10), V5 (GKPIPNPLLGLDST; SEQ ID NO:11 ), E-tag (GAPVPYPDPLEPR; SEQ ID NO:14), VSV-g (YTDIEMNRLGK; SEQ ID NO:15), 6xHis (HHHHHHH; SEQ ID NO:16), HSV (QPELAPEDPEDC; SEQ ID NO:17), CD34 enrichment tag, Glutathione-S- Transferase (GST), calmodulin binding protein (CBP), protein C tag, HaloTag, and biotin tag. An antibody, such as a monoclonal antibody, can specifically bind to an epitope tag and be used to purify a polypeptide comprising the epitope tag.
A fusion protein can comprise two or more different amino acid sequences operably linked to each other. A fusion protein construct can be synthesized chemically using organic compound synthesis techniques by joining individual polypeptide fragments together in fixed sequence. A fusion protein can also be chemically synthesized. A fusion protein construct can also be expressed by a genetically modified host cell (such as E. coli or Staphylococcus) cultured in vitro, which carries an introduced expression vector bearing specified recombinant DNA sequences encoding the amino acids residues in proper sequence. The heterologous polypeptide, e.g., a therapeutic protein can be fused, for example, to the N-terminus or C-terminus of an AcrIIIAI polypeptide. More than one polypeptide can be present in a fusion protein. Fragments of polypeptides can be present in a fusion protein. A fusion protein can comprise, e.g., an AcrIIIAI polypeptide, fragments thereof, or variants thereof.
In an aspect, an isolated fusion protein comprises (1 ) one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:1 , and (2) one or more heterologous polypeptides. A heterologous polypeptide can be, e.g., a therapeutic polypeptide, an affinity tag, an epitope sequence tag, a detectable marker polypeptide, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, an amino acid spacer, an amino acid linker, or a cleavable linker.
A fusion protein can further comprise e.g., one, two, three, four, five, six, seven or more of a heterologous protein (e.g., a marker polypeptide). Polypeptides can be in a multimeric form. In other words, a polypeptide can comprise two or more copies (e.g., two, three, four, five, six, seven or more) of an AcrIIIAI protein, fragments or variants thereof, a heterologous polypeptide, fragments thereof, or a combination thereof. A polypeptide can include, e.g., a fusion protein of two, three, four, five, six, seven or more polypeptides having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1 ; or a fusion protein of at least two polypeptides having about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:1. A polypeptide can be a fusion protein that can include one or more linkers between the individual proteins making up the fusion protein. Alternatively, no linkers can be present between the individual proteins making up the fusion protein. A fusion polypeptide can contain other amino acid sequences, such as amino acid linkers, amino acid spacers, signal sequences, TMR stop transfer sequences, transmembrane domains, as well as ligands useful in protein purification, such as glutathione-S-transferase, histidine tag, epitope tags, and staphylococcal protein A, or combinations thereof.
Polypeptides can be lyophilized, desiccated, or dried, for example freeze-dried. A lyophilized polypeptide can be obtained by subjecting a preparation of the polypeptides to low temperatures to remove water from the sample. A desiccated polypeptide composition can be obtained by drying out a preparation of the polypeptides by removal of water. A dried polypeptide preparation can refer to a polypeptide preparation that has been air dried (e.g., lyophilized).
Promoters
A recombinant polynucleotide described herein can comprise a promoter. The term “promoter” and “promoter sequence” as used herein means a control sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or a transgene, are controlled. Promoters can be constitutive, inducible, repressible, or tissue-specific, for example. Promoters can contain genetic elements at which regulatory proteins and molecules such as RNA polymerase and transcription factors may bind. A promoter can be operably linked to a polynucleotide encoding a secretion carrier.
The term “operably linked” refers to the expression of a polynucleotide that is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a polynucleotide under its control. A promoter can be positioned 5' (upstream) of a gene under its control. The distance between a promoter and a polynucleotide can be approximately the same as the distance between that promoter and the polynucleotide it controls in the polynucleotide from which the promoter is derived. Variation in the distance between a promoter and a polynucleotide can be accommodated without loss of promoter function.
A promoter can be a constitutive promoter, a repressible promoter, or an inducible promoter. The expression of the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex, AcrIIIAI , and/or a polynucleotide encoding one or more crRNA molecules be under control of the same promoter. Alternatively, each polynucleotide can be under the control of an individual promoter, which can be a same or different promoter.
A constitutive promoter can be, e.g., a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, a SFFV promoter, or any other suitable promoter. An inducible promoter can be a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, a UAS inducible promoter, or any other suitable promoter. In an aspect a promoter is responsive to conditional stimuli, including but not limited to temperature, nutrients, antibiotics, oxygen concentration, cell cycle phases, quorum sensing, chemokines, cytokines, growth factors, or any other stimulus or compound that can drive gene expression from the particular promoter in question. In an aspect, a promoter is a prokaryotic promoter. In an aspect, a promoter is an RNA-polymerase II eukaryotic promoter.
Vectors
A vector is a polynucleotide that can be used to introduce polynucleotides or expression cassettes into one or more host cells. A vector can be an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the polynucleotide sequence. An expression vector can have a promoter sequence operably linked to a polynucleotide sequence (e.g., transgene) to drive expression in a host cell, and in some aspects, also comprises a transcription terminator sequence. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs). A plasmid vector can be a circular double-stranded DNA construct used as a cloning and/or expression vector. Plasmid vectors can be linear or closed circular plasmids.
In an aspect, a vector is a phagemid. A phagemid can be provided as a component of a bacteriophage. Any suitable vector can be used to deliver polynucleotides or expression cassettes to a population of host cells.
In an aspect, expression vectors can comprise a recombinant polynucleotide encoding a recombinant AcrIIIAI polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some aspects, the various nucleic acid and control sequences described herein are joined together (i.e. , operably linked) to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the recombinant AcrIIIAI polypeptide at such sites.
Expression vectors can comprise a recombinant polynucleotide encoding a recombinant Casi o system or complex optionally including one more crRNA polynucleotides, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some aspects, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the recombinant Casi o system polypeptides and crRNA polynucleotides at such sites.
In some aspects, an expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). In some aspects, a vector can be integrated into the genome of the host cell and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some aspects, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.
In some aspects, an expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A selectable marker can be a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Casio Complexes and Systems
Provided herein are compositions and methods that provide for the recombinant expression of a Casi o gene (also known as Csm1 ), which is present in type III CRISPR systems. One or more polynucleotides that encode Casio, Cas6 and a Csm protein such as Csm2, Csm3, Csm4, Csm5, Csm6, and combinations thereof can be used in the disclosed compositions and methods. In some aspects, one or more recombinant polynucleotides that encode of Casio, Cas6, Csm2, Csm3, Csm4, Csm5 and Csm6 are provided. In some aspects, polynucleotides encoding two, three, or four Csm proteins are provided.
A Casio polypeptide complex can comprise Casi o and one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5. A Casi o polypeptide complex can further be complexed with a crRNA. A Casio system can comprise Cas10; one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5; Csm6; Cas6; and combinations thereof.
In an aspect, a Casi o system comprises Casi o, Csm2, Csm3, Csm4, Csm5, Csm6, and Cas6. In an aspect, isolated Cas10-Csm complexes can comprise Cas10-Csm complexes that are already loaded with the guide RNA, and Casi o, Csm2, Csm3, Csm4, and Csm5.
Casi o and Csm polynucleotides can be any naturally occurring, modified, or variant polynucleotides. For example, polynucleotides can be those as shown in GenBank accession numbers: Cas10: AAW53330.1 ; Csm2: AAW53329.1 ; Csm3: AAW53328.1 ; Csm4: AAW53327.1 ; Csm5: AAW53326.1 ; Csm6: AAW53325.1 ; Cas6: AAW53324.1. Other Cas10 and Csm polynucleotides and polypeptides can be used. For example Cas10 and Csm polynucleotides and polypeptides from other Staphylococcus sp., Streptococcus sp., Pyrococcus furiosis, Thermococcus onnurineus, Roseifluxus sp. RS-1, Psuedothermotoga lettingae, Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., or any other suitable organism, and archaeal species, such as Sulfolobus solfataricus, Methanopyrus kandleri, and Thermus thermophilus sp. or any organism having type III CRISPR-Cas10 systems can be used herein.
Representative Cas and Csm sequences are disclosed in, for example, Chou- Zheng & Hatoum-Aslan, Expression and Purification of the Cas10-Csm Complex from Staphylococci. Bio Protoc. 2017 Jun 5;7(11 ):e2353; Wiegand et al., Functional and Phylogenetic Diversity of Cas10 Proteins. CRISPR J. 2023 Apr;6(2): 152-162. Cas10 sequences are shown in Table 1 .
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Examples of Csm2 polypeptide sequences include:
MILAKTKSGKTIDLTFAHEWKSNVKNVKDRKGKEKQVLFNGLTTSKLRN LMEQVNRLYTIAFNSNEDQLNEEFIDELEYLKIKFYYEAGREKSVDEFLK KTLMFPIIDRVIKKESKKFFLDYCKYFEALVAYAKYYQKED (SEQ ID NO:24).
Examples of Csm3 polypeptide sequences include:
MYSKIKISGTIEWTGLHIGGGGESSMIGAIDSPWRDLQTKLPIIPGSS IKGKMRNLLAKHFGLKMKQESHNQDDERVLRLFGSSEKGNIQRARLQISD AFFSEKTKEHFAQNDIAYTETKFENTINRLTAVANPRQIERVTRGSEFDF
VFIYNVDEESQVEDDFENIEKAIHLLENDYLGGGGTRGNGRIQFKDTNIE TWGEYDSTNLKIK (SEQ ID NO:25).
Examples of Csm4 polypeptides include:
MTLATKVFKLSFKTPVHFGKKRLSDGEMTITADTLFSALFIETLQLGKDT DWLLNDLIISDTFPYENELYYLPKPLIKIDSKEEDNHKAFKKLKYVPVHH YNQYLNGELSAEDATDLNDIFNIGYFSLQTKVSLIAQETDSSADSEPYSV
GTFTFEPEAGLYFIAKGSEETLDHLNNIMTALQYSGLGGKRNAGYGQFEY EIINNQQLSKLLNQNGKHSILLSTAMAKKEEIESALKEARYILTKRSGFV QSTNYSEMLVKKSDFYSFSSGSVFKNIFNGDIFNVGHNGKHPVYRYAKPL WLEV (SEQ ID NO:26).
Examples of Csm5 polypeptides include:
MTI KN YEVVI KTLG P I H I GS GQVM KKQ DYI YD FYN S KVYM I N G N KLVKF L KRKNLLYTYQNFLRYPPKNPRENGLKDYLDAQNVKQSEWEAFVSYSEKVN QGKKYGNTRPKPLNDLHLMVRDGQNKVYLPGSSIKGAIKTTLVSKYNNEK NKDIYSKIKVSDSKPIDESNLAIYQKIDINKSEKSMPLYRECIDVNTEIK FKLTIEDEIYSINEIEQSIQDFYKNYYDKWLVGFKETKGGRRFALEGGIP DVLNQNILFLGAGTGFVSKTTHYQLKNRKQAKQDSFEILTKKFRGTYGKM KEIPSNVPVALKGTTNQSRHTSYQQGMCKVSFQELNNEVL
(SEQ ID NO:27).
Examples of Csm6 polypeptides include:
MKILFSPIGNSDPWRNDRDGAMLHIVRHYNLDKVVLYFTRTIWEGNENRK GHKIYEWEKIIQTVSPNTEVEIIIENVDNAQDYDVFKEKFHKYLKIIEDS YEDCEIILNVTSGTPQMESTLCLEYIVYPENKKCVQVSTPTKDSNAGIEY SNPKDKVEEFEIVNEVEKKSEKRCKEINILSFREAMIRSQILGLIDNYDY EGALNLVSNQKSFRNGKLLRKKLLSLTKQIKTHEVFPEINEKYRDDALKK SLFHYLLLNMRYNRLDVAETLIRVKSIAEFILKTYIEIHWPTLIIEKDGK PYLNDEDNLSFVYKYNLLLEKRKQNFDVSRILGLPAFIDILTILEPNSQL LKEVNAVNDINGLRNSIAHNLDTLNLDKNKNYKKIMLSVEAIKNMLHISF PEIEEEDYNYFEEKNKEFKELL (SEQ ID NO:28).
Examples of Cas6 polypeptides include:
MINKITVELDLPESIRFQYLGSVLHGVLMDYLSDDIADQLHHEFAYSPLK QRIYHKNKKIIWEIVCMSDNLFKEVVKLFSSKNSLLLKYYQTNIDIQSFQ IEKINVQNMMNQLLQVEDLSRYVRLNIQTPMSFKYQNSYMIFPDVKRFFR SIMIQFDAFFEEYRMYDKETLNFLEKNVNIVDYKLKSTRFNLEKVKIPSF TGEIVFKIKGPLPFLQLTHFLLKFGEFSGSGIKTSLGMGKYSII (SEQ ID NO:29).
A Casio system can comprise polynucleotides encoding Casi o and one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5. A Casi o system can further comprise one or more crRNAs. A Cas10 system can comprise polynucleotides encoding Cas10; one or more (e.g., 1 , 2, 3, or 4) of Csm2, Csm3, Csm4, and Csm5; Csm6; Cas6; and combinations thereof. The polynucleotides can be present in one or more vectors that can be delivered to cells.
As described by Burmistrz et al., Casio systems can have three nuclease activities. (Burmistrz et al. Inf. J. Mol. Sci. 2020, 27(3), 1122). The first is a sequence specific RNA cleavage performed by the Cas7. The targeted RNA is positioned along the crRNA in the RNP and digested by the multiple copies of Cas7 present in the complex. As a result the RNA molecule is cleaved at fixed, 6-nt long intervals. Complementarity between crRNA and targeted RNA does not have to be strict, and the presence of mismatches does not abolish nuclease activity. The second nuclease activity is non-specific ssDNA cleavage. This requires transcription of the protospacer sequence. The RNA polymerase opens the DNA double helix to start transcription exposing the antisense making it accessible for the HD domain of Casi o of the RNP complex. The DNase is activated by complementarity between crRNA and targeted RNA. The safety switch for this activity relies on complementarity between the 5'crRNA handle and the 3' protospacer region of targeted RNA. Such complementarity inhibits cleavage, and thus prevents the host’s CRISPR array from being targeted. The third nuclease activity is non-specific RNA degradation. Similarly to non-specific ssDNA cleavage, this activity also depends on Casi o. The non-specific RNA degradation is triggered by the binding of the RNP complex with targeted RNA with simultaneous non-complementarity between crRNA handle and targeted RNA. crRNAs
Systems described herein can comprise one or more polynucleotide sequences that encode a CRISPR RNA (crRNA) that can specifically target a polynucleotide of interest. The one or more crRNAs (e.g., 1 , 2, 3, 4, 5, 6, 7, or more crRNAs) can be operatively linked to a promoter.
The structure of a naturally occurring CRISPR locus includes a number of short repeating sequences (“repeats”). Repeats can occur in clusters and can be regularly spaced by unique intervening sequences called “spacers.” CRISPR repeats can vary in length and are partially palindromic. Repeats can be arranged in clusters of repeated units. Spacers are located between two repeats and can have a unique sequence of about 20- 72 bp in length. Repeat/spacer arrays can be transcribed as a long precursor that is cleaved within repeat sequences and processed into smaller crRNAs by Casio. crRNAs retain spacer sequences that specify the targets of CRISPR interference. crRNAs function as complementary guides in Cas/crRNA ribonucleoprotein complexes that cleave the nucleic acids carrying a cognate sequence (“the protospacer” or “spacer”). The spacer- derived sequence of the crRNA can anneal to its DNA target (i.e. , the spacer). In type III CRISPR systems, targeting can be prevented by excessive base pairing between the repeat-derived crRNA and the corresponding DNA sequence. For targeting to occur, the crRNA comprises sufficient mismatching (e.g., about 1 , 2, 3, 4, 5, 6, 7, 8, or more mismatches) between the crRNA tag region and corresponding DNA sequence adjacent to the spacer.
