Patent Application
20230193409
The present disclosure relates generally to CRISPR inhibition, and more specifically to proteins and derivatives thereof for use in inhibiting Cas13.
Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems and CRISPR-associated (Cas) proteins are prokaryotic adaptive immune systems that protect their hosts from invasion by viruses (1) and plasmids (2). CRISPR loci contain short DNA repeats separated by spacer sequences of foreign origin (3-5). To achieve immunity, the locus is transcribed and processed into small CRISPR RNAs (crRNAs), which associate with RNA-guided Cas nucleases (6) to locate and cleave complementary nucleic acid sequences (protospacers) (7-11). CRISPR systems are categorized into six types (I-VI) and 33 subtypes, which differ in their cas gene content and mechanism of immunity (12). While most types neutralize invaders through destruction of their DNA, Cas13, the RNA-guided nuclease of type VI systems, unleashes non-specific RNA degradation (trans-RNase activity) upon recognition of a phage target transcript (7, 13, 14). The cleavage of host transcripts leads to a growth arrest that prevents further propagation of the phage, allowing the uninfected cells in the population to survive and proliferate (14). Because the phage genome is not directly affected by Cas13, it continues to produce target transcripts, leading to a persistent activation of the nuclease and growth arrest (14).
Presumably in response to the pressure imposed by CRISPR-Cas immunity, phages evolved anti-CRISPR (Acr) proteins, small proteins (usually<150 aa) that are produced during infection and inactivate Cas nucleases (15). Acrs also exhibit exceptional diversity of sequences and mechanisms and, with few exceptions, specifically inhibit one CRISPR subtype. About 50 families of Acrs have been discovered that inhibit types I, II, III or V CRISPR-Cas systems (15-20). A characteristic of type I and II Acrs is that they rely on multiple rounds of phage infection to completely inactivate their cognate CRISPR system (21, 22). Expression of the phage-encoded Acr occurs shortly after infection, but it is not sufficient to neutralize all the active Cas nucleases inside the cell. Instead, only a fraction of the nucleases is inhibited per infective cycle, resulting in host immunosuppression rather than a complete halt of the CRISPR-Cas immune response (21, 22). As a consequence, the success of the Acr is highly dependent on the strength of the CRISPR-Cas immune response and on the multiplicity of infection (MOI): the presence of Cas nucleases programmed to target multiple sites in the viral genome, or a low concentration of phage prevent Acrl and AcrII inhibitors from overcoming immunity. But it is believed that no Acrs have been previously reported to inhibit type VI CRISPR-Cas immunity. Thus, there is an ongoing and unmet need to identify Acrs that have this function so that they can be adapted for use in deliberate inhibition of the pertinent cas enzymes, and for other proposes. The present disclosure is pertinent to these needs.
The crRNA-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. Whether and how viruses can counteract this immunity is not known. We describe a listeriophage (ΦLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri. Using genetics and biochemistry we demonstrate that AcrVIA1 interacts with the guide-exposed face of Cas13a to prevent access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion can completely dismantle type VI-A CRISPR-mediated immunity.
ArcfVIA1 has the following amino acid sequence:
The disclosure provides compositions comprising proteins comprising this sequence, or derivatives thereof, fusion proteins comprising this sequence, or derivatives thereof, expression vectors that encode this sequence, and methods of making and using proteins that comprise this sequence, or derivatives thereof, for inhibiting the function of Cas13 and/or protein complexes and/or ribonucleoprotein complexes that comprise Cas13. The disclosure further includes use of the described inhibitor protein in diagnostic assays that include Cas13. Inclusion of the inhibitor is expected to provide certain improvements in diagnostic tests where samples containing or suspected of containing RNA signatures are evaluated, and may preclude a requirement to reverse transcribe and/or create cDNA amplifications of the particular RNA that is the subject of the analysis.
Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. The disclosure includes all polynucleotide and amino acid sequences described herein, and all DNA and RNA sequences that encode any polypeptide as described herein. Each RNA sequence includes its DNA equivalent, and each DNA sequence includes its RNA equivalent. Complementary and anti-parallel polynucleotide sequences are included. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included. Sequences of from 80-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included. If reference to an amino acid or nucleotide sequence is made to by way of a database entry, the sequence corresponding to that database entry as it exists on the effective filing date of this application or patent is incorporated herein by reference.
In embodiments, a protein of this disclosure comprises SEQ ID NO:1, or a modified version thereof, wherein the modified version comprises a truncated protein, a fusion protein, or mutated version of said protein.
In embodiments, the disclosure provides compositions and methods for use in, for example, type VI CRISPR-Cas13 anti-CRISPR applications. As such, the proteins of this disclosure may be used to control Cas13 RNA editing activity. The disclosure therefore provides a means for controlling Cas13 activity in a variety of settings, including but necessarily limited to therapeutic, veterinary and agricultural, and research-based implementations. The proteins of this disclosure, and compositions comprising them, may be used in any cell type, including but not limited to prokaryotes and eukaryotes. In embodiments, a system comprising a Cas13 protein of this disclosure is used to modulate RNA editing in any of bacteria, archaea, plants, and animal cells, that latter of which include but are not necessarily limited to cells of insects, fish, avian animals, and mammals, including but not limited to humans. The proteins of this disclosure may also be used to modulate the activity of viruses, including but not limited to any virus having an RNA genome, whether single or double stranded, or a single strand or segmented genome, or any virus that uses an RNA intermediate, and any virus, such as virus with a DNA genome that is used to produce an RNA transcript. Further, the inhibition may be pertinent to Cas13 editing of any type of RNA, including but not necessarily limited to mRNA, hnRNA, miRNA, snoRNA, RNA produced within organelles, and the like. Inhibition of RNA editing by Cas13 may be performed in vitro or in vivo. Ex vivo modification of cells to express a protein of this disclosure for use in subsequent therapeutic or other approaches is also encompassed by this disclosure.
