Patent Application
20240131122
The Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 9, 2023 is named SEQ_LIST107648 002 and is 47.7 KB in size. The Sequence Listing does not go beyond the disclosure in the application as filed.
Lyme disease, also known as Lyme borreliosis, is the most prevalent vector-borne disease in North America and Eurasia. It is caused primarily by the spirochete Borrelia burgdorferi and the related Borrelia afzelii and Borrelia garinii species. The disease presents with various symptoms that can include fever, malaise, rash, arthritis, neurological dysfunctions, and cardiac manifestations. Humans are accidental hosts. In nature, B. burgdorferi is typically maintained through a transmission cycle between a vertebrate host reservoir (e.g., white footed mice and other small mammals, but also birds) and an ixodid tick vector. During feeding, B. burgdorferi-colonized tick vectors deliver the spirochetes into vertebrate hosts, where the spirochetes can replicate, disseminate, and often establish persistent infection.
Members of the Borreliaceae family contain the most segmented bacterial genomes known to date. For instance, the genome of the B. burgdorferi type strain B31 is composed of a linear chromosome and 21 linear and circular plasmids. During growth in culture under abundant nutrient condition, the Borreliaceae could be polyploid with each cell carrying multiple copies of both the chromosome and plasmids. The chromosome encodes the vast majority of essential housekeeping and metabolic functions. In contrast, the plasmids primarily encode lipoproteins that mediate the spirochetes' interaction with the vertebrate and tick host environments and help them evade host immune defenses. Additionally, each strain hosts several highly similar plasmid members of the cp32 class, which are prophages. In the B. burgdorferi type strain B31, which is the most well studied genetically, only plasmid cp26 has been shown to be required for growth in axenic culture. Several other plasmids are known to be required in the vertebrate or tick hosts. However, much remains unknown about the roles of B. burgdorferi plasmids. Furthermore, as the number of distinct plasmid types and the genes carried by any given plasmid type vary significantly among Borreliaceae species and strains, strain-to-strain inferences of plasmid function are not always possible.
An effective way to investigate plasmid function is to remove it from a given strain. Spontaneous plasmid loss during extended passaging in axenic culture has been known since the early days of Lyme disease research, but this approach is not specific to a particular plasmid of interest and often results in loss of multiple plasmids. Curing a specific plasmid can be achieved through transformation of B. burgdorferi with a shuttle vector that carries the plasmid maintenance locus of the endogenous plasmid of interest. The incompatibility that arises between the endogenous plasmid and the introduced shuttle vector leads to displacement of the endogenous plasmid by the shuttle vector. However, this approach requires knowledge of the plasmid maintenance locus of the targeted endogenous plasmids.
What is needed are new methods of cleaving genomic DNA such as endogenous virulence plasmids in a bacterial species with a segmented genome such as a Borrelia sp. Such methods could provide an alternative or supplement to antibiotic therapy which is generally less than fully effective and can cause severe side effects.
In one aspect, a method of reducing virulence of a bacterial species with a segmented genome which has infected a mammalian host or mammalian host cell comprises exposing the bacterial genome to an RNA-guided nuclease and a guide RNA (gRNA) and generating a double-stranded or single-stranded break in the bacterial genome, wherein the gRNA base pairs with a target sequence in the bacterial genome. In an aspect, the target sequence in the bacterial genome is in a bacterial chromosome or an endogenous virulence plasmid, such as an endogenous virulence plasmid.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
As described herein, in one aspect, the inventors determined a method to eliminate endogenous plasmids from B. burgdorferi strains would be to generate site-specific DNA lesions. In the absence of efficient DNA repair, those lesions were predicted to lead to degradation of the targeted endogenous plasmid. In the absence of a recombinational donor sequence, exogenously induced double-stranded DNA breaks (DSB s) in the chromosome can be lethal in several bacteria, including Escherichia coli, streptococci, Clostridium cellulolyticum, and the spirochete Leptospira biflexa. Repair of a site-specific DSB in Neisseria gonorrhoeae, when there are no homologous sequences to provide a template for recombinational repair, occurs at such low frequencies that less than one cell in ten thousands survives this type of genome lesion. In contrast, the presence of short (5 to 23 base pairs) homologous sequences flanking an endonuclease-induced DSB led to RecA-mediated repair in a small fraction of cells. Since most B. burgdorferi plasmids are not needed for growth in axenic culture, induction of DNA lesions in B. burgdorferi plasmids should cause plasmid loss if DNA repair is inefficient.