One of ordinary skill in the art can design crRNA polynucleotides to target a polynucleotide or gene of interest. In an aspect, a crRNA comprises a segment that is the same as or complementary to a DNA target sequence (a spacer) in the targeted cell. A crRNA can be operably linked to a promoter. A crRNA that is complexed with Cas10 during cleavage of a DNA target sequence can be about 25, 30, 35 or more nucleotides in length. In some aspects, a crRNA has at its 5' terminus an eight nucleotide sequence derived from an upstream repeat sequence, followed by a variable length of nucleotides derived from the spacer, and in some cases additional nucleotides derived from downstream sequences. Type III CRISPR systems can generally recognize their target despite mismatches (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more) mismatches within the target sequence as described above. The eight nucleotide sequence can be well conserved. An example is ACGAGAAC.
Recombinant Cells
Recombinant cells are provided herein. The recombinant cells can comprise a recombinant AcrIIIAI polynucleotides, wherein the recombinant Acrl I IA1 polynucleotides are heterologous to the cell. A recombinant AcrIIIAI polynucleotide encodes a polypeptide that has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1. An AcrIIIAI polynucleotide can have greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:2. Recombinant cells can also comprise a recombinant AcrIIIAI polypeptide, wherein the recombinant AcrIIIAI polypeptide is heterologous to the cell and the recombinant AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1.
Recombinant cells can further comprise a polynucleotide encoding a gene or gene cluster encoding a Cas10 polypeptide system, wherein the gene or gene cluster is operably linked to a promoter; or a Casi o polypeptide system. Recombinant cells can naturally express a Casi o polypeptide system or can be genetically engineered to express a gene or gene cluster encoding a Casio polypeptide system. Recombinant cells can further comprise a polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide system to a target polynucleotide. The polynucleotide encoding one or more crRNA molecules can be present on part of the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system. Alternatively, the polynucleotide encoding one or more crRNA molecules can be separate from the polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system.
Expression of a recombinant AcrIIIAI polynucleotide, a polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide system, and/or a polynucleotide encoding one or more crRNA molecules can be each independently under the control of a constitutive promoter, a repressible promoter, or an inducible promoter. The expression of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide system, and/or the polynucleotide encoding one or more crRNA molecules can be under control of the same promoter.
A constitutive promoter can be, e.g., a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, a SFFV promoter, or any other suitable promoter. An inducible promoter can be a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, a UAS inducible promoter, or any other suitable promoter.
AcrIIIAI polypeptides in the recombinant cells can modulate the activity of a Casio polypeptide system (either naturally occurring or recombinantly added to a cell). For example the expression of a recombinant AcrIIIAI polypeptide can be used to prevent or interfere with the activity of the Casio polypeptide system as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay or a Cas10-Csm DNA cleavage assay. In an aspect AcrIIIAI polypeptides can reduce the biological activity of a Casio polypeptide system by about 20, 30, 40, 50, 60, 70, 80, 90% or more as measured using a suitable assay. A recombinant cell can be a eukaryotic cell, a mammalian cell, a human cell, or a prokaryotic cell. A Casio polypeptide system can derived from, e.g., Pyrococcus furiosis, Thermococcus onnurineus, Sulfolobus solfataricus, Roseifluxus sp. RS-1, Psuedothermotoga lettingae, Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., Methanopyrus kandleri, or Thermus thermophilus or any other or any organism harboring a Type III CRISPR-Cas system.
Systems
Provided herein are systems that can comprise a polynucleotide encoding an AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the first polynucleotide is operably linked to a promoter. Another system can comprise a polynucleotide encoding one or more crRNA molecules that are capable of guiding a Casio polypeptide complex to a target polynucleotide or gene in a cell, wherein the polynucleotide is operably linked to a promoter; and a polynucleotide encoding an AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID N0:1 , wherein the polynucleotide is operably linked to a promoter.
Yet another system can comprise a first polynucleotide encoding a gene or gene cluster encoding a Casi o polypeptide complex or system, wherein the first polynucleotide is operably linked to a promoter; a second polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casi o polypeptide system to a target polynucleotide or gene in a cell, wherein the second polynucleotide is operably linked to a promoter; and a third polynucleotide encoding an AcrIIIAI polypeptide, wherein the Acrl I IA1 polypeptide has greater than 70, 80, 85, 90, 95, or 99% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the third polynucleotide is operably linked to a promoter. A polynucleotide encoding a Casi o polypeptide complex or system can encode a Casi o enzyme, a Cas6 protein, and a Csm protein selected from a Csm2 protein, a Csm3 protein, a Csm4 protein, a Csm5 protein, a Csm6 protein, and combinations thereof. In an aspect, a single polynucleotide encodes the crRNA, the Casi o protein, the Cas6 protein, and the Csm protein(s).
Each of the polynucleotides of these systems can be operably linked to the same promoter or different promoters. Each of the polynucleotides can be present in a one vector or all polynucleotides can be present in the same vector.
In some aspects, polynucleotides encoding a CRISPR Casi o system and/or AcrIIIAI polypeptides can be transiently present or expressed in a cell. Polynucleotides encoding CRISPR Casi o systems and/or AcrIIIAI polypeptides can be stably present in a cell, and can be integrated into a chromosome. A vector can be maintained in a cell using a selectable marker, which can also be encoded by the vector, such as an antibiotic resistance gene, or a gene that subjects the cells comprising the vector to a nutritional selection. Vectors can be introduced into cells using any suitable technique and delivery system, such as electroporation, lipid-based transfection systems, plasmid transformation systems, e.g., using competent cells, phage or viral transduction, micro-injection, including direct injection of a vector or crRNA. In some aspects, Casio and Csm proteins and/or AcrIIIAI polypeptides can be directly injected into a cell configured to express a suitable crRNA.
A polynucleotide encoding an AcrIIIAI polypeptide can be operably linked to an inducible promoter, or to a promoter that is sensitive to another stimulus. Therefore, the expression of an AcrIIIAI polypeptide can be controlled by switching expression on and off using the inducible or controllable promoter. When it is desired to shut off Cas10 activity in a cell the promoter operably linked to the polynucleotide encoding a Acrl I IA1 polypeptide can be activated so that AcrIIIAI is expressed. Once expressed the AcrIIIAI polypeptide can reduce or eliminate the activity of the Casi o system in the cell. To re-activate the Casi o system the promoter the promoter operably linked to the polynucleotide encoding a AcrIIIAI polypeptide can be deactivated so that AcrIIIAI is not expressed. The Casi o system can then return to operation.
Delivery Vehicles
Any of the polynucleotides, polypeptides, fusion proteins, or vectors described herein can be present with a delivery vehicle. A polynucleotide, polypeptide, or vector can be combined with or encapsulated within a delivery vehicle such a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform.
Methods of introducing a nucleic acid molecule, polypeptide, vector, or combinations thereof into a host cell are known can be used to introduce the nucleic acid molecule, polypeptide, or vector into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Methods for introduction of a nucleic acid molecule, polypeptide, vector, or combinations thereof include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery.
In some cases, a polypeptide described herein is provided as a nucleic acid molecule (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, etc.) that encodes the polypeptide. A polypeptide as described herein can be introduced into a cell (provided to the cell) by any convenient method, e.g., injection directly into a cell, nucleofection; via a protein transduction domain (PTD) conjugated to one or more components of the Casi o system.
In an aspect, a nucleic acid molecule, polypeptide, vector, or combination thereof can be delivered to a cell in a particle or associated with a nanoparticle or particle. A delivery nano particle or particle can comprise a lipid, a lipidoid (see, U.S. Pat. Publ. 20110293703), or a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, a cationic lipid.
Sugar-based particles, for example GalNAc (see WO2014118272 and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961 ) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell. In some aspects, lipid nanoparticles (LNPs) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell. In some aspects, Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (e.g., gold nanoparticles) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell.
A nanoparticle can be any particle having a diameter of less than 1000 nm. In some cases, nanoparticles suitable for use in delivering a nucleic acid molecule, polypeptide, vector, or combination thereof to a target cell can have a diameter of 500 nm or less, e.g., from 25 nm to 35 nm, from 35 nm to 50 nm, from 50 nm to 75 nm, from 75 nm to 100 nm, from 100 nm to 150 nm, from 150 nm to 200 nm, from 200 nm to 300 nm, from 300 nm to 400 nm, or from 400 nm to 500 nm.
Nanoparticles suitable for use in delivery can comprise, e.g., solid nanoparticles (e.g., metal such as silver, gold, iron, titanium, non-metal, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof), metal, dielectric, and semiconductor nanoparticles, hybrid structures (e.g., core-shell nanoparticles). Semi-solid and soft nanoparticles are also suitable for use in delivery e.g., liposomes, exosomes. Liposomes can be made from several different types of lipids, e.g., phospholipids. A stable nucleic-acid-lipid particle (SNALP) can be used for delivery. A SNALP formulation can contain the lipids 3-N-[(methoxypoly(ethylene glycol) 2000) carbamoyl]-1 ,2-dimyristyloxy- propylamine (PEG-C-DMA), 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40: 10:48 molar percent ratio. A SNALP liposome can be prepared by formulating D-Lin-DMA and PEG-C- DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C- DMA. A resulting SNALP liposomes can be about 80-100 nm in size.
A nucleic acid molecule, polypeptide, vector, or combination thereof can be delivered encapsulated in PLGA microspheres (see, e.g., US Pat. Publ. 20130252281 ; US Pat. Publ. 20130245107; US Pat. Publ. 20130244279). Supercharged proteins can also be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo.
Cell Penetrating Peptides (CPPs) can be used to deliver a nucleic acid molecule, polypeptide, vector, or combination thereof. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids.
Methods of Inhibiting
Provided herein are methods of inhibiting a Casi o polypeptide or Casi o polypeptide system in a cell. The method can comprising introducing an AcrIIIAI polypeptide into the cell, wherein the AcrIIIAI polypeptide is heterologous to the cell, and the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1 . In another aspect, the method can comprise introducing an AcrIIIAI polynucleotide into the cell, wherein the AcrIIIAI polynucleotide is heterologous to the cell, and the AcrIIIAI polypeptide has greater than 70, 80, 85, 90, 95, 96, 97, 98, 99% or more sequence identity to the sequence set forth in SEQ ID NO: 1 . The methods can inhibit a Cas10 polypeptide or Cas 10 polypeptide system in the cell. The methods can cause a reduction of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more of the biological activity of the CRISPR-Cas10 system in the cell as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay, or a Cas10-Csm DNA cleavage assay. In an aspect, a cell can naturally express the Casi o polypeptide or Casi o polypeptide system. Alternatively, a cell can be genetically engineered to express a heterologous Casi o polypeptide or Casi o polypeptide system.
In an aspect, a polynucleotide encoding the AcrIIIAI polypeptide is operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter. The cell can be contacted with an agent or condition that induces expression of the AcrIIIAI polypeptide in the cell. Therefore, the expression of the AcrIIIAI polypeptide can be switched on or off. In an aspect a cell can be engineered to express a CRISPR Cas10 system and that system can be switched off by switching on the expression of an AcrIIIAI polypeptide. The cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell. The methods can occur in vivo, ex vivo or in vitro.
In an aspect, methods of reducing activity of a CRISPR-Cas10 system are provided. An AcrIIIAI polypeptide, an AcrIIIAI polynucleotide, or vector capable of expressing an AcrIIIAI polypeptide, as described above, can be delivered to a cell. The delivery of the polypeptide, polynucleotide, or vector to the cell can cause a reduction of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more of the biological activity of the CRISPR-Cas10 system in the cell as measured by a suitable assay, e.g., a Cas10-Csm cOA production assay, or a Cas10-Csm DNA cleavage assay. The cell can naturally express a Casi o polypeptide system. Alternatively, the cell can be genetically engineered to express a heterologous Casi o polypeptide system. The cell can be a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell. The methods can occur in vivo, ex vivo, or in vitro.
An aspect provides methods of improving administration of whole phage therapeutics. Whole phage therapy is the administration of bacteriophage (i.e., phage), which can infect and kill bacteria, to a patient as an anti-bacterial therapy. Intact, whole phage or a mixture of many different phages to target bacteria can be used in phage therapy. Generally, specific phage that are effective against particular strains of infectious bacteria are selected and administered e.g., orally, topically, or intravenously, to a subject. Whole phage therapy can be improved by Introducing a polynucleotide encoding AcrIIIAI into a therapeutic phage genome to improve the phage’s ability to kill bacteria when delivered to a patient during whole phage therapy. By harboring AcrIIIAI , the phage can overcome the bacterial CRISPR defenses and overall do better at clearing out the bacterial infection. The AcrIIIAI can reduce the ability of the infectious bacteria (e.g., Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Clostridium, Salmonella, Enterococcus, and others) to destroy or reduce the effectiveness of the administered phage with their Cas10 systems. In addition, AcrIIIAI on its own is toxic to some bacteria, so even if a CRISPR system is not present in the bacteria, the AcrIIIAI may still provide a fitness advantage to the phage. The efficacy of a whole phage therapy can be improved by about 10, 20, 30, 40, 50, 60, 70, 80, 90% or more as compared to a system that does not include AcrIIIAI as described herein. Another aspect provides methods of treatment of a bacterial infection (e.g., Staphylococcus, Streptococcus, Pseudomonas, Escherichia, Clostridium, Salmonella, Enterococcus, and others) comprising administering a whole phage therapeutic comprising genetically engineering phage making up the whole phage therapeutic to express a recombinant AcrIIIAI polypeptide and administering the whole phage therapeutic to a subject in need thereof. A subject can be a mammal such as a human, a non-human mammal, or a non-human primate.
Pharmaceutical Compositions and Administration
A therapeutic or pharmaceutical composition can include at least one of the polypeptides, polynucleotides, phage, or vectors described herein in a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” refers to at least one component of a pharmaceutical preparation that is normally used for administration of active ingredients. As such, a pharmaceutically acceptable carrier can contain any pharmaceutical excipient used in the art and any form of vehicle for administration. The compositions can be, for example, injectable solutions, aqueous suspensions or solutions, non-aqueous suspensions or solutions, solid and liquid oral formulations, salves, gels, ointments, intradermal patches, creams, lotions, tablets, capsules, sustained release formulations, and the like. Additional excipients can include, for example, colorants, tastemasking agents, solubility aids, suspension agents, compressing agents, enteric coatings, sustained release aids, and the like. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference. The pharmaceutical composition can further comprise an additional active ingredient(s), such as an antibiotic.
The form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate- buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation can also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Pharmaceutical compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97, 1997).
An effective amount of a pharmaceutical composition as described herein will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the methods in use. An effective amount can comprise one or more administrations (1 , 2, 3, 4, 5 or more) of a composition depending on the aspect. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment.
A viral vector, e.g., an AAV viral vector, can be administered to a subject at a dose ranging from about 1013 to about 1016 viral vector particles. Polypeptide compositions can be delivered at about 0.001 , 0.01 , 0.1 , 0.5, 1.0, 5.0 mg/kg, or any other suitable amount. A vector such as a viral vector, a polynucleotide, a polypeptide, or a pharmaceutical composition as described herein can be administered to a subject intravenously, intrathecally, intracistema-magna, intracerebrally, intraventricularly, intranasally, intratracheally, intra- aurally, intra-ocularly, or peri-ocularly, orally, rectally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intracistemally, intranervally, intrapleurally, topically, intralymphatically, intracistemally or intranerve.