In embodiments, the disclosure provides for using a protein of this disclosure to inhibit, for example, crRNA from properly complexing with Cas13a, and/or inhibits binding of Cas13 or a complex comprising Cas13 to a complementary target RNA. In embodiments, the disclosure provides for inhibition of conformational changes required for the activation of the RNase function of Cas13a.
In embodiments, the disclosure provides a contiguous segment of the amino acids of SEQ ID NO:1 that is sufficient to partially or fully inhibit the RNA cleavage function of Cas13a, such as by preventing association of the nuclease with a targeted protospacer RNA. Thus, in embodiments, the disclosure provides a polypeptide comprising a contiguous segment of SEQ ID NO:1 that comprises from 10-232 contiguous amino acids of SEQ ID NO:1, including all integers and ranges of integers there between, or a contiguous polypeptide that is at least 80% identical to such a segment of SEQ ID NO:1. In embodiments, a contiguous segment of a protein of this disclosure consists of SEQ ID NO:1. In embodiments, a protein comprising a contiguous sequence that consists of SEQ ID NO:1 is functional relative to a shorter, or a mutated version of SEQ ID NO:1.
In embodiments, pharmaceutical compositions comprising an inhibitor protein of this disclosure are provided. In embodiments, a pharmaceutical composition comprises at least one pharmaceutically acceptable additive.
In embodiments, the disclosure provides an expression vector encoding the described protein. In embodiments, a sequence encoding the described inhibitor protein is operably linked to an inducible promoter so that expression of the inhibitor protein can be controlled, such as to inducibly express the protein in order to inhibit or completely stop Cas13-based RNA editing.
In embodiments, the disclosure provides for administering to cells, tissues, or an organism, or a combination thereof, an inhibitor protein of this disclosure, or a polynucleotide encoding the inhibitor protein. In embodiments, an effective amount of the inhibitor protein that is sufficient to inhibit or stop Cas13-based RNA editing is introduced into cells, tissue, or an organism. In embodiments, and without intending to be limited by any particular theory, it is considered, at least for use in bacteria, that a single copy of the gene encoding the described inhibitor protein will be sufficient to stop Cas13a RNA degradation in a single bacterium. In embodiments, an amount of the described inhibitor protein that is administered and is sufficient to inhibit or stop Cas13-based RNA editing is less than the amount of Cas-enzyme inhibition determined from any suitable reference, e.g., the amount of inhibitor protein is less than a control value. Suitable control values can be obtained from other proteins, which may include known protein inhibitors of other Cas-enzymes, including but not necessarily limited to Cas9 enzyme protein-based inhibitors. Thus, it is considered that the presently provided proteins are more potent than previously described Cas-enzyme inhibitors, insofar as their capacity to inhibit Cas13a nuclease activity. Accordingly, in embodiments, the disclosure comprises introducing or causing the expression of an inhibitor protein described herein such that the inhibitor protein functions to inhibit or stop RNA editing within a cell that also comprises a Cas13-based RNA editing system, which may comprise an engineered system that is designed to specifically target any particular RNA, or target more than one RNA. As such, the system comprises at least a Cas13 protein, and a guide RNA targeted to a target RNA. In embodiments, the Cas13 activity that is inhibited using a described inhibitor protein functions comprises an L. seeligeri type VI-A Cas13a protein. The sequence of this VI-A Cas13a protein is known in the art and is available from, for example, GenBank accession number WP_012985477.1, the sequence from which is incorporated herein as of the effective filing date of this application or patent.
In embodiments, the disclosure provides for editing RNA in one or more cells using a Cas13 protein, such as the L. seeligeri type VI-A Cas13a, protein, as a component of a Cas13-CRISPR RNA editing system. The RNA editing system comprises the Cas13a protein or a vector encoding it, and may further comprise one or more crRNAs and/or guide RNAs, or one or more vectors encoding crRNAs and/or guide RNAs so that respective RNA is expressed in the cell. Additional CRISPR proteins may be included, such as any additional protein that is required for Cas13a RNA editing to function. In general, the guide RNA is designed to target a protospacer present in a targeted RNA. The protospacer is not particularly limited. In embodiments, Cas13a targeting efficiency decreases substantially if the 3′ end of the target RNA is flanked by nucleotides homologous to a CRISPR repeat sequence, such as a sequence comprising GTTTAGT (SEQ ID NO:2), and thus suitable modifications of the target RNA can be taken into account when implementing aspects of the disclosure.
In embodiment, the method comprises allowing RNA editing catalyzed at least in part by the Cas13a protein, and at a desired time, causing the RNA editing to be inhibited or stopped by introducing into the cell an inhibitor protein of this disclosure, such as by introducing the protein into the cell directly, or using a delivery system, or by inducing its expression from a controllable promoter. In embodiments, nuclease activity of the Cas13a is suppressed in the cells of an organism wherein an adverse result is experienced by the individual as a consequence of the Cas13a RNA editing. In embodiments, the individual experiencing the adverse event is being treated for viral infection. In embodiments, the individual is being treated with a Cas13 used as an anti-viral therapeutic against an infection by an RNA virus. In embodiments, the individual is infected with a coronavirus.