As described herein, to generate such site-specific lesions, the clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 system derived from Streptococcus pyogenes was employed. Cas9 is the endonuclease component of a type of bacterial innate immunity defense against invading foreign DNA molecules. It has two catalytic residues, D10 and H840, each cutting one of the strands of the targeted double stranded DNA sequence. Cas9 targeting to a specific DNA sequence can be achieved by co-expression of a short guide RNA molecule, or gRNA. Base pairing between the Cas9-bound sgRNA and the target DNA sequence next to a protospacer-adjacent motif (PAM) directs the Cas9 activity to the genome location specified by the sgRNA. While wild-type Cas9 (Cas9WT) generates a DSB in the target DNA sequence, single active site mutants (Cas9D10A and Cas9H840A) are nickases that generate single-stranded DNA breaks (SSBs). Finally, the double mutant, catalytically dead Cas9 D10A/H840A, or dCas9, does not create DNA lesions and thus serves as a negative control. dCas9, however, can interfere with transcription when targeted to promoters and promoter-proximal coding region. A dCas9-based CRISPR interference (CRISPRi) platform in B. burgdorferi has been previously reported. Described herein are the effects of targeting Cas9WT and its nickase versions to several B. burgdorferi endogenous plasmid loci.
In an aspect, method of reducing virulence of a bacterial species with a segmented genome which has infected a mammalian host or mammalian host cell comprises exposing the bacterial genome to an RNA-guided nuclease and a guide RNA (gRNA) and generating a double-stranded or single-stranded break in the bacterial genome, wherein the gRNA base pairs with a target sequence in the bacterial genome. In an aspect, the target sequence in the bacterial genome is in a bacterial chromosome or an endogenous virulence plasmid. For example, the genome of B. burgdorferi type strain B31 is composed of a linear chromosome and 21 linear and circular plasmids.
As used herein, a bacterial species with a segmented genome is a bacterial species in which the genome is segmented into several different pieces of genetic material instead of being on one piece of DNA. Segmented genomes can include circular and linear chromosomes, chromids, megaplasmids, as well as smaller plasmids. Exemplary bacterial species with segmented genomes include Borrelia sp., Vibrio sp., Agrobacteria sp., Bacillus sp., Brucella sp., Burkholderia sp., Leptospira sp., Rhizobium sp., and Rhodobacteria sp. Exemplary Borrelia sp. include Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia hermsii, Borrelia turicatae, and Borrelia parkeri. Translation of the methods described herein across bacterial phyla is expected due to the successful implementation of CRISPR-based methods across species.
In an aspect, reducing virulence of a bacterial species comprises targeting a virulence plasmid of the bacterial species. Virulence plasmids are plasmids inside of a bacterium that comprise virulence determinants which enhance the pathogenicity of the bacterium. In the case of Borrelia burgdorferi, virulence plasmids include the lp25, lp28-1, lp36 and cp32 plasmids. Exemplary target genes on lp25 include bbe10 and bbe17. Exemplary target genes on lp28-1 include the vlsE lipoprotein gene, a vls2-vls16 silent cassette, or a non-vls locus, such as bbf03.
The method includes targeting the genome or a bacterial virulence plasmid of the bacterial species with an RNA-guided nuclease and a guide RNA (gRNA) to generate a double-stranded or single-stranded break in the genome or bacterial virulence plasmid. RNA-guided nucleases include, but are not limited to, Class 2 CRISPR nucleases such as Cas9 and Cpf1, as well as other nucleases derived or obtained therefrom. As used herein, the terms Cas9 and Cpf1 include active derivatives of these RNA-guided nucleases. RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, to cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, typically, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM”. RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity.
Various RNA-guided nucleases may require different sequence and spatial relationships between PAMs and protospacers. In general, Cas9 nucleases recognize PAM sequences that are 3′ of the protospacer. Cpf1 generally recognizes PAM sequences that are 5′ of the protospacer.
In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for instance, recognizes a PAM sequence of NNGRRT (R is A or G and N is any nucleotide) or NNGRRV (V is G, C or A), wherein the N residues are immediately 3′ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. And F. novicida Cpf1 recognizes a TTN PAM sequence. PAM sequences have been identified for a variety of RNA-guided nucleases, and a strategy for identifying novel PAM sequences has been described in the art. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of reference molecules.
Examples of polypeptide sequences of Cas9 molecules that may be used according to the embodiments herein are set forth in SEQ ID NOs: 29 (Streptococcus mutans), 30 (Streptococcus pyogenes), 31 (Streptococcus thermophilus), 32 (Listeria innocua), 33 (Staphylococcus aureus), and 34 (Neisseria meningitidis). In certain embodiments, the Cpf1 protein may comprise a sequence selected from the group consisting of SEQ ID NOs. 35 (Acidaminococcus sp. strain BV3L6), 36 (Lachnospiraceae bacterium ND2006), and 37 (Lachnospiraceae bacterium MA2020).