As used herein, the terms “treating”, “treat” or “treatment” include administering polypeptides, polynucleotides, vectors, and/or pharmaceutical compositions described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.
In certain aspects, a composition can be administered to an individual for a prophylactic or therapeutic purpose, and thus can be provided as a pharmaceutical preparation by mixing a polynucleotide, polypeptide, phage, or vector described herein with a pharmaceutically acceptable carrier. Examples of prophylactic or therapeutic purpose purposes include but are not necessarily limited to inhibiting the growth of, reducing the amount of and/or killing undesirable and/or pathogenic microorganisms, or to reduce the pathogenicity and/or antibiotic resistance of such microorganisms. For administering to a subject in need thereof, the pharmaceutical composition can be administered using any suitable route and type of formulation, and the dosing of the formulation can be determined by those skilled in the art given the benefit of the present disclosure. In an aspect, the compositions and methods of the invention relate to reducing pathogenic bacteria. In aspects, the compositions and methods are adapted for veterinary purposes.
In another approach, the compositions and methods of this disclosure are adapted for use in eliminating pathogenic bacteria from a non-living surface, such as a the surface of a device, or the surface of an area used for any procedure wherein the reduction of pathogenic bacteria is important, including but not necessarily limited to surfaces used for food preparation or medical purposes.
The compositions and methods are more particularly described below and the Examples set forth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).
All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The aspects illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of' can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claims. Thus, it should be understood that although the present methods and compositions have been specifically disclosed by aspects and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.
Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein can each be specifically excluded from the claims.
Whenever a range is given in the specification, for example, a temperature range, a time range, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements or steps that are included in the description herein can be excluded from the claimed compositions or methods
In addition, where features or aspects of the compositions and methods are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
The following are provided for exemplification purposes only and are not intended to limit the scope of the aspects described in broad terms above.
EXAMPLES
Example 1 EXPERIMENTAL MODEL AND SUBJECT DETAILS
Materials and Methods
Bacterial strains and growth conditions. S. epidermidis RP62a (32), Acrispr (known as LAM 104 in (33)) and Acrispr-cas (known as LM1680 in (34)) were generous gifts from Luciano Marraffini. S. epidermidis strains and S. aureus RN4220 (35) were grown in Brain Heart Infusion (BHI, BD Diagnostics) and Tryptic Soy Broth (TSB, BD Diagnostics), respectively. E. coli DH5a was grown in Luria Bertani (LB) broth (VWR), and E. coli Rosetta 2 (BL21 (DE3)) was grown in Terrific Broth (TB, VWR). These media were supplemented with the following antibiotics: 10 pg/ml chloramphenicol (to select for pC194-based plasmids), 10 pg/ml erythromycin (to select for pTet-based plasmids), 15 pg/ml anhydrotetracycline (to activate the inducible promoter), 15 pg/ml neomycin (to select for S. epidermidis), 30 pg/ml chloramphenicol (to select for E. coli Rosetta2 plasmids), and 50 pg/ml kanamycin (to select for pET28b-10HisSmt3-based plasmids). All bacterial strains were grown at 37 °C, except for protein induction at 17 °C. Liquid cultures were propagated with constant agitation in an orbital shaker set to 180-200 rpm.
Phage propagation and enumeration. S. epidermidis phage PhiIBB (Genbank: NC_041928.1 ) was a generous gift from Luis Melo (36). PhiIBB, Terranova (Genbank: MH542234.1 ), Twillingate (Genbank: MH321491.1 ), and Quidividi (Genbank: MH321490.1 ) (37) were propagated on S. epidermidis RP62a Acrispr-cas. Concentrated phage stocks were prepared by picking 1 -5 purified phage plaques and resuspending them into 500 pl of TSB by vortexing for 30 sec. Suspensions were then subjected to centrifugation at ~15,000 x g for 2 min to pellet agar and cells. The resulting supernatant was passed through a 0.45 pm syringe filter to create a low-titer phage lysate. Low-titer lysates (300 pl) were combined with overnight host culture (diluted 1 :100) in 7 ml of Heart Infusion Agar (HIA, Hardy Diagnostics, prepared at 0.3 x concentration) supplemented with 5 mM CaCL. Phage-host mixtures were poured atop plates containing a solid layer of TSA supplemented with 5 mM CaCL. The top agar layer was allowed to solidify ~10 min at room temperature and then incubated overnight at 37 °C. The following day, the entire top agar layer was harvested with a sterile cell scraper and resuspended in 20 mL fresh TSB. Phages were released into suspension by vortexing for 5 min. Suspensions were then pelleted via centrifugation at 10,000 x g for 10 min, and the supernatant was passed through a 0.45 pm bottle filter to obtain high concentrations of each phage. Phage concentrations were determined using the double-agar overlay method as described in (38). Briefly, overnight cultures of host bacteria were diluted 1 :100 in molten HIA (prepared at 0.5 x concentration) supplemented with 5 mM CaCL, and the agar was poured atop a plate containing solidified TSA supplemented with 5 mM CaCL. The top layer was allowed to solidify at room temperature for 10 min, and ten-fold dilutions (10°-10-7) of concentrated phage lysate were prepared. 5 pl spots of each dilution were dropped atop the agar, spots were allowed to air dry, and plates were incubated overnight at 37 °C. The phage concentration (i.e., titer) in plaque-forming units per ml (pfu/ml) was determined by the following formula: [(number of plaques counted on the most diluted spot) I (dilution factor)]*200. Phage stocks were stored at 4 °C. Phage identities were routinely confirmed/authenticated via PCR amplification and sequencing of genomic regions unique to each phage.
Phage challenge assays. For all phage challenge plate assays, concentrated phage stocks were diluted in 10-fold increments (10°-10-7), and dilutions were spotted atop lawns of cells. Following overnight incubation, plaques were enumerated using the protocol described in the section above titled “phage propagation and enumeration”. For assays using the anhydrotetracycline- (aTc-) inducible system, plates and top agar were supplemented with 15 pg/ml aTc. For the phage challenge assays in liquid culture, overnight cultures were diluted 1 :100 in fresh BHI broth, and phages were added to reach multiplicities of infection (i.e., phage:cell ratios) of 0.1 , 0.5, or 1.0. Phage-host mixtures (200 pl) were then aliquoted into triplicate wells of a 96-well plate. Cells were grown at 37 °C with agitation for 15 hours in a SpectraMax M2e microplate/cuvette reader (Molecular Devices), and OD600 (optical density at 600 nm) measurements were taken every 15 min. For all phage challenge assays, graphs show an average of triplicate measurements (+S.D.) as a representative of at least two independent trials.
Constructing pcrispr-spct/)-, pcrispr-cas-, pcrispr-cas-spcl-, and pTet- plasmids. All plasmids were constructed using either inverse PCR or Gibson Assembly (39) with the primers and templates listed in Table 2. pAH011 (40), pcrispr-cas (41 ), and pcrispr-cas-spcl (42), all of which are derivatives of pC194 (43), were used as the backbones for plasmids designated as pcrispr-spc , pcrispr-cas-csm6H6C, and pcrispr- cas-spcl-, respectively, in this study.
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
pTarget (15), a derivative of pE194 (44), was used as the backbone for all plasmids designated as pTet- in this study. All assembled plasmids were first introduced into S. aureus RN4220 via electroporation (described in the section below), and inserted sequences were confirmed by PCR amplification and Sanger sequencing (performed by Eurofins MWG Operon). At least two transformants were confirmed by sequencing and at least one of each construct was purified using the EZNA Plasmid Mini Kit (Omega Bio-tek). Where indicated, plasmids were introduced into S. epidermidis RP62a Acrispr or Acrispr- cas via electroporation.
Electroporation into Staphylococci. Electrocom petent cells were prepared as described in (45). Briefly, fresh media was used to dilute 10 mL of overnight culture until OD600 of 0.5. Diluted cultures were incubated at 37 °C for 30 min, then placed on ice for 10 min. All subsequent steps were performed on ice. Cells were pelleted at 4000 x g for 10 min, and then washed with an equal volume ice-cold water two times. Cell pellets were washed twice more with 10% glycerol (ice-cold) using 1/20- and 1/25- the volume of culture, respectively. Cells were finally resuspended in 10% glycerol (at 1/200 the volume of initial culture) and stored at -80 °C in 50 pl aliquots. For electroporation, constructs/plasmids were dialyzed against sterile water using the drop dialysis method on a 0.022 m filter for 20 min. At the same time, competent cells were thawed on ice for 5 min, and then left at room temperature for another 5 min. Cells were then pelleted for 1 min at 5,000 x g. Pellets were resuspended in 50 pl of sterilized 10% glycerol supplemented with 500 mM sucrose. Then the entire volume of dialyzed construct/plasmid was added into the cell suspension. Mixtures were transferred into a 2 mm electroporation cuvette (VWR) and pulsed at 29 kV/cm, 100W, and 25 mF with a GenePulser Xcell (BioRad). Cells were then placed in 1 ml of sterile TSB containing 500 mM sucrose and allowed to recover at 37 °C with agitation for at least 1 h (S. aureus) or 2 h (S. epidermidis). Next, 200 pl recovered cells were plated on TSA or BHI agar supplemented with appropriate antibiotics. Plates were incubated at 37 °C overnight and transformants were recovered on the following day.
Phage hybridization and genotyping. Fresh overnight culture of S. epidermidis RP62a Acrispr-cas was diluted 1 :100 in BHI (2 ml) supplemented with 5 mM CaCL. Cells were grown to an OD600 of 0.5, and then infected with two phages: Twillingate at an MOI of 0.05 and PhiIBB at a MOI of 8. The bacteria : phage mixtures were incubated in a 37 °C water bath for 5 minutes (without agitation) to allow phage absorption. Then, infected cells were collected via centrifugation at 17,000 x g for 5 min. The pelleted cells were washed with 1 mL of fresh BHI and centrifuged again at 17,000 x g for 5 min to remove unabsorbed phages. The final pellet was resuspended in 150 mL fresh BHI. 10-fold dilutions (out to 1 O’ 5) of the cell suspension were spotted atop a semisolid layer of 0.5 x HIA agar containing 5 mM CaCL and a 1 :100 dilution of overnight culture of S. epidermidis RP62a Acrispr bearing pcrispr-spc<f>-polA. Spots were air-dried, and plates were incubated overnight at 37 °C. The following day, ten well-isolated plaques, now referred to as putative hybrid phages, were picked from plates and placed into separate tubes containing 0.5 mL fresh BHI. The tubes were vortexed, centrifuged, and filtered as described in “Phage propagation and enumeration” to separate the phages from the agar. Ten-fold dilutions (10°-10’7) of the supernatant were plated atop a semisolid HIA agar layer containing 5 mM CaCL and a 1 :100 dilution of overnight S. epidermidis RP62a Acrispr cells bearing pcrispr-spc<f>-polA. This process was repeated three times to obtain pure phages. These putative hybrids were grown to high titer according to the protocol described in “Phage propagation and enumeration”. Genomic DNA was extracted from the high-titer lysates by combining 300 mL with an equal volume of phenokCHC isoamyl alcohol, vortexing for 15 sec, and subjecting to centrifugation at 10,000 x g for 2 min. The upper aqueous phase was removed into a fresh tube and DNA was ethanol precipitated and resuspended into nuclease-free water. For phage genotyping, forward and reverse primer sets were designed to anneal immediately outside the 10 ‘gap’ regions for both phages Twillingate and PhiIBB (Table 2). PCR products generated from putative recombinants were resolved on a 1 % agarose gel and imaged alongside the 1 Kb Plus DNA ladder (NEB). Since the ten gap regions in phages PhiIBB and Twillingate have different lengths, a determination of which gaps originated from which phage could be made based on PCR product length.
Genomic DNA extraction of phage Twill-BB H8 for whole genome sequencing. Phage genomic DNA was extracted from a high titer lysate as described in (38). Briefly, phage lysate (20 ml) was digested with DNase I and RNase A (10 pg/ml of each) for 30 min at 37 °C. Digested lysate was combined with 10 ml of precipitant solution (30% [wt/vol] polyethylene glycol [PEG] 8000 and 3 M NaCI) and incubated at 4°C overnight to allow phages to precipitate. Precipitated phages were pelleted by centrifugation for 10 min at 10,000 x g and 4 °C. The phage pellet was then resuspended in 250 pl of resuspension buffer (5 mM MgSCM, 10 mM EDTA (pH 8.0)), and incubated with proteinase K (100 pg/ml) at 50 °C for 30 min. The phage suspension was then combined with 500 pl of resin contained in the Promega Wizard DNA cleanup kit. The mixture was inverted several times and applied to the mini-column contained within the kit. The resin was then washed with 2 ml of 80% isopropanol and dried by centrifugation for 2 min at 13,000 x g. The DNA was finally eluted from the resin with 100 pl of distilled water preheated to 80 °C.
Phage DNA sequencing and assembly. Library preparation and sequencing were performed at the SeqCenter (Pittsburgh, PA) using the Illumina Library Prep Tagmentation kit. Sequencing was performed on an Illumina NextSeq 2000. Adapters and indexes were removed by bcl2fastq v. 1.8.4 (Illumina) and FastQC v. 0.11.9 was used to confirm data quality. Sequencing reads were assembled using SPAdes v. 3.15.5 (46) in isolate mode with the kmer values of 21 , 33, 55, 77 and 99. The resulting assembly graph was inspected using Bandage v. 0.8.1 (47) and the highest coverage contig with the proper length representing the phage genome was extracted as a fasta file.
Phage genome sequence alignments with MAUVE. Genome sequences of wildtype and/or the newly-assembled hybrid phage (TwillBB-H8) were first were re-opened such that position 1 in all phages occurs at homologous loci. Re-opened genomes were then saved as fasta files and multiple genome alignments were generated using MAUVE by downloading the software and following the instructions on the website (darlinglab.org/mauve/mauve.html)
Generation of phage escapers (E1-E10) and genotyping. Ten-fold dilutions (10°- 10’7) of phage Twillingate were spotted atop a lawn of S. epidermidis RP62a Acrispr-cas cells bearing pcrispr-spc(/)-gp18. Spots were air-dried, and plates were incubated overnight at 37 °C. The following day, ten well-isolated plaques, now referred to as escaper phages (E1 -E10), were picked and purified as described for phage hybrids in the section above titled “Phage hybridization and genotyping”. Genomic extracts from escapers were subjected to PCR amplification using primers OH016/OH013 which flank the gap 3 region (Table 2). PCR products were resolved on a 1 % agarose gel containing ethidium bromide and visualized under UV transillumination using an Azure 400 imager (Azure Biosystems). For samples that gave visible products (E1 -E4, E6, E9, and E10), PCR products were purified and subjected to Sanger sequencing to delimit the precise regions that were deleted.
Constructing pET28b-10His-Smt3- plasmids. pET28b-10His-Smt3-acr///A7, pET28b-10His-Smt3-ac/7//A7/-/63A,/-/64A, and pET28b-10His-Smt3-cs/7?2 were each constructed using a two-piece Gibson assembly with primers and templates listed in Table 2. The PCR products derived from the plasmid backbone were treated with 1 ,000 Units of Dpnl (NEB) for 1 h at 37 °C, followed by heat inactivation at 80 °C for 20 min. Then, inserts and backbones were purified using the EZNA Cycle Pure Kit (Omega Bio-tek) and Gibson assembled. Assembled products were introduced into E. coli DH5a by chemical transformation (described in the section below). Four transformants were selected and confirmed via PCR and DNA sequencing with T7P/T7T. Confirmed plasmids were extracted using the EZNA Plasmid DNA Mini Kit (Omega Bio-tek) and transformed into E. coli Rosetta 2 (BL21 (DE3)) for protein purification.