In embodiments, a Cas13a CRISPR editing system, and/or a protein of this disclosure, is administered to bacteria using a modified bacteriophage, or by packaged phagemids. In embodiments, a Cas13a CRISPR editing system, and/or an inhibitor protein of this disclosure, is encoded by a conjugative plasmid. In embodiments, providing a conjugative plasmid encoding an inhibitor protein of this disclosure may cause the inhibitor protein to be expressed in other bacteria by horizontal plasmid transfer. In embodiments, Cas13a system and a means for controllable inhibitor protein expression may be introduced into bacteria (or eukaryotic cells) that are used for industrial purposes, such as in the food or beverage industry, or for the production of biological agents. In embodiments, bacteria that are modified as described herein comprise lactic acid bacteria. In additional and non-limiting embodiments, the Cas13a and a means for controllable inhibitor protein expression are introduced into pathogenic bacteria, including but not limited to Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, or Enterobacter spp. In embodiments, the system is introduced into biofuel producing bacteria, such as Zymomonas mobilis. In embodiments, the system is introduced into plant-associated bacteria, such as Agrobacterium tumiefaciens. Thus, in embodiments, the disclosure includes expression of the described inhibitor protein in a heterologous host. A heterologous host means any cell that does not comprise a polynucleotide that encodes the described inhibitor protein prior to being modified to express the described inhibitor protein as described herein.
In embodiments, the targeted RNA that is edited by the Cas13a system in the absence of a described inhibitor protein encodes and is translated into any protein of interest, which may include but is not limited to a selectable or detectable marker. In embodiments, the targeted RNA encodes a protein that produces a detectable signal, thereby permitting analysis of targeting using a system of this disclosure by detecting an absence or a reduction in the detectable signal when the inhibitor protein is present and functional within the cell. In embodiments, the detectable signal is produced by a fluorescent protein. In embodiments, the targeted RNA encodes an antibiotic resistance protein, or a virulence factor. Thus, in embodiments, a change in antibiotic resistance or virulence can be determined by operation of a functional inhibitor protein. In embodiments, an inhibitor protein of this disclosure is used during analysis of any of RNA editing, knock-down and/or RNA visualization applications.
For use in eukaryotic cells, the described inhibitor protein can be modified to enhance its utility, such as by including a nuclear localization signal as a component of the inhibitor protein. Thus the disclosure includes use of at least one nuclear localization signal (NLS) in the described inhibitor protein. In general, a suitable NLS includes one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Further, the described inhibitor proteins may also be modified by including, for example, a suitable purification tag, such as a poly-histidine tag.
In embodiments, a system of this disclosure is introduced into eukaryotic cells using, for example, one or more expression vectors, or by direct introduction of ribonucleoproteins (RNPs). In embodiments, expression vectors comprise viral vectors. In an embodiment, adenoviral vectors may be used, and many such vectors are known in the art and can be adapted for use with eukaryotic cells when provided the benefit of this disclosure.
The following description and examples are intended to illustrate embodiments of, but not limit the disclosure.
In arriving at aspects of the present disclosure, we first obtained temperate phages from a collection of 62 environmental isolates of Listeria spp., a natural host for type VI-A CRISPR-Cas systems. We treated cultures with mitomycin C and looked for the ability of the supernatants to form plaques on an agar lawn seeded with a mutant of Listeria seeligeri SLCC3954 (23) lacking its two restriction-modification systems and the type VI-A CRISPR array (L. seeligeri ΔRM Δspc),
Sequencing of the ΦLS46 genome revealed a similar organization to a previously characterized temperate phage of L. seeligeri, ΦRR4 (14), which harbors four independent transcriptional units: an early lytic region encoding predicted replicases and recombinases involved in phage circularization and genome replication; a late lytic region carrying phage structural genes; a lysogeny cassette containing transcriptional regulators and a predicted site-specific integrase; and a region containing six genes, two of them with homology to the Cas9 inhibitors AcrIIA1 and AcrIIA2, for the evasion of type II CRISPR-Cas immunity by ΦRR4. In ΦLS46, however, this region contains four genes, none of which display strong homology to known inhibitors (
Collectively, data presented in this disclosure indicate that acrVIA1 is necessary and sufficient to inhibit Cas13a-induced growth arrest and thus thwart type VI CRISPR immunity against plasmids and phages.
The inhibition of Cas13a-induced growth arrest suggests that AcrVIA1 inhibits the trans-RNase activity of Cas13a. To investigate this, we purified both proteins (
We performed site-directed mutagenesis of the acrVIA1-3xflag (carried by plasmid pgp2) to modify the amino acid residues predicted to mediate the nuclease-inhibitor contacts, and thus test the contribution of different putative interactions to the inhibition of type VI-A immunity against plasmid conjugation. Alanine substitutions at predicted Cas13acrRNA-interacting residues in AcrVIA1 (quintuple mutant Y39A, S40A, N43A, S93A, Q96A) caused nearly complete loss of inhibitory function (
Previously described anti-CRISPRs that inhibit type I and II CRISPR systems require multiple rounds of infection to completely inhibit anti-phage immunity, and fail in conditions of strong CRISPR immunity or low viral load (21, 22). To investigate if AcrVIA1 also displayed limited inhibition capabilities, we first tested its efficacy in conditions of weak or strong type VI CRISPR-Cas immunity, by infecting cells harboring either one or three targeting spacers, respectively (
In this disclosure, we used genetic and biochemical approaches to isolate and characterize a phage-encoded inhibitor of L. seeligeri Cas13a: AcrVIA1. This inhibitor interacts with the crRNA-exposed face of Cas13a and makes specific contacts with both the nuclease and its guide RNA that prevent the binding of a complementary target RNA and the conformational changes required for the activation of Cas13a's RNase function. We analyzed whether AcrVIA1 could inhibit other Cas13 family members. Addition of AcrVIA1 had no effect on protospacer RNA cleavage by purified L. buccalis Cas13acrRNA (
We also found that AcrVIA1 can completely neutralize type VI-A CRISPR-Cas immunity against ΦLS46, even in unfavorable conditions for inhibition such as multiple protospacer targeting and low viral load. We believe this to be a consequence of the lack of phage DNA clearance during the type VI response (14). This would lead to a continuous transcription and translation of AcrVIA1 and progressive neutralization of Cas13a. Assuming that the collateral RNA degradation generated by activation of Cas13a in Listeria hosts allows a low level of AcrVIA1 transcription and translation, enough inhibitor will accumulate to inactivate all the Cas13a molecules inside the bacterial cell. This is in contrast to type I and II Acrs, whose initial production inhibits only a fraction of Cascade-Cas3 and Cas9 molecules, respectively, and the Acr-harboring phage is destroyed by the nucleases that remain active (21, 22). Gradual inhibition of Cas13a after phage infection would require AcrVIA1 to constantly capture the Cas13acrRNA molecules that disengage from the target RNA and prevent them from finding their targets again. Alternatively, the inhibitor could displace the target RNA molecules from activated Cas13a′crRNA nucleases, to de-activate them. Such a mechanism would be especially effective when the target RNA is a transcript that is produced, and therefore activates Cas13a, before AcrVIA1 is generated. Indeed, spcA1-mediated immunity, which targets the first gene of the acr operon (gp1) and should activate Cas13a before production of the inhibitor (encoded by the second gene of the operon, gp2) is effectively abrogated by AcrVIA1. And moreover, many of the spacers used in this disclosure target phage transcripts that are abundantly produced shortly after infection (those targeted by spcA1, spcE1, and spcE2 for example,
Notwithstanding the foregoing description, the disclosure further comprises use of the described protein inhibitor to improve certain diagnostic assays which are used to analyze RNA, such as in biological samples. In embodiments, the disclosure includes use of the inhibitor in any diagnostic assay that is intended to determine the presence or absence of a particular RNA polynucleotide, and quantitative approaches are also included.
In embodiments, a biological sample analyzed according to this disclosure comprises any suitable biological sample, including but not limited to blood, urine, mucosa, mucosal secretions, saliva, and lacrimal secretions. In embodiments, a biological sample is tested directly. In embodiments, the biological sample is subject to a processing step before testing, a non-limiting example of which comprises RNA extraction. In embodiments, a diagnostic assay of this disclosure may exhibit increased sensitivity to the presence or absence of a particular RNA, and in embodiments may obviate the requirement for cDNA synthesis and amplification and still provide a test with sufficient sensitivity and specificity. Accordingly, in certain embodiments, a diagnostic test of this disclosure may be performed without using reverse transcriptase, and/or may be performed without a PCR amplification step. In embodiments, a diagnostic test of the disclosure may be performed without transcription of a PCR-amplified template.
In embodiments, in any diagnostic assay used to detect and/or quantify RNA using a Cas13-related approach, the disclosure includes adding the described inhibitor to biological sample obtained from an individual that is either tested directly, or is processed before testing, such as to separate RNA from the sample. In embodiments, the inhibitor is added a short time (e.g., within 1 second to 60 minutes) after Cas13 in sample has associated with the target RNA, if the target RNA is present, in the patient sample. Without intending to be bound by any particular theory, and as in part illustrated by
In embodiments, the disclosure includes adding the described inhibitor to an assay that comprises Cas13, and a guide RNA targeted to a particular RNA polynucleotide sequence of interest, and at least one reporter RNA, wherein the reporter RNA is configured to permit Cas13-mediated detection of its degradation, or lack of degradation by the Cas13, e.g., the reporter RNA can be detectably cleaved when the non-specific RNA nuclease activity of Cas13 is triggered.
In non-limiting embodiments, a reporter RNA polynucleotide is not targeted by the Cas13-related guide RNA, and is labeled at one position with a detectable label, and also with a moiety that quenches a detectable signal from the detectable label at another position. In embodiments, a fluorophore and a quencher moiety are conjugated to the reporter RNA in sufficient proximity to one another such that the detectable signal is quenched when the RNA is intact. Accordingly, when and if the RNA reporter is cleaved by the non-specific nuclease activity of the Cas13, which is considered to only become active once the Cas13 has engaged a target in a guide-RNA directed manner, the detectable label is liberated from the intact reporter RNA, and a signal from it can be detected using any suitable approach.