Transformation of E. coli. For preparation of chemically-competent E. coli, overnight cultures were diluted in LB (1 : 100) and incubated with agitation at 37 °C until the OD600 reached ~0.5. The culture was placed on ice for 10 min, and subjected to centrifugation at 4000 x g for 5 min. The resulting pellet was resuspended in transformation and storage (TSS) buffer (85% LB medium, 10% (w/v) PEG MW 8000, 5% (v/v) DMSO, 50 mM MgCL) at 1/10 the starting culture volume. Cells were distributed in 50 ml aliquots and stored at -80 °C. For transformation, aliquots were thawed for 10 min on ice and combined with 5 ml Gibson assembled product or 1 ml purified plasmid. The mixture was allowed to cool on ice for 30 min and then heat-shocked at 42 °C for 30 seconds. Cells were immediately placed on ice for 2 min. 1 ml of fresh LB was added into the tube and cells were allowed to recover at 37 °C for 1 h with agitation. Following recovery, 200 ml cells were plated on LB-agar containing appropriate antibiotics and incubated overnight at 37 °C.
Purification ofAcrIIIAI, Csm2, Csm5, PNPase, RNase R, and RNase J2 from pET28b-based plasmids. Recombinant AcrIIIAI , Csm2, Csm5, PNPase, RNase R, and RNase J2 encoded in pET28b-10His-Smt3- plasmids were overexpressed and purified from E. coli Rosetta 2 (BL21 (DE3)) as described previously (27, 29, 30, 48). Briefly, overnight cultures of E. coli Rosetta 2 harboring pET28b-based plasmids were diluted 1 :100 in 1 L TB supplemented with appropriate antibiotics. At an OD600 of 0.5-0.6, cells were induced with 0.3 mM isopropyl-1 -thio-b-d-galactopyranoside (IPTG) and 2% ethanol, followed by 20 min incubation in an ice-water bath. Cells were then allowed to grow at 17 °C with agitation for 16-18 h. Grown cells were harvested and washed with pre-chilled PBS buffer (137 mM NaCI, 2.7 mM KCI, 10 mM Na2HPO4, 1 .8 mM KH2PO4, pH 7.4). Final pellets were stored at -80 °C until purification. All purification steps were conducted at 4 °C. For purification of AcrIIIAI and AcrIIIAI H63A H64A, all buffers were set to a pH of 6.5; for purification of Csm5 and Csm2, all buffers were set to a pH of 7.0; and for purification of PNPase, RNase R, and RNase J2, all buffers were set to a pH of 7.5. For the first step of purification, each 1 L pellet was suspended in 30 ml of Buffer A (50 mM Tris-HCI, 1 .25 M NaCI, 200 mM Li2SO4, 10% sucrose, 25 mM Imidazole) containing one complete EDTA- free protease inhibitor tablet (Roche), 0.1 mg/ml lysozyme, and 0.1 % Triton X-100. After 1 h rotation, lysed cells were sonicated on ice (four 30 sec pulses with 1 min rest in between). Insoluble materials were removed via centrifugation at 10,000 x g and 13,500 x g for 20 min, respectively, followed by filtration with a 0.22 pm PES membrane. Then, 3 ml of Ni- NTA agarose resin slurry (Thermo Fisher Scientific) was equilibrated with Buffer A and mixed with the filtered lysates for 1 h. The resin was pelleted via centrifugation and washed with 40 ml of Buffer A. Resins were then resuspended with 5 ml of Buffer A, transferred to a 5-ml gravity column (G-Biosciences), and further washed with 25 ml of Buffer A. Proteins were collected in 1 ml fractions using a stepwise elution of 3 ml of IMAC buffer (50 mM Tris-HCI, 250 mM NaCI, 10% glycerol) containing 50, 100, 200 and 500 mM imidazole, respectively. For the second step of purification, the 200 mM and 500 mM imidazole aliquots were pooled and mixed with SUMO Protease (Mclab, 1000 U) and the supplied salt-free buffer to remove the 10His-Smt3 tag. Digested samples were injected into a 10K MWCO Pierce™ Slide-A-Lyzer® Dialysis Cassette (Thermo Fisher Scientific) and dialyzed against 2 L IMAC buffer containing 25 mM Imidazole for 3 h. Then, 2 ml of Ni-NTA agarose resin slurry (Thermo Fisher Scientific) was equilibrated with the dialysis buffer and incubated with the dialysate samples for 1 h. The resins were collected in a 5-ml gravity column, and tag-free proteins were collected and, if necessary, concentrated using a 10K MWCO centrifugal filter (PALL) to obtain desired concentrations. Prior to size exclusion chromatography, proteins were stored in IMAC buffer containing 25 mM imidazole. AcrIIIAI was further purified via size exclusion chromatography (SEC) using a Superdex 75 Increase 10/300 GL column on an AKTA pure™ instrument (Cytiva) at 4 °C. One column volume was collected with Buffer B in 1 ml fractions and stored at -80 °C. All fractions were resolved on an 10-15% SDS PAGE and visualized with 0.1 % Coomassie G-250. Protein sizes were estimated with PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific). Gels were imaged using an Azure 400 imager (Azure Biosystems).
Extraction and visualization of AcrIIIAI -associated nucleic acids (acrlllAI- RNAs). The acrlllA1 -RNA extraction protocol was adapted from the protocol used for extracting total RNAs from Cas10-Csm in (30) with minor modifications. Briefly, each size exclusion fraction of AcrIIIAI was resuspended in 1.5 ml of TRIzol Reagent (Invitrogen). After 5 min incubation, 200 pl of chloroform was added to the mixture and vortexed for 15 sec, followed by 10 min incubation at room temperature. Samples were then centrifuged for 15 min at 12,000 x g at 4 °C. The top aqueous phase containing the RNA was transferred to a new tube and mixed with 0.5 ml of isopropanol to precipitate the RNA. Mixtures were incubated at room temperature for 10 min, followed by 10 min centrifugation at 12,000 x g at 4 °C. RNA pellets were washed with 1 mL of 75% ethanol, followed by 5 min centrifugation at 12,000 x g at 4 °C. RNA pellets were air-dried and re-dissolved with 25 pl RNase-free water. The entire extracted acrlllA1 -RNAs were end-labeled with T4 Polynucleotide Kinase in a reaction containing y-[32P]-ATP (PerkinElmer) for 1 h at 37 °C. Then, radiolabeled acrlllA1 -RNAs were subjected to ethanol precipitation to remove excess y-[32P]-ATP. Purified acrlllA1 -RNAs were mixed 1 :1 with 95% formamide loading dye and resolved on 12% Urea PAGE gels. The sizes were estimated with a Decade™ Marker RNA ladder (Thermo Fisher Scientific). Gels were exposed to storage phosphor screens and visualized using Amersham Typhoon biomolecular imager (Cytiva).
Nuclease digestion ofacrlllA1-RNAs. 200 ng of radiolabeled acrl I IA1 -RNAs were treated with 1 unit of DNase I (NEB), 5 pg of RNase A (Thermo Fisher Scientific), or 1 pl of 0.5 M EDTA to serve as control. Reactions were incubated at 37 °C for 30 min, quenched by adding an equal volume of 95% formamide loading dye, and resolved on a 15% Urea PAGE gel. Gels were exposed to storage phosphor screens and visualized using an Amersham Typhoon biomolecular imager (Cytiva). Two independent trials were performed.
RNA-seq analysis of acrlllA1-RNAs. Extracted acrlllA1 -RNAs (100-300 ng) were submitted to the Roy J Carver Biotechnology Center at the University of Illinois at Urbana- Champaign for RNA-seq. The small RNAs were treated with Antarctic Phosphatase and Polynucleotide Kinase, and libraries were constructed with the Next Small RNA Sample Prep kit (NEB) according to the manufacturer’s instructions. The library pool was quantitated by qPCR and sequenced on a MiSeq flowcell for 151 cycles from each end of the fragments using a MiSeq 300-cycle sequencing kit version 2. Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina), and adaptors were trimmed from the 3’-end of the reads. For analysis, RNA-seq reads were first mapped to the entire E. coli Rosetta 2 (BL21 (DE3)) genome (NZ_CP083274.1 ). Subsequent to the identification of coverage peaks, reads were aligned to individual tRNA and tmRNA sequences, which were extracted from the GenBank file and reverse complemented if necessary to match the strand orientation. Coverage profiles were calculated as a function of position using Bowtie2 (v. 2.4.4) and Samtools (v. 1.13) (49). Furthermore, using pysam (https://github.com/pysam-developers/pysam, a python wrapper around HTSlib (50) and the Samtools), the starting and ending positions of every aligned read were binned into respective position histograms and were overlaid atop the coverage profile which was calculated using IGVtools (v. 2.13.2.) (51 ). For AcrIIIAI purified from E. coli, three independent RNA-seq analyses of acrlllA1 -RNAs extracted from three different AcrIIIAI preps were performed. Constructing tRNA read coverage heatmaps. To generate heatmaps, all tRNA sequences were extracted from the GenBank file, reoriented, and combined into a single FASTA file and aligned using the cmalign program (part of the Infernal suite) with the “-g - notrunc” flags. Infernal (v. 1.1.4) (52) is a suite of programs for RNA sequence analysis using profiles of RNA sequence and secondary structure consensus. The covariance model utilized in this analysis was created and used by tRNAviz4 (53) (using cmbuild and cmcalibrate programs, part of the Infernal suite). Using a custom Python bioinformatics library developed for this study, each tRNA raw coverage profile previously generated was first normalized to one, absolute value of its derivative (with respect to position) calculated, and mapped onto its corresponding alignment parsed from the cmalign output file. Only unique tRNA sequences (differing at most by two nucleotides) which had a peak raw coverage above 2000 were used in the analysis. The tRNA type, isotype and loop positions were also independently verified using tRNAscan-SE (54).
Synthesis of tmRNA for nuclease assays. The S. epidermidis tmRNA substrate was synthesized using in vitro transcription. Briefly, the transcription template was amplified from S. epidermidis RP62a using primer pairs L328/L329 (Table 2). The PCR product was purified using the EZNA Cycle Pure Kit (Omega Bio-tek), and 0.5-1.0 pg of purified PCR fragment was used as template. The tmRNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). The reaction was carried out at 37 °C for 2 h. Following tmRNA synthesis, the DNA template was digested with DNasel (NEB), and the synthesized RNA was extracted using Trizol, followed by ethanol precipitation. The S. epidermidis tmRNA pellet was rehydrated in RNase-free water and 25 pg of synthesized RNA was subjected to quick CIP reaction (NEB) to dephosphorylate its ends. The dephosphorylated RNA was extracted again using Trizol, followed by ethanol precipitation. The final S. epidermidis tmRNA pellet was rehydrated in RNase-free water.
AcrIIIAI and RNase R nuclease assays. For nuclease assays with Acrl I IA1 , the in vitro synthesized tmRNA (described above) and a 31 nt linear RNA (Table 3) were used as substrates. For nuclease assays with RNase R, acrlllA1 -RNAs extracted from AcrIIIAI (described above) and a 43 nt linear RNA (ssRNA-01 , Table 3) were used as substrates. Substrates were radiolabeled on their 5’-ends using T4 polynucleotide kinase and y-[32P]- ATP, and purified over a G25 column (IBI Scientific). Substrates were combined with various AcrIIIAI fractions or RNase R (1 pmol) in nuclease buffer (25 mM Tris-HCI pH 7.5, 2 mM DTT, 10 mM MgCl2). The nuclease reactions were carried out at 37 °C for 1 h (Acrl I IA1 ) or indicated time points (RNase R), then quenched with an equal volume of 95% formamide loading buffer. Samples were resolved on an 8% Urea PAGE gel at 55 W for 1.5 h. The RNA product lengths estimated with a Decade™ Marker RNA ladder (Thermo Fisher Scientific). Gels were exposed to storage phosphor screens and visualized using an Amersham Typhoon biomolecular imager (Cytiva).
Cas10-Csm and Csm6 expression and purification. Cas10-CsmCsm2H6N, Cas10- CsrnACsm2 Csm4H6N, Cas10-CsrnCsm2H6N Csm3D32A, and Csm6H6C were overexpressed and purified from S. epidermidis RP62a Acrispr-cas cells bearing pcrispr-cas-csm2H6N, pcrispr- cas-Acsm2-csm4H6N , pcrispr-cas-csm2H6N-csm3D32A, or pcrispr-cas-csm6H6C respectively, as previously described (55). Briefly, overnight cultures were diluted 1 :100 in 1 L BHI supplemented with appropriate antibiotics. Cells were incubated at 37 °C with agitation until an OD600 of 2.0 was achieved. Cells were pelleted and washed with cold water, and final pellets were stored at -80 °C until purification. To purify Cas10-Csm complexes, thawed pellets were resuspended in 10 ml lysis buffer (22 mM MgCl2 and 44 pg/ml lysostaphin) and incubated in a 37 °C water bath for 1 h. Subsequently, lysed cells were combined with 10 ml of 2x resuspension buffer (600mM NaCI, 100 mM NaH2PO4, pH 8.0) supplemented with 20 mM imidazole, 0.1 % Triton X-100, and one tablet of EDTA-free protease inhibitor (Thermo Fisher Scientific). The mixture was homogenized by inverting the tube gently several times and then sonicated on ice (three 30 sec pulses with 1 min rest in between). Insoluble materials were removed via centrifugation at 10,000 x g and 13,500 x g for 20 min, respectively. Lysates were then filtered through a 0.2 pm PES membrane. Filtered lysates were passed through pre equilibrated Ni-NTA columns to capture Cas10-Csm complexes. The Ni-NTA columns were packed in 5 ml gravity columns (G-Biosciences) using 1 ml Ni-NTA agarose resin slurry (Thermo Fisher Scientific) and equilibrated with 10 ml of 1x resuspension buffer (300 mM NaCI, 50 mM NaH2PO4, pH 8.0). Ni-NTA columns with bound Cas10-Csm complexes were washed with 10 ml Wash Buffer 1 (resuspension buffer supplemented with 20 mM imidazole) and then 10 ml Wash Buffer 2 (resuspension buffer supplemented with 20 mM imidazole and 10% glycerol). Cas10-Csm complexes were eluted with five 0.5 ml fractions of elution buffer (resuspension buffer supplemented with 250 mM imidazole and 10% glycerol). Samples from each fraction were resolved in 15% SDS PAGE gels and stained with 0.1 % Coomassie G-250. Protein sizes were estimated with PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific). Gels were imaged using an Azure 400 imager (Azure Biosystems). Protein samples were stored in elution buffer at -80 °C.