In embodiments, any detectable label can be used with the reporter RNA, non-limiting examples of which include fluorophores, metals or chemiluminescent moieties, fluorescent particles, quantum dots, etc., provided the signal from the detectable label can be quenched, or its intensity shifted to a different wavelength in, for example, a fluorescence resonance energy transfer (FRET) process by a suitable quencher moiety conjugated to the reporter RNA.
In embodiments, an inhibitor of this disclosure is added to an assay such as the so-called SHERLOCK (for Specific High Sensitivity Enzymatic Reporter UnLOCKing) assay, described in PCT publication WO2017219027, published Dec. 21, 2017, and SHERLOCK: nucleic acid detection with CRISPR nucleases, Kellner M J, Koob J G, Gootenberg J S, Abudayyeh O O, and Zhang F. Nature Protocols. 2019, October;14(10):2986-3012. doi: 10.1038/s41596-019-0210-2. (NATURE PROTOCOLS, VOL 14, OCTOBER 2019, 2986-3), the disclosures of each of which are incorporated herein by reference. In embodiments, the SHERLOCK assay is adapted to omit reverse-transcriptase cDNA synthesis and subsequent amplification using PCR-based approaches. In embodiments, less PCR amplification products are required to detect the presence or absence of RNA, relative to a control assay wherein the inhibitor is not included.
In a non-limiting embodiment, the disclosure provides for use of the described inhibitor for detecting RNA viruses, including but not limited to the coronavirus referred to in the art the time of this disclosure as SARS-CoV-2, which causes COVID-19. In an embodiment, the assay is performed using a lateral flow device. In embodiments, the testing is performed by testing for the presence or absence of RNA encoded by the viral S gene and/or the Orflab gene. In embodiments, the Cas13 used in this approach or related approaches is LwaCas13a. In embodiments, liberated label can be detected in the lateral flow device at a predetermined position. Suitable controls may be included, such as a predetermined amount of synthetically produced viral target RNA.
In the present disclosure, it is revealed that after Cas13 has already engaged target RNA, addition of AcrVIA1 enhances the longevity of Cas13 activity. Thus, it is expected that this aspect of the inhibitor may make Cas13-based diagnostics more sensitive, and may obviate the need for reverse transcription and PCR amplification of patient samples.
In more detail,
The following materials and methods were used to produce results described in this disclosure.
All genetically modified L. seeligeri strains generated as described herein are derived from L. seeligeri SLCC3954 (23). Environmental L. seeligeri isolates and L. monocytogenes strains are listed in Table S2. Unless otherwise stated, L. seeligeri and L. monocytogenes strains were cultured in Brain Heart Infusion (BHI) medium at 30° C. Where appropriate, BHI was supplemented with the following antibiotics for selection: nalidixic acid (50 μg/mL) chloramphenicol (10 μg/mL), erythromycin (1 μg/mL), or kanamycin (50 μg/mL). For cloning, plasmid preparation, and conjugative plasmid transfer, E. coli strains were cultured in Lysogeny Broth (LB) medium at 37° C. Where appropriate, LB was supplemented with the following antibiotics: ampicillin (100 μg/mL), chloramphenicol (25 μg/mL), kanamycin (50 μg/mL). For conjugative transfer of E. coli—Listeria shuttle vectors, plasmids were purified from Turbo Competent E. coli (New England Biolabs) and transformed into the E. coli conjugative donor strains SM10 λkpir or S17 λpir (33).
Temperate listeriophages were isolated by prophage induction via stimulation of the SOS response with the DNA-damaging agent mitomycin C, followed by plaque isolation on the L. seeligeri ΔRM Δspc indicator strain. Each strain of L. seeligeri and L. monocytogenes was cultured overnight and diluted to OD600=0.1, then treated with 1 μg/mL mitomycin C to activate the phage lytic cycle. Prophage induction was carried out overnight at 30° C., then culture supernatants were passed through 0.45 μm filters. Each filtrate was screened for phages by infection of ΔRM Δspc using the top agar overlay method: 100 ul of serially diluted induction filtrate was used to infect 100 μL of saturated ΔRM Δspc culture in a 5 mL overlay of BHI containing 0.75% agar, in the presence of 5 mM CaCl2. Infection plates were incubated at 30° for 24 hrs. Single plaques were resuspended in BHI, then propagated three times on ΔRM Δspc, a single plaque was isolated each time to ensure phage purity. High titer phage lysates were obtained by preparing top agar infections of ΔRM Δspc with plaques at near-confluent density, then soaking the agar with SM buffer (100 mM NaCl, 10 mM MgSO4, 50 mM Tris-HCl pH 7.5).
All genetic constructs for expression in L. seeligeri were cloned into the following three compatible shuttle vectors, each of which contains an origin of transfer sequence for mobilization by transfer genes of the IncP-type plasmid RP4. These transfer genes are integrated into the genome of the E. coli conjugative donor strains SM10 λpir and S-17 λpir (33). All plasmids used in this disclosure, along with details of their construction, can be found in Table S2. pPL2e—single-copy plasmid conferring erythromycin resistance that integrates into the tRNAArg locus in the L. seeligeri chromosome (34). pAM8—E. coli—Listeria shuttle vector conferring chloramphenicol resistance (35). pAM326—E. coli—Listeria shuttle vector conferring kanamycin resistance (produced according to this disclosure). To express crRNAs containing engineered spacers, a minimal type VI CRISPR array containing the native promoter and a single repeat-spacer-repeat unit with BsaI entry sites was cloned into BamHI/SalI-digested pPL2e to generate pAM305. To clone new spacers, pAM305 was digested with BsaI, and ligated to spacer inserts consisting of annealed oligos with cohesive overhangs compatible with the sticky ends generated by BsaI-cleavage of pAM305.
All plasmid targeting assays described herein use the pAM8-derived plasmid pAM54 (35), in which a protospacer matching the endogenous type VI spc4 was cloned into the 3′ untranslated region of a chloramphenicol resistance cassette. The negative control for plasmid targeting assays is pAM8, which contains the chloramphenicol cassette without a protospacer.
Putative anti-CRISPR constructs were assembled by cloning into HindIII/EagI-digested pAM326.
All genetic constructs for expression in L. seeligeri were introduced by conjugation with the E. coli donor strains SM10 λpir, S-17 λpir (33), or for allelic exchange (see below), β2163 ΔdapA (36). Donor cultures were grown overnight in LB medium supplemented with the appropriate antibiotic (25 μg/mL chloramphenicol for pPL2e-derived plasmids, 100 μg/mL ampicillin for pAM8-derived plasmids, or 50 μg/mL kanamycin for pAM326-derived plasmids) at 37° C. Recipient cultures were grown overnight in BHI medium supplemented with the appropriate antibiotic (1 μg/mL erythromycin for pPL2e-derived plasmids, 10 μg/mL chloramphenicol for pAM8-derived plasmids, 50 μg/mL kanamycin for pAM326-derived plasmids) at 30° C. 100 μL each of donor and recipient culture were diluted into 10 mL of BHI medium, and concentrated onto a filter disc (Millipore-Sigma, HAWP04700) using vacuum filtration. Filter discs were laid onto BHI agar supplemented with 8 μg/mL oxacillin (which weakens the cell wall and enhances conjugation) and incubated at 37° C. for 4 hr. Discs were removed, cells were resuspended in 2 mL BHI, and transconjugants were selected on medium containing 50 μg/mL nalidixic acid (which kills donor E. coli but not recipient L. seeligeri) in addition to the appropriate antibiotic for plasmid selection. Transconjugants were isolated after 2-3 days incubation at 30° C.
Allelic exchange plasmids were generated by cloning 1 kb homology arms flanking the genomic region to be deleted into the suicide vector pAM215 (14), which does not replicate in Listeria, and contains a chloramphenicol resistance cassette and lacZ from Geobacillus stearothermophilus. These plasmids were then transformed into the E. coli donor strain □2163 AdapA (36), which is auxotrophic for diaminopimelic acid (DAP), selecting on LB medium supplemented with the appropriate antibiotic and 1.2 mM DAP. Conjugation was carried out as described above, except all steps were carried out in the presence of 1.2 mM DAP. Transconjugants were selected on media lacking DAP and containing 50 μg/mL nalidixic acid, to ensure complete killing of donor E. coli, as well as 10 μg/mL chloramphenicol to select for integration of the pAM215-derived plasmid. Chloramphenicol-resistant colonies were patched on BHI supplemented with 100 μg/mL 5-Bromo-4-Chloro-3-Indolyl β-D-Galactopyranoside (X-gal) and confirmed lacZ+ by checking for blue colony color. Plasmid integrants were passaged 3-4 times in BHI at 30° in the absence of antibiotic selection, to permit loss of the integrated plasmid. Cultures were screened for plasmid excision by dilution and plating on BHI+X-gal. White colonies were checked for chloramphenicol sensitivity, then chromosomal DNA was prepared from each, and tested for the desired deletion by PCR using primers flanking the deletion site. Deletions were finally confirmed by Sanger sequencing.
The ΦLS46 genome was sequenced by whole-genome sequencing and assembly of its parent lysogen, L. seeligeri LS46. Chromosomal DNA was prepared from LS46 by lysozyme digestion of the cell wall, followed by cell lysis with 1% sarkosyl, then phenol-chloroform extraction and ethanol precipitation. 1 ng of chromosomal DNA was used to make an NGS library using the Illumina Nextera XT DNA Library Preparation Kit according to the manufacturer's instructions. Library quality was confirmed by analysis on Agilent
TapeStation, then 2×150 bp paired-end sequencing was carried out on the Illumina NextSeq platform. Raw reads were quality-trimmed using Sickle (github.com/najoshi/sickle) using a quality cutoff of 30 and length cutoff of 45. Trimmed reads were assembled using SPAdes (cab.spbu.ru/software/spades/) with the default parameters, which resulted in 140 assembled contigs with an N50 of 2841899. These contigs were mapped onto the completed reference genome of L. seeligeri SLCC3954 using Medusa (combo.dbe.unifi.it/medusa/) with the default parameters, which resulted in 105 scaffold assemblies. In our draft genome assembly, one scaffold (Scaffold 1) represents a 2.8 Mbp assembly, Scaffold 7 contains 46 Kbp, and each of the remaining 103 scaffolds contains between 100-1300 bp. To identify putative prophages in the assembled genome, we used Phaster (phaster.ca), which predicted a single prophage element, occupying the entirety of Scaffold 7. We confirmed that this prophage (ΦLS46) was the one isolated by mitomycin C induction of LS46 using PCR of the ΔRM Δspc-passaged phage stock with ΦLS46-specific primers.