Native gel electrophoresis. For testing interactions between Cas10-Csm and AcrIIIAI , Cas10-CsrnCsm2H6N or Cas10-CsrnACsm2 Csm4H6N complexes (30 pmol) were combined with increasing amounts of AcrIIIAI FT (flow-through containing both RNA-free and RNA-bound fractions) (30, 150, 300, and 1500 pmol) and allowed to chill on ice for ~5 min. Cas10-Csm complexes (30 pmol) and AcrIIIAI (1500 pmols) were also loaded individually on the same gel as controls, alongside the Native Mark Protein Standard (Thermo Fisher Scientific). For testing the interactions between cellular nucleases and AcrIIIAI FT, 60 pmols of exonucleases (PNPase, RNase J2 or RNase R) were combined with 1200 pmols AcrIIIAI (or an equivalent volume of protein buffer as a negative control) and allowed to chill on ice for 5 min. In an additional experiment, RNase R (60 pmols) was combined with increasing amounts of Acr (600, 1200, and 2400 pmol) and allowed to chill on ice for 5 min. RNase R (60 pmols) and AcrIIIAI (2400 pmols) were also loaded individually on the same gel as controls, alongside the native protein standard. Proteins were resolved in a 6% native polyacrylamide gel (29:1 acrylamide/bisacrylamide) with 0.75 mm thickness. Tris-Glycine Buffer (25 mM Tris, 250 mM glycine, pH 8.5) was used to cast the native gels and also used as running buffer. Gels were run for 80 min at 100 V in a Mini-PROTEAN Tetra Cell (Bio-Rad) submerged in an ice water bath. For visualization, gels were stained with 0.1 % Coomassie G-250 for 10-20 min, and then submerged in destaining solution (50% methanol and 10% acetic acid) for 1 -2 h. Destained gels were imaged with an Azure 400 imager (Azure Biosystems). To visualize acrl 11 A1 -RNAs in native gels, the Coomassie Blue Destaining Solution was completely removed from the gel by soaking gels in dH2O (with agitation) for at least three hours, while changing out the water every hour. Gels were then soaked in TAE buffer (40 mM Tris Base, 20 mM acetic acid, 1 mM EDTA) containing 5% ethidium bromide with agitation for 30-45 min. Gels were then imaged with an Azure 400 imager (Azure Biosystems) under UV light.
Size exclusion chromatography to test protein-protein interactions. For detecting interactions between AcrIIIAI and Csm2/5, the AcrIIIAI flow-through (containing both RNA-free and RNA-bound forms) was combined with Csm2 (7 nmol) or Csm5 (14 nmol) at 4:1 and/or 8:1 molar ratios in their respective storage buffers. For detecting interactions between AcrIIIAI and RNase R, the AcrIIIAI flow-through containing both RNA-free and RNA-bound forms (30 and 60 nmols) was combined with 3 nmols of RNase R in their respective storage buffers. As controls, the highest molar amount of each protein was passed through the column alone. The mixtures containing Csm2/5 were fractionated on a Superdex S75 Increase 10/300 GL column (Cytiva), while the mixtures containing RNase R were fractionated on a Superdex 200 Increase 10/300 GL column (Cytiva). Columns were pre-equilibrated and run with Buffer B (50 mM Tris-HCI, 250 mM NaCI, 5% glycerol, pH 6.5) using an AKTA pure™ instrument (Cytiva) at 4 °C. As the proteins were passed through the column, one column volume was collected in 1 ml fractions. Fractions were resolved on 15% SDS PAGE gels and visualized with 0.1% Coomassie G-250. Protein sizes were estimated with PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Thermo Fisher Scientific). Gels were imaged using an Azure 400 imager (Azure Biosystems). For each pairwise interaction, at least two independent trials were performed.
Cas10-Csm RNA cleavage assays. The Cas10-Csm RNA cleavage assay was adapted from (24). Briefly, a target RNA that bears complementarity to the crRNA encoded by the first spacer in the S. epidermidis RP62a CRISPR-Cas locus (ssRNA-01 , Table 3), was radiolabeled on its 5’ end with T4 polynucleotide kinase and y-[32P]-ATP and purified over a G25 column (IBI Scientific). Next, Cas10-CsmCsm2H6N complexes (20 pmols) and AcrIIIAI (200 pmols) were pre-incubated alone or combined for 30 min at room temperature.
Table 3. Accompanies Methods. PAGE purified oligonucleotides and synthetic constructs
Figure imgf000058_0001
Figure imgf000059_0001
Indicates position of radiolabel
To start the reaction, tubes were placed at 37 °C and a master mix containing nuclease buffer (25 mM Tris-HCI pH 7.5, 10 mM MgCl2, and 2 mM DTT) and radiolabeled ssRNA-01 were added to each tube. Aliquots (10 pl) were withdrawn from reaction mixes at various time points (0.5, 1 , 2, and 4 min) and quenched by adding an equal volume of 95% formamide loading buffer. Reactions were resolved on a 15% UREA PAGE gel at 55 W for 2 h. Gels were exposed overnight to a storage phosphor screen and visualized using an Amersham Typhoon biomolecular imager. ImageQuant software was used for densitometric analysis. Fractions of substrates cleaved were determined using the following equation: density of uncut substrates for each timepoint were divided by the density of uncut substrate in the lane where no protein was added. Three independent trials were performed, and averages (+S.D.) were calculated.
Cas10-Csm cOA production assays. The cOA production assay was carried out as described in (24) with minor modifications. Briefly, Cas10-CsrnCsm2H6N (20 pmols) and AcrIIIAI (20, 100, or 200 pmols) were combined and pre-incubated at room temperature for 1 h. As controls, Cas10-Csm (20 pmols) and AcrIIIAI (200 pmols) were incubated alone for the same amount of time. cOA production was initiated in these reactions by adding TNG Buffer (50 mM Tris HCI pH 8.0, 150 mM NH4CI, 5% (v/v) glycerol, 500 pM ATP and 10 mM Mg2+), 5 pCi a-[32P]-ATP, and unlabeled ssRNA-01 (Table 3). Reactions were incubated at 37 °C for 10 min and quenched by adding an equal volume of 95% formamide loading buffer. Produced cOAs were resolved on a 15% UREA PAGE gel at 55 W for 1 .25 h alongside A10, an oligonucleotide marker which consists of ten consecutive adenine residues. Gels were exposed overnight to a storage phosphor screen and visualized using an Amersham Typhoon biomolecular imager. ImageQuant software was used for densitometric analysis. Fractions of cOAs produced were determined using the following equation: density of cOAs from each reaction containing AcrIIIAI were divided by the density of cOAs produced in the absence of AcrIIIAI . Three independent trials were performed, and averages (+S.D.) were calculated.
AcrIIIAI cOA degradation assay. Radiolabeled cOAs were first produced by Cas10-Csm complexes according to the protocol described above. cOAs were then extracted from reaction mixtures by adding an equal volume of phenol: CHCh: isoamyl alcohol (25:24:1 ), vortexing for 15 sec, and subjecting to centrifugation at 10,000 x g for 2 min. The upper aqueous phase was removed into a fresh tube and extracted as above with an equal volume of CHCh. The aqueous phase was removed into a fresh tube and purified over a G25 column (IBI Scientific). To test for AcrIIIAI -mediated cleavage of cOAs, purified cOAs were added into a reaction mixture containing nuclease buffer (25 mM Tris-HCI pH 7.5, 10 mM MgCh, and 2 mM DTT) and AcrIIIAI (200 pmols). Reactions were allowed to proceed for 1 h at 37 °C and then quenched with an equal volume of 95% formamide loading buffer. Reactions were resolved on a 15% UREA PAGE gel at 55 W for 1.25 h alongside A10, an oligonucleotide marker which consists of ten consecutive adenine residues. ImageQuant software was used for densitometric analysis. Fractions of cOAs cleaved were determined using the following equation: density of cOAs from each reaction containing AcrIIIAI was divided by the starting density of cOAs added into the reaction. Three independent trials were performed, and averages (+S.D.) were calculated.
Cas10-Csm DNA cleavage assay. For DNA cleavage assays, Cas10- CsrnCsm2H6N Csm3D32A (30 pmols) was pre-incubated with AcrIIIAI (30, 150, or 300 pmols) in nuclease buffer (20 mM Tris pH 8.5, 40 mM KOI, 5 mM MgCh, 1 mM DTT) at 37 °C for 5 min. As controls, Cas10-CsrnCsm2H6N Csm3D32A (30 pmols) and AcrIIIAI (300 pmols) were pre-incubated alone under the same conditions. Next, a target RNA that bears complementarity to the crRNA encoded by the first spacer in the S. epidermidis RP62a CRISPR-Cas locus (ssRNA-01 , Table 3) was added to a final concentration of 200 nM. A reaction mixture containing the Cas10-Csm complex without ssRNA-01 was included as a control. To start reactions, (|)X174 DNA (0.05 mg) was added to each tube. Reaction mixtures were incubated at 37 °C for 4 h and then quenched by the addition of quench buffer (2:2:1 vol/vol/vol Proteinase K:water:0.5 M EDTA, pH 8.0). The samples were then treated with RNase A (20 mg) and incubated at room temperature for 5 min. Samples were resolved on a 1.5% agarose gel at 90 V for 70 min. Gels were stained with Invitrogen™ SYBR™ Gold Nucleic Acid Gel Stain (Fisher Scientific) according to the manufacturer’s instructions and imaged using an Azure 400 imager (Azure Biosystems). Imaged software was used for densitometric analysis. Fractions of substrates cleaved were determined using the following equation: density of uncut substrates for each reaction containing AcrIIIAI divided by the density of uncut substrate in the lane where no target RNA was added. Three independent trials were performed, and averages (+S.D.) were calculated.
AcrIIIAI crRNA cleavage assay. To test for AcrIIIAI -mediated cleavage of complex-associated crRNAs, reactions were set up exactly as above using the maximum amount of AcrIIIAI (300 pmols) in the absence of target RNA and (|)X174 DNA. Reactions were allowed to proceed for 4 h at 37 °C. Next, RNAs contained within each reaction mixture were extracted and visualized using 750 ml TRIzol Reagent (Invitrogen) and following the rest of the protocol described in “Extraction and visualization of AcrIIIAI - associated nucleic acids (acrlllA1 -RNAs)”. Two independent trials were performed.
One-step growth curve for Twillingate WT and E6. The protocol for the one-step growth curves was adapted from (56) with minor modifications. Briefly, 50 ml of mid-log S. epidermidis RP62a Acrispr was combined with phage lysate to achieve an MOI of 0.1. Phages were allowed to adsorb to the host for 5 min at room temperature without agitation. Cells were pelleted by centrifugation at 8,000 x g for 10 min and washed with 50 ml of TSB twice. Following washes, cells were incubated with agitation at 37 °C, and 200 pl samples were taken at 10 min intervals and passed through 0.45 pm syringe filters. Supernatant titers were determined as described in the section above titled “Phage propagation and enumeration” for each time point. The experiment was carried out in triplicate, and averages (+S.D.) were calculated. The latent period (30 min) was defined as the time period post-adsorption to the beginning of the first burst.
Infection assays with Twillingate WT and E6 for genomic DNA sequencing by Nanopore. Overnight cultures of S. epidermidis RP62a Acrispr bearing pcrispr-spc^-EV or pcrispr-spc(/)-polA were diluted (1 :100) in BHI broth supplemented with 5 mM CaCh Cells were incubated in a 37 °C orbital shaker until an OD600 of 1 .0 was reached. Cells were distributed into separate tubes and phages were added to reach an MOI of 1.0. Phages were allowed to adsorb to cells for 5 min at room temperature, and placed back into the incubated shaker for 20 min to allow for a single cycle of infection. Cells were pelleted at 5000 x g for 5 minutes at 4°C, and the pellets were washed with an equal volume of BHI broth, followed by another wash with PBS. Supernatants were discarded, and the pellets were stored at -80 °C. For DNA extraction, cell pellets were resuspended in 200 pL of sterile water and transferred into microtubes. Resuspended pellets were then incubated with lysostaphin (100 pg/mL) and MgCl2 (5 mM) at 37 °C for 2 hours. The Wizard® Genomic DNA Purification Kit (Promega) was used to extract the genomic DNA according to the manufacturer’s instructions. The final DNA pellets were dissolved in 50-60 pL of prewarmed DNase-free water. DNA concentrations were measured using the NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific), and the samples were stored at -20 °C until being sent for sequencing.
Phage DNA cleavage analysis via Nanopore sequencing. Library preparation and Nanopore sequencing were performed at the SeqCenter (Pittsburgh, PA). For library preparation, the PCR-free Ligation Sequencing Kit (Oxford Nanopore Technologies, SQK- NBD1 14.24) was used. Sequence reads within the resultant fastq files were aligned to the corresponding phage genomes using Minimap2 aligner (v. 2.26-r1175) (57). Subsequent alignment files were sorted and indexed into bam format using Samtools program with the respective "sort" and "index" commands. Normalized read-length histograms (i.e., probability distribution function) were then generated for each alignment file utilizing the Pysam Python library with only those reads that were successfully mapped to the reference. Then we proceeded to calculate and plot the complementary cumulative distribution functions (CCDF) on a semi-logarithmic scale. Comparing CCDF curves highlights the distribution of 'rare' events (i.e., very long reads), allowing for easier identification of discrepancies in tail behavior between the data sets.
Large-scale infection with Twillingate WT and E6 for Cas10-Csm purification and RNA-seq analysis. Overnight cultures of S. epidermidis RP62a Acrispr-cas bearing pcrispr-cas-csm2H6N were diluted 1 :100 into 1 L of BHI broth. Flasks were incubated in a 37 °C orbital shaker until an OD600 of 1 .80-2.00 was obtained. Flasks were supplemented with 5 mM CaCL and subjected to infection by Twillingate WT or E6 at an MOI of 1 .0. One flask was left uninfected with an equal volume of BHI added as negative control. Phage adsorption was allowed to proceed at room temperature for 10 min. Subsequently, flasks were transferred to the orbital shaker for an additional 20 minutes to allow for a single round of infection. Cultures were then pelleted, and Cas10-Csm complexes were purified according the protocol above titled “Cas10-Csm expression and purification”. Next, RNAs were extracted from ~500 pmols of Cas10-Csm complex using 750 ml TRIzol Reagent (Invitrogen) as described in the section titled “Extraction and visualization of AcrIIIAI - associated nucleic acids (acrlllA1 -RNAs)”. Seven biological replicates were performed for untreated samples and samples treated with Twillingate WT. Three biological replicates were performed for Twillingate E6. Cas10-Csm associated RNAs were analyzed using DESeq2 according to the section below.
Analysis of Cas10-Csm associated RNAs following phage infection. RNA extracted from Cas10-Csm complexes (~300 ng) were submitted to the Roy J Carver Biotechnology Center at the University of Illinois at Urbana-Champaign for RNA-seq. RNA- seq was performed exactly as described in the section above titled “RNA-seq analysis of acrlllA1 -RNAs”. Raw Illumina reads in fastq format were aligned to S. epidermidis RP62a Acrispr-cas genome using Bowtie2 (v. 2.4.4), producing a Sequence Alignment Map (SAM) file. Following alignment, SAM files were sorted and indexed to generate Binary Alignment Map (BAM) files using Samtools (v. 1.13). Utilizing the -pC and --countReadPairs flags, the alignment outputs, along with an annotation file were given to featureCounts (v. 2.0.3) program which quantified the number of fragments mapped to each genomic feature of the reference genome for every replicate and condition. The -pC flag ensured only read pairs that mapped to the same chromosome and strand were counted, while the -- countReadPairs flag treated each fragment pair as a single unit of evidence. The raw counts derived from featureCounts were compiled into a singular CSV file. This, in conjunction with another CSV file outlining the experimental design and conditions, served as the input for the R script that runs the DESeq2 (v. 1 .34.0) analysis pipeline in order to perform a differential gene expression analysis. The resulting DESeq2 outputs were then employed to create Volcano plots, which showcase the significantly differentially expressed genes between conditions.