Gene deletions in ΦLS46 were constructed in two ways. One group of deletions was obtained by selection of spontaneous escapers of Cas9 targeting of the anti-CRISPR locus in ΦLS46. A Cas9 spacer targeting the anti-CRISPR region (gp4) was cloned into the vector pAM307, which carries Cas9 from Streptococcus pyogenes along with a repeat-spacer-repeat construct with BsaI entry sites. This plasmid (pAM379) was introduced into ΔRM Δspc, which was then infected with ten-fold serial dilutions of ΦLS46 in a plaque assay on BHI top agar. Cas9-targeting reduced the efficiency of ΦLS46 plaquing by several orders of magnitude, but spontaneous Cas9-resistant escaper plaques were isolated and checked for deletions by PCR using primers flanking the anti-CRISPR locus. The deletions were then precisely mapped by Sanger sequencing. To generate an in-frame deletion of the acrVIA1 gene, we first assembled a homology repair template (pAM386) containing 1 kb homology arms flanking an in-frame deletion of acrVIAL In the deletion construct, the first six and last six codons of acrVIA1 remain, both to avoid Rho-dependent termination of untranslated RNA, as well as to preserve the Shine-Dalgarno sequence for the gp3 gene predicted to be present in the last six codons of acrVIA1. The repair template plasmid was introduced into ΔRM Δspc, this strain was infected with ΦLS46 in BHI top agar (allowing recombinants to be generated), and a phage stock was harvested. A Cas9 spacer targeting acrVIA1 was cloned into pAM307 to generate pAM377 and introduced into ΔRM Δspc. The ΦLS46 stock passaged on ΔRM Δspc carrying the pAM386 repair template was used to infect ΔRM Δspc carrying pAM377, and Cas9-resistant escaper mutants were isolated. Two mutant phage isolates were Sanger sequenced across the acrVIA1 gene, and found to contain the precise deletion.
10 μM synthetic RNA substrates (listed in Table S7) were labeled with ATP [γ-32P] for 30 min at 37° with 1 ul NEB T4 Polynucleotide Kinase, then purified using GE MicroSpin G-50 columns. In a 10 μL reaction, 1 nM purified L. seeligeri Cas13-His6:crRNA complex was combined with 10 nM synthetic target RNA, in buffer containing 10 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 5 mM β-mercaptoethanol, and 5% glycerol, at room temperature for the indicated time. Reactions were quenched by addition of an equal volume of loading dye (95% formamide, 14 mM EDTA, 0.025% SDS, 0.04% bromophenol blue, 0.04% xylene cyanol), then denatured by boiling 5 min, then crash cooled on ice for 1 min before loading on denaturing TBE-Urea PAGE gels with 15% acrylamide. Reactions were exposed to phosphoscreen 1 hour and imaged with Beckman Coulter FLA7000IP Typhoon storage phosphorimager.
ΔCRISPR strains of L. seeligeri harboring pAM364 (Cas13-his6 cloned into a pPL2e backbone) and pAM395 (Ptet-AcrVIA1-3xFlag cloned into a pAM326 backbone) along with empty vector controls, were cultured in 50 mL BHI supplemented with 50 μg/mL kanamycin and 100 ng/mL aTc at 30° C. until the OD600 reached 0.7. 30 mL culture samples were harvested, pelleted by centrifugation at 8,000 rpm for 2min, and frozen at −80° C. Pellets were resuspended in 0.5 mL ice-cold lysis buffer (50 mM HEPES pH 7.0, 200 mM NaCl, 5 mM MgCl2, 5% glycerol, 1 mg/mL lysozyme, supplemented with Roche cOmplete EDTA-free protease inhibitor cocktail. Samples were incubated at 37° C. for 5 min, then placed on ice and lysed by sonication. Insoluble material was pelleted by centrifugation at 15,000 rpm for 1 hr at 4° C. A “load” sample was harvested, then the remaining soluble fraction was applied to 30 μL of pre-equilibrated ANTI-FLAG M2 Affinity Gel (Millipore-Sigma) for 4 hr at 4° C. The resin was pelleted by centrifugation at 2,000 rpm for 1 min, then the “unbound” sample was harvested. The resin was washed three times by centrifugation and resuspension in 1 mL wash buffer (20 mM HEPES pH 7.0, 200 mM NaCl, 5 mM MgCl2, 5% glycerol). All wash buffer was then removed, and the resin was resuspended in 40 μL 2× Laemmli SDS-PAGE loading buffer lacking β-mercaptoethanol, and boiled for 5 min. The resin was pelleted and supernatant was harvested as the “IP” sample. 5% β-mercaptoethanol was added to all samples before separation on 4-20% acrylamide SDS-PAGE gels. For immunoblot analysis, proteins were transferred to a methanol-activated PVDF membrane, blocked with 5% nonfat milk, and probed with anti-His6 (Genscript), anti-Flag (Sigma) and anti-σABacillus subtilis (37) primary antibodies, then with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad). Proteins were detected using Western Lightning chemiluminescence reagent.
Synthetic RNA substrates were radiolabeled as described for RNA cleavage assays. In vitro RNP assembly was performed for 30 min in a 10 μL reaction at room temperature in the presence of 5mM HEPES pH 7, 10 mM NaCl, 1 mM BME, 5 mM MgCl2, 1 μg/mL bovine serum albumin, 10 μg/mL salmon sperm DNA, and 5% glycerol. Labeled RNA substrates were added at a final concentration of 10 nM, dCas13a (R445A, H450A, R1016A, H1021A) at 500 nM, and AcrVIA1 at 1800 nM. Reactions were placed on ice 1 min, then 10 μL of non-denaturing loading dye (25% glycerol, 0.05% xylene cyanol, 0.05% bromophenol blue, 50 mM HEPES pH 7.0) was added, and samples were electrophoretically separated by 10% acrylamide native PAGE at 4° C. Gels were exposed and imaged as described for RNA cleavage assays.