Codon usage analysis ofS. epidermidis RP62a and Twillingate. Raw frequency data for codons and corresponding amino acids in S. epidermidis RP62a and phage Twillingate were generated using the online application at bioinformatics.org/sms2/codon_usage.html. The frequency data for synonymous codons — those that code for the same amino acid — were first aggregated, and then absolute counts and the relative percentages of each codon within its respective amino acid group were calculated and exported into a table. Quantification and statistical analysis. Student’s Wests were performed to determine if observed drops in plaque counts (Fig. 2) or enzymatic activities (Fig. 5) were statistically significant. Tests were performed using Microsoft Excel, and a difference was deemed significant if the p-value was below 0.05. Details for numbers of replicates etc. for specific experiments can be found in the corresponding figure legends and methods.
Example 2: A subset of staphylococcal phages exhibit Type lll-A anti-CRISPR activity
With a goal of identifying new Type III Acrs, we screened a diverse collection of phages for the ability to evade the Type lll-A CRISPR-Cas system in the clinical isolate S. epidermidis RP62a (32). This system employs the Cas10-Csm effector complex (composed of Cas10, Csm2/Cas11 , Csm3, Csm4, Csm5, and a crRNA) (41 ), an accessory nuclease that responds to cOA signaling (Csm6) (31 , 58), and three non-Cas cellular nucleases (PNPase, RNase J, and RNase R) (29, 30) to mount a successful defense (Figure 1 A). To screen for anti-CRISPR activity, we used S. epidermidis RP62a Acrispr, in which the native repeat-spacer array has been deleted, but harbors pcrispr-spc , a plasmid with a single repeat downstream of which spacer(s) targeting phage(s) of interest can be inserted (33, 40). This system typically exhibits robust immunity against diverse phages with a single targeting spacer (42). However, while screening through a group of related phages with myovirus morphology from the family Herelleviridae (PhiIBB, Terranova, Quidividi, and Twillingate), we noticed that some have the ability to actively escape immunity. To demonstrate this, spacers complementary to three conserved genes within these phages [hyp (hypothetical), holin, and polA] were designed in both forward and reverse directions (Figure 1 B). Two of the targeted genes (polA and hyp) have consensus s70 promoters that are known to drive gene expression early during phage infection, while the third gene (holin) has a promoter with an extended -10 motif and lacks a -35 element, which are hallmarks of genes expressed late in the life cycle of these phages (59). As expected, strains bearing pcrispr-spc encoding a non-targeting spacer (EV) or spacers producing crRNAs that are identical to the target sequence (polA-RC, hyp-RC, and holin- RC) are completely susceptible to phage infection, as evidenced by the appearance of billions of plaques (zones of bacterial death) when dilutions of phage are plated on lawns of each strain (Figure 1 C). In contrast, strains expressing crRNAs complementary to the transcript of each targeted gene were completely protected against PhiIBB and Terranova, as evidenced by the absence of plaques. However, these same targeting strains remained sensitive to Quidividi and Twillingate, suggesting that these phages have the capacity to resist immunity. To rule out the possibility that escape from CRISPR targeting occurred due to the passive acquisition of mutation(s) in targeted loci in the latter phages, we purified surviving phages and sequenced the targeted regions to confirm that the three loci indeed maintained the wild-type (WT) genotype. Further, to test the limits of this anti-CRISPR activity, we performed phage challenge experiments in liquid culture with representative sensitive and resistant phages PhiIBB and Twillingate, respectively, at different phage: host ratios (multiplicities of infection, or MOIs) (Figure 1 D). The bacterial growth curves revealed that even when Twillingate is outnumbered by bacteria 10:1 (MOI=0.1 ), the phage remains completely resistant to CRISPR activity, irrespective of whether the target is located in early- or late- expressed genes. Altogether, these data demonstrate that a subset of S. epidermidis phages employ a robust proactive strategy to overcome Type lll-A CRISPR- Cas immunity.
Example 3 Twillingate gp17 {acrIIIAI) is necessary and sufficient for anti-CRISPR activity
We next sought to identify the gene(s) responsible for anti-CRISPR activity. We started by taking advantage of the fact that the CRISPR -sensitive and -resistant phages share a high degree of sequence similarity. For instance, PhiIBB and Twillingate share ~88% identity at the nucleotide level in a pairwise alignment; however, there are ten ‘gaps’ in which the resistant phage Twillingate possesses genes that PhiIBB lacks (Figure 9A). Thus, we reasoned that gene(s) conferring anti-CRISPR activity must reside within these gaps. To begin to narrow down which gap region confers the CRISPR resistance phenotype, a two-step hybridization strategy was employed (Figure 9B). First, PhiIBB and Twillingate were combined and allowed to co-infect and recombine within S. epidermidis RP62a Acrispr-cas, which bears a large genomic deletion encompassing the CRISPR-Cas locus (34). The resulting phage lysate enriched with recombinants was then passaged through S. epidermidis RP62a Acrispr bearing pcrispr-spc^-polA in order to recover phage hybrids that acquired anti-CRISPR activity. Eight hybrid phages were purified, and their DNA was extracted and subjected to PCR amplification across the 10 gap regions to determine from which phage (PhiIBB or Twillingate) each gap region was derived. This analysis revealed that all eight CRISPR-resistant hybrids shared a short stretch of Twillingate-derived DNA (<10,000 nucleotides) encompassing gaps 1 -3 (Figure 9C). We further sequenced the whole genome of ‘Twill-BB H8’, the hybrid phage that acquired the shortest genomic segment from Twillingate, and confirmed that its sequence was identical to PhiIBB across the majority of its genome, with the exception of the short region encompassing gaps 1 -3 (Figure 9D). These observations narrowed our search for the acr to the six candidate genes encoding hypothetical/uncharacterized proteins in gaps 1 -3 — Twillingate gp11, gp14, gp16, gp17, gp18, and gp19 (Figure 9E).
We next took several approaches to further narrow the gene(s) required for Acr activity. In silico analyses were first performed using the webtools HHPred (60) and I- TASSER (61 ) to identify potential structural homologs; however, this analysis failed to produce high-confidence hits across the entire proteins. We next attempted to introduce each of the six candidate genes into separate plasmids; however, multiple attempts failed to produce viable transformants, suggesting the gene products are toxic to the host. We also performed additional hybridization experiments using Twill-BB H8 and PhiIBB to further narrow the list, but we were unable to isolate hybrids with shorter Twillingate- derived segments. Finally, we reasoned that since the gene encoding the Acr must necessarily be transcribed and translated before it can perform its function, then CRISPR targeting of the acr itself (or genes nearby within the same transcript) might allow CRISPR to exhibit some activity and thus reveal the achs location. To test this, we began by designing spacers targeting the candidates within gap 3 — gp16-gp19 (Figure 2A). Spacers were introduced into pcrispr-spct/), and S. epidermidis RP62a Acrispr strains harboring the plasmids were challenged with Twillingate (Figure 2B). The results showed that Twillingate produces 10-1000 - fold fewer plaques when plated atop strains targeting acr candidates versus strains that target the conserved genetic loci (polA, hyp, and holin). In addition, many plaques on the former strains appeared slightly larger than those produced when targeting the conserved genetic loci and were comparable in size to plaques on the EV control plate, suggesting that these surviving ‘escaper’ phages might have subverted immunity by losing the targeted locus altogether. To test this, we purified ten such escapers, extracted their DNA, and PCR-amplified across the gap 3 region. PCR products were obtained for only seven escapers, and the products were all shorter than expected (Figure 2C). Accordingly, sequencing of the PCR products revealed that these phages harbor large deletions within/beyond gap 3, resulting in complete loss of the targeted locus, and possibly the acr itself (Figure 2D). To test the latter possibility, we plated the escaper with the smallest deletion (Escaper 6 (E6), Dgp17-gp19) atop the same set of targeting strains and found that the phage had indeed completely lost anti-CRISPR activity (Figure 2E). This analysis allowed us to further narrow our search for the acr to three of the candidates — gp17, gp18, and gp19.
Finally, to pinpoint which of these gene(s) confer Acr activity, we developed an inducible system in S. aureus (Figure 2F). In this system, S. aureus RN4220 lacks a chromosomal CRISPR-Cas system, but harbors a multi-copy plasmid (pcrispr-cas-spcl) encoding the Type lll-A CRISPR-Cas system of S. epidermidis RP62a with a single spacer targeting polA in phage ISP (Bari et al, 2017). In addition, a second plasmid was introduced into this strain (pTet-gpx), which has an anhydrotetracycline- (aTc-) inducible promoter downstream of which candidate gene(s) were inserted. To test for Acr activity, ISP dilutions were plated atop these strains in the presence and absence of aTc. As expected, strains bearing the EV versions of each plasmid permitted the phage to form millions of plaques, and the CRISPR system on its own completely eliminated plaque production regardless of aTc addition (Figure 2G). Additionally, in the presence of gp16, gp18, or gp19, the CRISPR system maintained its ability to block ISP plaque formation. However, when the gp17-gp18 operon, or gp17 alone were introduced into pTet-gpx, ISP formed millions of plaques under inducing conditions despite the presence of the targeting CRISPR system. Altogether, these data demonstrate that Twillingate gp17 is necessary and sufficient in this genetic context to subvert Type lll-A CRISPR-Cas immunity. Here onward, we refer to its protein product as AcrIIIAI (the first Type lll-A specific anti-CRISPR), according to the recommended naming convention for newly-discovered Acrs (6).
Example 4 AcrIIIAI co-purifies with fragmented t(m)RNAs
We next sought to elucidate the biochemical features of AcrIIIAI . To do so, acrIIIA I was introduced into the pET28b vector, overexpressed in E. coli Rosetta 2 (BL21 (DE3)), and purified using a three-step process consisting of tandem Ni2+-affinity purifications followed by size exclusion chromatography (Figure 3A). AcrIIIAI has a theoretical molecular weight of ~10.2 kDa (Figure 10A); however, it was found to elute off of the sizing column earlier than expected in fractions 10-12 (Figure 3B). Additionally, we observed that the A254 peak was more prominent than the A280 peak in these fractions, suggesting that the protein likely co-purifies with nucleic acids. To investigate further, we extracted nucleic acids from each fraction, labelled their 5’-ends with 32P, and resolved them on a denaturing gel. This analysis revealed that fractions 10-12 are indeed enriched with nucleic acids ranging from ~30-80 nucleotides (nt), while fractions 13 and 14, which contain some Acrl 11 A1 , are devoid of nucleic acids (Figure 3C). Based on the elution profile of the nucleic acid-free fractions, we speculate that Acrl I IA1 may form a dimer on its own.
We then further explored the nature of the co-purifying nucleic acids. The nucleic acids were first subjected to nuclease digestion and found to be completely sensitive to RNase A (Figure 3D). These data indicate that AcrIIIAI associates with small RNAs. To determine their identity, purified RNAs were sequenced using an Illumina MiSeq system, and the reads were mapped to the E. coli genome. The results showed prominent coverage peaks corresponding to diverse tRNAs as well as the tmRNA (Figure 3E). Importantly, three independent trials from different AcrIIIAI preps showed similar results. Since intact tRNAs are typically ~70-100 nt, while most AcrIIIAI -associated RNAs are about half those lengths (Figure 3D), we speculated that the latter are likely fragmented. To investigate further, we examined read coverage profiles across individual tRNAs. Since the Illumina reads (150 bp) are longer than intact tRNAs, cleavage sites can be inferred from the pileup of shorter reads terminating at similar internal positions. This analysis revealed abrupt changes in coverage in discrete regions corresponding to single-stranded loops, especially the anti-codon loops (Figure 3 F and G). A similar analysis of the tmRNA showed a steep drop in coverage in an analogous region (the single-stranded mRNA-like region) (Figure 3H). Taken together, these data demonstrate that AcrIIIAI associates with t(m)RNA fragments, here onward referred to as acrl I IA1 -RNAs.
The close association of AcrIIIAI with fragmented t(m)RNAs led us to speculate that AcrIIIAI itself may facilitate/catalyze their cleavage. To test this, AcrIIIAI was incubated together with a radiolabeled t(m)RNA substrate, and degradation was observed in the presence of divalent metals (Figure B). We noted that the tmRNA cleavage products generated in this assay range between ~70-100 nts, while the major tmRNA fragments that co-purify with AcrIIIAI are much more defined at ~90 nts (inferred by the read coverage profile, Figure 3H). This length discrepancy may be due to the sequence differences between S. epidermidis tmRNA (which was used in the nuclease assay) and E. coli tmRNA (which co-purifies with AcrIIIAI ), and/or additional tmRNA trimming by cellular nucleases in E. coli that might occur during the protein purification process. Nonetheless, the generation of fragments of the approximate expected lengths in this in vitro assay inspired us to investigate further. Thus, we searched for candidate active site residues in Acrl I IA1 that might be responsible for metal coordination, specifically aspartate, glutamate, and histidine residues, which are commonly found in nuclease active sites (62- 64). Due to the abundance of aspartate and glutamate residues in this small protein (nine of each, Figure 10A), we settled upon mutating its only two histidine residues to create Acrl I IA1 H63A’ H64A. We overexpressed and purified this mutant, and observed that it remains stable during purification, with an elution profile comparable to that of wild-type AcrIIIAI (Figure 10C). Subsequent nuclease assays revealed that the RNA-free fractions of AcrIIIAI (13 and 14) harbor nuclease activity, while the RNA-bound fractions (10-12) are devoid of this activity. This would be the expected result if cleaved RNAs are indeed the products of AcrlllATs activity. Moreover, the activity of AcrIIIAI H63A’ H64A is diminished when compared to that of wild-type (Figure 10D), lending further support to the notion that AcrIIIAI is the cause of tmRNA cleavage. Notably, a similar assay using a linear RNA substrate showed more modest degradation (Figure 10E), suggesting a preference for structured RNAs. We also tested for AcrIIIAI H63A’ H64A function in our inducible system and observed diminished anti-CRISPR activity (Figure 10F). Altogether, these data support the hypothesis that AcrIIIAI catalyzes t(m)RNA cleavage, and suggest that this activity is important for robust anti-CRISPR function.
Example 5: AcrIIIAI is unlikely to function by impacting translation dynamics
Our observation that AcrIIIAI binds/cleaves t(m)RNAs led us to speculate that AcrIIIAI might function by modulating translation dynamics. We first considered the possibility that t(m)RNA cleavage may be a way for the phage to leverage codon usage bias to cripple translation of cellular machinery while enhancing translation of phage- encoded proteins. Although Twillingate does not carry its own t(m)RNAs (37), subtle differences in codon usage in the phage might facilitate such a mechanism. To test this, we examined the codon usage statistics of S. epidermidis RP62a and Twillingate, and found that all codons are used by both entities with similar frequencies. This observation argues against the notion that codon usage bias comes into play in AcrlllATs function.
In addition, since the Type III CRISPR-Cas system must compete with ribosomes for access to targeted transcripts, we also considered the possibility that tRNA fragmentation may be a way to slow translation and induce ribosomal queuing, which in turn might prevent Cas10-Csm from accessing targeted RNAs. To test this compelling hypothesis, we took advantage of the fact that Twillingate harbors multiple introns, particularly within the gene that encodes the large terminase subunit (terL) (37). We reasoned that if ribosomal queueing is necessary for Acrll IA1 activity, then AcrIIIAI should only function when the CRISPR target is located in exons, but not within untranslated regions of introns. Accordingly, we tested CRISPR activity in strains that target terL in both intron and exon regions, and found that Twillingate maintains CRISPR resistance irrespective of target location. This observation argues against the notion that AcrIIIAI activity relies upon protection of targeted transcripts by slowed translation machinery.
Example 6: AcrIIIAI binds to Cas10-Csm via interactions with Csm2
We next investigated the direct impacts of AcrIIIAI on CRISPR activity. Since the majority of known Acrs bind directly to Cas effector complexes (5), we tested for binding to Cas10-Csm using different protein-protein interaction assays. First, the complex was overexpressed and purified from S. epidermidis RP62a Acrispr-cas bearing pcrispr-cas- csm2H6N, a multi-copy plasmid that encodes the native CRISPR locus of S. epidermidis RP62a plus a 6-His tag on the N-terminus of Csm2 (41 ). Complexes were resolved by denaturing SDS-PAGE to assess purity, and then by native PAGE to assess integrity. As expected, all five subunits are represented in the complex (Figure 4A), and it exhibits an approximate molecular weight of 270-312 kDa (Figure 4B), which agrees with predicted stoichiometries — Cas10i:Csm22/3:Csm33/4:Csm4i:Csm5i (65). However, when increasing amounts of AcrIIIAI were pre-incubated with the complex and then resolved on a native gel, a prominent shifted band with slower migration appeared, suggesting that AcrIIIAI binds to the complex. Intriguingly, the band corresponding to unbound complex failed to completely shift upward, even when AcrIIIAI was added in excess (up to 50:1 molar ratio). In light of this observation, and the fact that Cas10-Csm exists in vitro as a heterogenous mixture of subcomplexes, some of which may be devoid of Csm2 and/or Csm5 (65, 66), we speculated that AcrIIIAI may specifically bind to Csm2 or Csm5.
To test this, we overexpressed and purified Csm2 and Csm5, and passed them through a size exclusion column individually or combined with a mixture of RNA-bound and RNA-free AcrIIIAI (i.e. the FT following the second purification step, Fig. 3A). Although AcrIIIAI and Csm5 failed to show binding (Figure 11 A and B), we observed a stable interaction between AcrIIIAI and Csm2 (Figure 4 C and D). This is evidenced by the pronounced leftward shift in the elution profile when protein mixtures are passed through the column as compared to when they are fractionated on their own. Notably, both RNA- free and RNA-bound versions of AcrIIIAI completely shift into the Csm2-bound fractions when combined, suggesting that binding is mediated by protein-protein interactions, rather than by RNA-bridging interactions. Interestingly, while Acrl I IA1 is predicted to form a dimer on its own (according to the size exclusion chromatogram, Fig. 3B), the predicted binding stoichiometry to Csm2 is 8:1 (AcrIIIAI :Csm2). The reason for this discrepancy is unclear, although it is conceivable that Csm2 might alter the stoichiometry of AcrIIIAI .
The data thus far excludes Csm5 as a binding partner for AcrIIIAI , but does not rule out other members of the complex. Thus, to explore the possibility that additional subunits within the complex may interact with AcrIIIAI , we purified Cas10-CsrnACsm2 Csm4H6N from S. epidermidis RP62a Acrispr-cas bearing pcrispr-cas-Acsm2-csm4H6N, which harbors an inframe deletion of csm2 and encodes a 6-His tag on the N-terminus of Csm4. Complexes were resolved by denaturing SDS-PAGE (Figure 4E), and then resolved by native PAGE in the absence/presence of AcrIIIAI (Figure 4F). The results showed that AcrIIIAI is unable to produce a shifted band when combined with Cas10-CsrnACsm2 Csm4H6N. Altogether, these data indicate that AcrIIIAI binds to Csm2 alone within the Cas10-Csm complex.
Example 7. AcrIIIAI attenuates cOA production and DNA cleavage by Cas10-Csm
After having established a direct interaction between AcrIIIAI and Cas10-Csm, we wondered if and how AcrIIIAI impacts the complex’s functions. Although Csm2 has no known catalytic activity, we reasoned that AcrIIIAI might allosterically inhibit one or more activities of other members in the complex — target RNA binding/cleavage by the Csm3 active site, nonspecific DNA cleavage by the Casi o HD domain, and cOA production by Casl O’s palm domain. Since the Casi o HD domain is dispensable for anti-plasmid immunity under certain conditions (70), we first sought to determine which of these activities are essential for anti-phage immunity in our inducible system (Figure 2F). To test this, pcrispr-cas-spcl variants were constructed that encode mutations in the Csm3 active site, Casi o HD, and Casi o palm domains (CS/T?3D32A, cas 70H14A D15A, and cas 70G584A G585A D586A D587A, respectively). These plasmids were introduced into S. aureus RN4220 cells that harbor pTet-EV or pTet-gp17 (-acrIIIAI), and transformants were challenged with ISP in the presence/absence of aTc to assess CRISPR activity (Figure 13). Consistent with previous observations (24), we found that the Csm3D32A mutant maintains robust immunity, thus making Csm3’s activity an unlikely target for AcrIIIAI . In contrast, CRISPR immunity was lost in the presence of Cas10H14A D15A or Cas10G584A G585A D586A D587A, supporting the hypothesis that Casi o may be the primary target of AcrIIIAI mediated inhibition.
To test this hypothesis, we sought to determine the impact of AcrIIIAI on the catalytic activities of Cas10-Csm using a series of in vitro biochemical assays. In these assays, both RNA-bound and RNA-free fractions of AcrIIIAI were tested to explore possible function(s) for acrlllA1 -RNAs. We first assayed for target RNA cleavage by Cas10-Csm. In this assay, purified complexes were combined with a radiolabeled target RNA, reactions were incubated for increasing amounts of time, and RNAs were resolved on a denaturing gel. Consistent with previous observations (15), complexes alone cleaved the RNA target at 6-nt increments, one cut for each copy of Csm3 within the complex (Figure 14A and C). In contrast, AcrIIIAI caused little/no cleavage of the RNA target, regardless of the presence/absence of acrlllA1 -RNAs. Importantly, when AcrIIIAI was preincubated with complexes at a 10:1 ratio, Cas10-Csm maintained full ability to cleave targeted RNA (Figure 14 B and D). These data demonstrate that AcrIIIAI has no effect on target RNA binding/cleavage by Cas10-Csm.
We next tested for Cas10-mediated cOA production. For this assay, Cas10- CsmCsm2H6N was combined with unlabeled target RNAs in the presence of a-32P-ATP, which is used by Casio as a substrate for cOA production, resulting in radiolabeled cOAs (Figure 5A). Interestingly, when complexes were pre-incubated with increasing amounts of AcrIIIAI , the cOA signal was diminished by up to 40%, and both RNA-free and RNA-bound versions of AcrIIIAI had a similar effect (Figure 5 A and B). To determine whether AcrIIIAI causes cOA degradation in a manner similar to Acrlll-1 (8), we purified radiolabeled cOAs and incubated them together in the same reaction buffer with both versions of AcrIIIAI for 1 hour. The results showed little/no decrease in the cOA signal (Figure 15 A and B). Taken together, these data demonstrate that rather than cleaving cOAs, AcrIIIAI dampens cOA production by Cas10-Csm.
We finally tested for Cas10-mediated DNase activity. For this assay, complexes were incubated for 4h with unlabeled target RNA in the presence of single-stranded circular DNA from fX174 as substrate. Additionally, we used Cas10-CsrnCsm2H6N Csm3D32A, which is unable to cleave the targeted RNA and thus exhibits constitutive DNase activity that can be visualized on an agarose gel (Figure 5C). As expected, in the absence of target RNA, little/no cleavage of the DNA substrate was observed, while in the presence of the target, complexes caused nearly complete DNA degradation. Interestingly, AcrIIIAI significantly attenuated DNase activity by up to 40% (Figure 5D). Again, both RNA-free and RNA-bound fractions of AcrIIIAI showed a similar effect. Since AcrIIIAI works against a constitutively active complex (Figures 5 C, D and 13), it is unlikely that AcrIIIAI functions by reducing the target RNA turnover rate in a manner similar to AcrlllB2 (9, 10). However, since AcrIIIAI likely harbors nuclease activity (Fig. 10), we envisioned that one mechanism by which it might render Cas10-Csm nonfunctional is by cleaving its crRNAs. Thus, to test this possibility, we assayed crRNA integrity after incubating complexes with 10-fold excess of AcrIIIAI under the same reaction conditions used for the DNase activity assays (Figure 15C). The results showed that neither RNA-bound nor RNA-free fractions of AcrIIIAI have the capacity to cleave complex-associated crRNAs. Taken together, these data demonstrate that AcrIIIAI attenuates DNA cleavage by Cas10-Csm through allosteric inhibition.
The results thus far show that AcrIIIAI dampens both of Casl O’s catalytic activities in a purified system. However, in light of the incomplete inhibition observed in our in vitro assays, we wondered whether AcrIIIAI provides more robust inhibition in vivo, where presumably, complexes are more likely to be intact, and additional cellular factors that might be necessary for full Acr function (e.g. ions, ligands, and/or proteins) are present. To test this, we designed an experiment to track DNase activity as a proxy for Casi o function in cells during phage infection (Figure 5E). In this experiment, DNA abundance and integrity were assayed following infection with Twillingate WT versus E6, which harbors a deletion encompassing acrIIIAI (Figure 2 D and E). Both phages were allowed to infect S. epidermidis RP62a Acrispr bearing pcrispr-spc^-polA or the EV control for 20 min, which is ~10 min shorter than the latent period of these phages (determined by one-step growth curve). This ensures a single round of infection. Following infection, cells were washed to remove free phage, DNA was extracted from cells, and DNA extracts were subjected to long-read sequencing by Nanopore. Since the Nanopore platform directly sequences DNA samples without prior fragmentation or amplification (71 ), the abundance and lengths of sequencing reads should reflect the abundance and lengths of DNA fragments in the original sample. We first analyzed this dataset for CRISPR function by mapping reads to the corresponding phage genomes and examining the depths of coverage. As expected, similar coverage was observed across both phages following infection of non-targeting (EV) strains, and significantly reduced depth of coverage was observed across Twillingate E6 following infection of the targeting strain as the phage succumbs to CRISPR immunity (Figure 5F). In contrast, the level of coverage across Twillingate WT following infection of the CRISPR targeting strain was identical to that observed with the non-targeting strains, consistent with the robust anti-CRISPR activity in this phage. We next examined the length distributions of reads mapping to both phage and bacterial genomes in this dataset (Figure 5 G and H). Consistent with direct DNA cleavage by the CRISPR system, Twillingate E6 showed a progressively diminishing representation of longer reads in its distribution, as evidenced by the complementary cumulative distribution functions (1 -CDF), when grown on the po/A targeting strain versus the EV non-targeting strain (Figure 5G). In contrast, the read length distributions of Twillingate WT on both strains appeared comparable. These results corroborate the reduction of Casi o DNase activity observed in biochemical assays and suggest that Casio repression by Acrl I IA1 may be more pronounced /n v/vo.
Example 8 Cas10-Csm co-purifies with acrlllA1-RNAs following Twillingate infection
Since acrlllA1 -RNAs have little/no impact on Cas10-Csm function in a purified system, we wondered whether they may play other roles /n v/vo. To explore this idea, we first sought to confirm that they co-purify with Cas10-Csm in the native S. epidermidis background. As mentioned earlier, multiple attempts to overexpress AcrIIIAI in S. epidermidis failed, thus preventing us from purifying it directly from the native host. However, now knowing that AcrIIIAI binds to Csm2 within the Cas10-Csm complex, we designed an experiment to capture acrlllA1 -RNAs through purification of the complex (Figure 6A). In this experiment, S. epidermidis RP62a Acrispr-cas cells harboring pcrispr- cas-csm2H6N were infected with Twillingate WT or E6 for 20 min, Cas10-CsmCsm2H6N complexes were extracted from infected cells, and RNAs were further extracted from complexes. Complex-associated RNAs were then subjected to Illumina sequencing and the reads were mapped to S. epidermidis RP62a Acrispr-cas. The results showed that t(m)RNAs are indeed significantly enriched in complexes purified from cells infected with Twillingate WT, but not E6 (Figure 6B and C). We also investigated the integrity of the tRNAs by examining their depth of coverage profiles (Figure 6D). This analysis revealed that complex-associated acrl 11 A1 -RNAs purified from S. epidermidis exhibit more extensive fragmentation than acrlllA1 -RNAs purified from E. coli in the absence of the complex (compare heatmaps in Figures 6D and 3G). This difference may be explained by the activities of nucleases specific to each host. Notably, since this experiment was performed under non-targeting conditions (i.e. pcrispr-cas-csm2H6N does not harbor a Twillingatetargeting spacer), the observed degradation is unlikely to have been caused by Csm6, which is activated by cOAs released by Casi o upon phage transcript recognition (26).
Example 9 AcrIIIAI -RNAs bind stably to RNase R in a purified system
Having established that acrlllA1 -RNAs indeed co-localize with Cas10-Csm during phage infection, we wondered whether their intended targets might be one or more of the non-Cas cellular nucleases involved in immunity (27, 29, 30). To explore this idea, we tested for direct interactions between AcrIIIAI (a mixture of RNA-bound and RNA-free versions) and all of the accessory nucleases known to promote immunity in this Type lll-A system — Csm6, PNPase, RNase R, and RNase J (Figure 7). Each of these nucleases were purified, combined with AcrIIIAI , and the mixtures were resolved by native gel electrophoresis. The results showed that while the migration of Csm6, PNPase, and RNase J remain unchanged in the presence of AcrIIIAI , a pronounced shift in migration of RNase R occurs when combined with AcrIIIAI (Figure 7 A-C). However, we noticed that the protein band corresponding to unbound AcrIIIAI in these gels persists in the presence of RNase R, leading us to speculate that rather than interacting with AcrIIIAI directly, RNase R may be interacting with acrlllA1 -RNAs, which are refractory to Coomassie staining. To test this, the native gels were soaked in ethidium bromide and imaged under UV light to visualize acrlllA1 -RNAs (Figure 7 D-F). The results revealed that RNase R indeed forms a stable complex with acrlllA1 -RNAs. To confirm this interaction, we passed RNase R and AcrIIIAI through a size exclusion column alone and combined in different ratios, and found that while the bulk of AcrIIIAI retains similar elution profiles whether in the presence or absence of RNase R, the A254 peak corresponding to acrlllA1 -RNAs co-localizes with RNase R (Figure 7 G and H). These data suggest that RNase R may be the intended target for acrlllA1 -RNAs.
One possibility is that the interaction between RNase R and acrlllA1 -RNAs might antagonize RNase R’s function, while another is that the interaction might facilitate AcrlllATs activity. As an example of the latter, our previous work showed that RNase R is recruited to Cas10-Csm through interactions with Csm5 (30), thus we wondered whether the interaction between RNase R and acrlllA1 -RNAs might facilitate AcrIIIAI recruitment to the complex and promote its function. To test this, we assayed for Twillingate’s ability to overcome CRISPR targeting in S. epidermidis cells harboring an in-frame deletion of rnr, the gene that encodes RNase R (30). The controls showed the expected results — while WT Twillingate maintains the ability to form plaques on WT S. epidermidis RP62a cells bearing plasmids with spacers that target early or late phage genes (pcrispr-spc^-polA and pcrispr-spc(/)-hoHn, respectively), Twillingate E6 was unable to plaque on these targeting strains (Figure 16). In addition, consistent with previous observations, RNase R is dispensable for CRISPR function against both phages in this system, presumably because targeted transcripts are expressed to sufficient levels to allow the CRISPR system to mount a strong enough immune response (30). Importantly, the results showed that anti-CRISPR activity is also maintained in S. epidermidis RP62a Drnr, indicating that RNase R is unlikely to facilitate AcrlllATs function.