L. seeligeri ΔRM Δspc was infected with ΦLS46 at OD600 of 0.5, MOI of 0.1 in BHI medium containing 5 mM CaCl2 at 30° C. At each time point, 1.5 mL of culture was harvested, pelleted by centrifugation at 8,000 rpm for 2 min, and frozen at −80° C. To harvest RNA, samples were resuspended in 90 μL of RNase-free phosphate-buffered saline containing 2 mg/mL lysozyme, and incubated at 37° C. for 3 min. 10 μL of 10% sarkosyl was immediately added to lyse the cells. 300 μL of TRI Reagent (Zymo Research Direct-Zol RNA Miniprep Plus Kit) was added to each sample, then RNA was prepared according to the manufacturer's instructions, eluting in 50 μL RNase-free water. Ribosomal RNA was removed from 1 μg of purified RNA using the NEBNext rRNA Depletion Kit (Bacteria) according to the manufacturer's instructions. After rRNA removal, samples were concentrated using the Zymo Research RNA Clean and Concentrator-5 Kit according to the manufacturer's instructions, eluting RNA in 6 μL RNase-free water. Libraries were prepared for deep sequencing using the Illumina TruSeq Stranded mRNA Library Preparation Kit, skipping mRNA purification and beginning at the RNA fragmentation step. Quality control of libraries was carried out on an Agilent TapeStation. Paired-end (2×75 bp) sequencing was performed on the NextSeq platform. Raw paired-end reads were mapped to the ΦLS46 genome using Bowtie2 with parameters “very-sensitive” and I=40. Using a custom script, the coverage at each position on the ΦLS46 genome was calculated by tallying a count for each of the positions covered by each mapped read. Read counts at each genomic position were normalized to the total number of reads in each library.
The L. seeligeri type VI CRISPR array alongside Cas13a-His6 or dCas13a-His6 (R445A, H450A, R1016A, H1021A) were cloned into pAM8 as described in Table S4, and conjugated into L. seeligeri Δspc Δcas13a. For expression, these strains were cultured at 30° C. in BHI supplemented with 10 μg/mL chloramphenicol for ˜24 hr. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5% glycerol, 20 mM imidazole, 7 mM β-mercaptoethanol). The harvested cells were then lysed by an EmulsiFlex-C3 homogenizer (Avestin) and centrifuged at 20,000 rpm for 30 min in a JA-20 fixed angle rotor (Avanti J-E series centrifuge, Beckman Coulter). The supernatant was applied to 5 mL HisPur™ Cobalt Resin (Thermo Fisher Scientific). The protein was eluted with lysis buffer supplemented with 500 mM imidazole after washing the column with 10 column volumes of lysis buffer. The elution fractions were further dialyzed against buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7 mM β-mercaptoethanol), and applied on a 1 mL HiTrap SP Fast flow column (GE Healthcare). Proteins were eluted by a linear gradient from 100 mM to 1 M NaCl in 20 column volumes, and then concentrated in 50 kDa molecular mass cut-off concentrators (Amicon) before further purification over a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT).
AcrVIA1 was cloned into a pRSF-Duet-1 vector (Novagen), in which the acrVIA1 gene was attached with N-terminal His6-SUMO tag following an ubiquitin-like protease (ULP1). The vector was transformed into Escherichia coli BL21 (DE3) strain and expressed by induction with 0.25 mM isopropyl-β-D-1-thiogalactopyranoside (GoldBio) at 16° C. for 20 hr. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5% glycerol, 20 mM imidazole, 7 mM β-mercaptoethanol). The harvested cells were then lysed by an EmulsiFlex-C3 homogenizer (Avestin) and centrifuged at 20,000 rpm for 30 min in a JA-20 fixed angle rotor (Avanti J-E series centrifuge, Beckman Coulter). The supernatant was applied to 5 mL HisTrap Fast flow column (GE Healthcare). The protein was eluted with lysis buffer supplemented with 500 mM imidazole after washing the column with 10 column volumes of lysis buffer and 2 column volumes of lysis buffer supplemented with 40 mM imidazole. The elution fractions were further dialyzed against buffer A (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 7 mM β-mercaptoethanol), and applied on a 5 mL HiTrap Q Fast flow column (GE Healthcare). Proteins were eluted by a linear gradient from 100 mM to 1 M NaCl in 20 column volumes, and then concentrated in 10 kDa molecular mass cut-off concentrators (Amicon) before further purification over a Superdex 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT). Leptotrichia buccalis Cas13 purification was conducted as previously described (35), and the same samples were used in this disclosure.
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The following reference listing is not an indication that any of the references are material to patentability:
While the disclosure has been particularly shown and described with reference to specific embodiments, it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
This application claims priority to U.S. Provisional patent application No. 63/004,940, filed on Apr. 3, 2020, the disclosure of which is incorporated herein by reference.
This invention was made with government support under grant no. 1DP1GM128184-01 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/24979 | 3/30/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63004940 | Apr 2020 | US |