In light of these results, we sought to explore the nature of the interaction between RNase R and acrlllA1 -RNAs. First, we modeled S. epidermidis RNase R in complex with Mg2+ using AlphaFold 3 (67), and observed that the model closely resembles the experimentally-determined structure of E. coli RNase R (PDB ID 5XGLI) (72) and has a confidence score that surpasses the 0.5 threshold (pTM=0.76) (Figure 17A and B). We next modeled RNase R-Mg2+ together with representative S. epidermidis t(m)RNAs, both full-length and 40-nt fragments starting from the 5’-end. Interestingly, the interface predicted template modeling (ipTM) scores for RNase R-Mg2+ in complex with full-length t(m)RNAs were consistently lower than those for RNase R-Mg2+ in complex with t(m)RNA fragments (averages of 0.39 ± 0.11 versus 0.75 ± 0.07, respectively) (Figure 17C and Table 4). Here, it bears mentioning that ipTM scores below 0.6 suggest a failed prediction (67-69). Moreover, the models with RNase R in complex with t(m)RNA fragments showed single-stranded RNA threaded through RNase R’s RNA channel, with the 3’-end poised at the active site Mg2+ (Figure 17D and E), similar to a model with RNase R bound to linear RNA (Figure 17F). In contrast, models with full-length t(m)RNAs bound to RNase R do not show such strand separation and RNA threading through the active site (Figure 17G and H). These data suggest that RNase R might have the capacity to cleave acrlllA1 -RNAs. To test this, we performed nuclease assays and found that RNase R can indeed degrade acrlllA1 -RNAs, albeit with less efficiency than it degrades a linear RNA substrate (Fig. 7 I and J). Altogether, this data allows us to speculate that RNase R is the intended target of acrlllA1 -RNAs, and the nature of their interaction is likely antagonistic. The precise impacts of acrlllA1 -RNAs on RNase R’s function are discussed in the section below. Table 4 Confidence scores for AlphaFold3 Models with S. epidermidis RNase R-
Mg2+ in complex with indicated t(m) RNAs
Figure imgf000077_0001
Figure imgf000078_0001
Table 4 continued
Figure imgf000078_0002
* ipTM interface predicted template modeling score, in which values higher than 0.8 indicate confident high-quality predictions, while values below 0.6 suggest a failed prediction.
** pTM predicted template modeling score, in which scores above 0.5 suggest the predicted structure might be similar to the true structure.
Example 10 Discussion
Here, we describe the first Type lll-A specific anti-CRISPR protein, AcrIIIAI . AcrIIIAI provides robust protection against immunity whether early- or late- expressed genes are targeted by the CRISPR system (Figure 1 ). This inhibition is due, at least in part, to direct interactions with the Cas10-Csm complex — AcrIIIAI binds to Csm2 (Figures 4 and 12) and causes diminished DNase activity and cOA production by Casi o (Figure 5). Reduced cOA production, in turn, would result in diminished activation of the cOA- responsive accessory nuclease Csm6. Thus, AcrIIIAI on its own has the capacity to inhibit collateral damage to phage nucleic acids by both core Cas and accessory nucleases. This may not be the whole story, as AcrIIIAI also co-purifies with small RNAs — t(m)RNA fragments herein referred to as acrlllA1 -RNAs (Figure 3). We found that acrlllA1 -RNAs associate with the Cas10-Csm complex in the native host during Twillingate infection (Figure 6), which establishes their physiological relevance. These t(m)RNAs are necessarily derived from the host since Twillingate is devoid of its own t(m)RNAs (37), and AcrIIIAI is very likely to be responsible for their fragmentation (Figure 10), although more experimentation is required to identify the precise active site. We show that t(m)RNA fragmentation is unlikely to impact translation dynamics, and these RNAs have little/no effect on AcrIIIAI ’s ability to bind Cas10-Csm and inhibit Cas10’s functions (Figures 4 and 5). However, our observation that acrlllA1 -RNAs bind stably to RNase R (Figures 7 and 17) suggests the latter is their intended target. Based on our collective findings, we propose a model in which AcrIIIAI and its associated RNAs mitigate collateral damage on multiple fronts to provide robust protection against Type III CRISPR immunity (Figure 8).
The impact of acrlllA1 -RNAs on RNase R remains to be determined. RNase R is a nonspecific 3’-5’ exoribonuclease that has the capacity to cleave through structured RNAs (73, 74). It plays critical roles in cellular RNA metabolism, including rRNA quality control (75) and the degradation of nonstop mRNAs following rescue of stalled ribosomes (76). In addition to these well-established ‘housekeeping’ functions, RNase R bolsters Type III CRISPR-Cas immunity — it interacts with Csm5 within the Cas10-Csm complex and collaborates with PNPase (another 3’-5’ exonuclease) to catalyze crRNA maturation and targeted transcript degradation, particularly when targeted transcripts are expressed at low levels (30). In light of our finding that RNase R has the ability to degrade acrlllA1 -RNAs (Figure 7I), we speculate one possible mechanism by which acrlllA1 -RNAs might interfere with RNase R’s functions is through competitive inhibition. In other words, acrlllA1 -RNAs might constitute excess nonspecific substrates that absorb collateral damage dealt by RNase R at the site of phage infection. Another possibility is that acrlllA1 -RNA binding might impact RNase R’s localization and/or stability. Indeed, RNase R is subjected to tight regulation in the cell through its interactions with tmRNA and associated protein SmpB, which confine RNase R to the ribosomes and reduce RNase R’s stability (reviewed in Bechhofer and Deutscher, 2019). We also cannot rule out the possibility that acrIIIAI - RNAs might have an impact on other accessory nucleases that are co-opted for immunity, including PNPase and RNase J2 (29, 30). Notably, PNPase and RNase J2 are members of the RNA degradosome, a large protein complex that catalyzes bulk RNA decay (78) and plays a critical role in quality control of tRNAs (79). Thus, it is possible that acrIIIAI -RNAs might impact degradosome-associated nucleases through competitive inhibition or some other mechanism yet to be identified.
AcrIIIAI is not widely distributed in the sequenced phage collection. Such an observation is typical of Acrs, which are highly variable and non-homologous (5). However, acr genes are often co-transcribed with more highly-conserved anti-CRISPR associated (aca) genes, which encode proteins that regulate transcription of the operon. Accordingly, we searched for homologs for the gene encoded directly downstream of acrIIIAI (gp18) and found them in a handful of other staphylococcal myophages. We also performed in silico analyses to determine whether gp18 resembles known transcriptional regulators. Specifically, we obtained a predicted structure with AlphaFold2 (80) (pLDDT=92.9, not shown) and submitted it to the DALI server (81 ) to identify structural homologs. Interestingly, there were no transcription regulators among the highest confidence hits. Although gp18 is not required for anti-CRISPR activity (Fig. 2G), its presence in the same operon as acrIIIAI implies that it may have a related function.
In sum, our results uncover a Type III anti-CRISPR with unique composition and mechanism of action. Here, it bears mentioning that the robust protection afforded by AcrIIIAI makes it amenable for use in therapeutic and biotechnological applications. For instance, according to CRISPRCasdb (82), Type lll-A CRISPR-Cas systems are found in many staphylococci, mycobacteria, and other pathogens that cause drug-resistant infections. As phage therapy gains traction as an alternative treatment modality (83), CRISPR-Cas and other anti-phage defenses threaten to undermine its effectiveness. Thus, therapeutic phages engineered with AcrIIIAI are more likely to be effective when targeting bacteria with Type lll-A CRISPR-Cas systems. In addition, AcrIIIAI can be used as a selectable marker (in conjunction with Type lll-A CRISPR targeting as a counterselection tool) to isolate phage variants that have taken up desired genetic payloads. These and other applications of AcrIIIAI have the potential to benefit both basic and applied research.
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Claims

CLAIMS We claim:
1 . A polynucleotide encoding an Acrl I IA1 polypeptide, wherein the Acrl I IA1 polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 .
2. The polynucleotide of claim 1 , wherein the polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2.
3. A polynucleotide comprising a heterologous promoter sequence operably linked to a polynucleotide encoding one or more Acrl I IA1 polypeptides, wherein the one or more Acrl I IA1 polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1.
4. The polynucleotide of claim 3, wherein the promoter is a constitutive promoter, a repressible promoter, or an inducible promoter.
5. The polynucleotide of claim 4, wherein the constitutive promoter is SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter.
6. The polynucleotide of claim 4, wherein the inducible promoter is a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal- regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
7. A polynucleotide encoding a fusion protein, wherein the polynucleotide comprises a heterologous polynucleotide and a polynucleotide encoding one or more Acrl I IA1 polypeptides, wherein the one or more Acrl IIA1 polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 .
8. The polynucleotide of claim 7, wherein the heterologous polynucleotide is or encodes an affinity tag, an epitope sequence tag, a detectable marker polypeptide, an amino acid spacer, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand, an amino acid linker, or a cleavable linker.
9. A vector comprising the polynucleotide of any of claims 1-8.
10. An isolated AcrIIIAI polypeptide, wherein the AcrIIIAI polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 .
11 . An isolated fusion protein comprising (1 ) one or more AcrIIIAI polypeptides, wherein the one or more AcrIIIAI polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and (2) one or more heterologous polypeptides.
12. The isolated fusion protein of claim 11 , wherein the heterologous polypeptide is an affinity tag, an epitope sequence tag, a detectable marker polypeptide, a signal sequence, a stop transfer sequence, a transmembrane domain, a protein purification ligand an amino acid spacer, an amino acid linker, a cleavable linker, or combinations thereof.
13. A delivery vehicle comprising the polynucleotide of any of claims 1-8, the vector of claim 9, the isolated AcrIIIAI polypeptide of claim 10, or the isolated fusion protein of claim 11 or 12.
14. The delivery vehicle of claim 13, wherein the delivery vehicle is a liposome or nanoparticle.
15. A CRISPR-Cas expression system comprising :
(a) a first polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, wherein the first polynucleotide is operably linked to a promoter;
(b) a second polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casio polypeptide complex to a target polynucleotide in a cell, wherein the second polynucleotide is operably linked to a promoter; and (c) a third polynucleotide encoding one or more Acrl I IA1 polypeptides, wherein the Acrl I IA1 polypeptides have greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , wherein the third polynucleotide is operably linked to a promoter.
16. The CRISPR-Cas expression system of claim 15, wherein the third polynucleotide is operably linked to an inducible promoter, repressible promoter, or constitutive promoter.
17. The CRISPR-Cas expression system of any of claims 15-16, wherein the gene or gene cluster encoding a Casio polypeptide complex comprises CRISPR-associated genes encoding Casio, Csm2, Csm3, Csm4, Csm5, Csm6, and/or Cas6.
18. A recombinant cell comprising:
(a) a recombinant AcrIIIAI polypeptide, wherein the recombinant AcrIIIAI polypeptide is heterologous to the cell and the recombinant AcrIIIAI polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 ; or
(b) a recombinant AcrIIIAI polynucleotide, wherein the recombinant AcrIIIAI polynucleotide is heterologous to the cell and the recombinant AcrIIIAI polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2.
19. The recombinant cell of claim 18, wherein the recombinant cell further comprises a polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, wherein the gene or gene cluster is operably linked to a promoter; or a Casio polypeptide complex.
20. The recombinant cell of claim 18, wherein the recombinant cell naturally expresses a Casio polypeptide complex.
21 . The recombinant cell of any of claims 18-20 further comprising a polynucleotide encoding one or more crRNA molecules that are capable of guiding the Casio polypeptide complex to a target polynucleotide.
22. The recombinant cell of any of claims 18-21 , wherein the AcrIIIAI polypeptide modulates an activity of a Casio polypeptide complex.
23. The recombinant cell of any of claims 18-22, wherein the cell is a eukaryotic cell.
24. The recombinant cell of any of claims 18-22, wherein the cell is a mammalian cell.
25. The recombinant cell of any of claims 18-22, wherein the cell is a human cell.
26. The recombinant cell of any of claims 18-22, wherein the cell is a prokaryotic cell.
27. The recombinant cell of claim 21 , wherein the polynucleotide encoding one or more crRNA molecules is present as part of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex.
28. The recombinant cell of claim 19, wherein the Casio polypeptide complex is derived from Pyrococcus furiosis, Thermococcus onnurineus, Sulfolobus solfataricus, Roseifluxus sp. RS-1, Psuedothermotoga lettingae, Staphylococcus epidermidis, Staphylococcus sp., Streptococcus sp., Methanopyrus kandleri, Thermus thermophilus, or any organism harboring a Type III CRISPR-Cas system.
29. The recombinant cell of any one of claims 18-28, wherein expression of the recombinant Acrl I IA1 polynucleotide, the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, and/or the polynucleotide encoding one or more crRNA molecules is independently under control of a constitutive promoter, a repressible promoter, or an inducible promoter.
30. The recombinant cell of claim 29, wherein the constitutive promoter is a SV40 promoter, a CMV promoter, a PGK promoter, a ubiquitin C (UBC) promoter, an EF1 A promoter, and a CAGG promoter, or a SFFV promoter.
31 . The recombinant cell of claim 29, wherein the inducible promoter is a TRE3G inducible promoter, a tetracycline-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, an estrogen receptor-regulated promoter, or a UAS inducible promoter.
32. The recombinant cell of any one of claims 19 to 31 , wherein the expression of the polynucleotide encoding a gene or gene cluster encoding a Casio polypeptide complex, and/or the polynucleotide encoding one or more crRNA molecules is under control of the same promoter.
33. A method of inhibiting a Casio polypeptide or Casio polypeptide complex in a cell, the method comprising introducing:
(i) an Acrl I IA1 polypeptide into the cell, wherein the Acrl I IA1 polypeptide is heterologous to the cell, and the Acrl I IA1 polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 , and/or
(ii) an Acrl I IA1 polynucleotide into the cell, wherein the Acrl IIA1 polynucleotide is heterologous to the cell, and the Acrl I IA1 polypeptide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:1 thereby inhibiting the Casio polypeptide or Cas 10 polypeptide complex in the cell.
34. The method of claim 33, wherein the cell naturally expresses the Casio polypeptide or Casio polypeptide complex.
35. The method of claim 33, wherein the cell has been genetically engineered to express a heterologous Casio polypeptide or Casio polypeptide complex.
36. The method of any of claims 33-35, wherein the polynucleotide encoding the Acrl I IA1 polypeptide is operably linked to an inducible promoter, a repressible promoter, or a constitutive promoter.
37. The method of claim 36, further comprising contacting the cell with an agent or condition that induces expression of the Acrl I IA1 polypeptide in the cell.
38. The method of any of claims 33-37, wherein the cell is a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell.
39. The method of any of claims 33-38, wherein the method occurs ex vivo or in vitro.
40. A method of reducing activity of a CRISPR-Cas10 system in a cell comprising delivering the polynucleotide of claims 1 to 8 to the cell, the vector of claim 9, or the polypeptide of claims 10 to 12 to the cell.
41 . A method for regulation of a CRISPR-Cas system, comprising delivering the CRISPR-Cas expression system of any of claims 15-17 to a target cell.
42. The method of regulation of a CRISPR-Cas system or claim 41 , wherein the cell is a eukaryotic cell, a prokaryotic cell, a mammalian cell, or a human cell.
43. A recombinant phage comprising a genetically modified genome comprising a heterologous Acrl I IA1 polynucleotide.
44. The recombinant phage of claim 43, wherein the AcrIIIAI polynucleotide has greater than 80% sequence identity to the sequence set forth in SEQ ID NO:2
45. A method of improving administration of a whole phage therapeutic comprising genetically engineering phage making up the whole phage therapeutic to express a recombinant AcrIIIAI polypeptide and administering the whole phage therapeutic to a subject in need thereof.
46. The method of claim 45, wherein the subject has a bacterial infection caused by Staphylococcus sp.
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