In an aspect, wild-type Cas9 (Cas9WT) generates a DSB in the target DNA sequence. Single active site mutants (Cas9D10A and Cas9H840A) are nickases that generate single-stranded DNA breaks (SSBs). Any of the foregoing Cas9 polypeptides can be used in the methods described herein.
Also included are polynucleotides encoding the RNA-guided nuclease. The polynucleotide can include operably linked nucleotide regulatory sequences (i.e., non-coding sequences) that produce a functional gene. Thus, an RNA-guided nuclease ORF (open reading frame) can be operably linked to regulatory sequences that lead to accurate and efficient transcription and translation of the ORF. These regulatory sequences can include, but are not limited to, enhancer elements, promoter elements, termination sequences (for either transcription or translation), polyadenylation signal sequences and intron/exon splicing sequences. In some embodiments, a polynucleotide encoding the RNA-guided nuclease is contained in an expression vector, which may have other nucleotide sequences including an origin of replication, one or more selectable markers, a visual marker, and/or a site that facilitates manipulation of the vector and insertion or subcloning of additional gene sequences, such as one or more restriction sites or a multiple cloning site (MCS).
In the methods described herein, the RNA-guided nuclease may be delivered as a polynucleotide expressing the RNA-guided nuclease ORF, or as a polypeptide. If the RNA-guided nuclease is delivered as a polynucleotide expressing the RNA-guided nuclease ORF, the polypeptide is expressed after delivery to a cell capable of expressing the ORF.
The terms “guide RNA” and “gRNA” refer to a nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 or a Cpf1 to a target sequence such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric or as a single-guide RNA (sgRNA)), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing).
Guide RNAs, whether unimolecular or modular, include a “targeting domain” that is fully or partially complementary to a target domain within a target sequence, such as a DNA sequence in the genome of a cell where editing is desired. Targeting domains are referred to by various names in the literature, including without limitation “guide sequences”, “complementarity regions”, “spacers” and generically as “crRNAs”. Targeting domains are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for instance, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5′ terminus of in the case of a Cas9 gRNA, and at or near the 3′ terminus in the case of a Cpf1 gRNA.
In addition to the targeting domains, gRNAs typically (but not necessarily, as discussed below) include a plurality of domains that may influence the formation or activity of gRNA/Cas9 complexes. For example, the duplex structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat:anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9 and can mediate the formation of Cas9/gRNA complexes.
In an aspect, an RNA-guided nuclease binds a guide RNA and is targeted to a specific sequence (a target site) in a target nucleic acid. An RNA-guided nuclease is targeted at a target site by the Cas9 guide RNA to which it is bound. The guide RNA comprises a sequence that is complementary to a target sequence within the target nucleic acid, thus targeting the bound RNA-guided nuclease to a specific location within the target nucleic acid (the target sequence) (e.g., stabilizing the interaction of RNA-guided nuclease with the target nucleic acid).
As used herein, when the bacterial virulence plasmid is the lp25 plasmid, the spacer sequence of the guide RNA may comprise SEQ ID NO: 1 or SEQ ID NO: 2. When the bacterial virulence plasmid is the lp28-1 plasmid, the spacer sequence of the guide RNA may comprise any of SEQ ID NOs. 3-6. The gRNA may comprise any sequence adjacent to a PAM that is not found elsewhere in the target genome.
The gRNA can be delivered in the form of an expression cassette or as RNA. RNA can be prepared in an in vitro transcription reaction. In some embodiments, generating in vitro transcribed RNA comprises incubating a linear DNA template with an RNA polymerase and a nucleotide mixture under conditions to allow (run-off) RNA in vitro transcription. The nucleotide mixture can be part of an in vitro transcription mix (IVT-mix). In some embodiments, the RNA polymerase is a T7 RNA polymerase. In some embodiments, the nucleotide mixture is composed of (chemically) non-modified ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP. In some embodiments, the in vitro transcription can include the presence of modified nucleotides as is known in the art.
In an aspect, a gene encoding the RNA-guided nuclease and an expression cassette for the gRNA are carried on a vector comprising the expression cassette for expressing the gRNA and an inducible expression cassette for expressing the RNA-guided nuclease. In an aspect the vector is a shuttle vector that can be replicated in E. coli and the bacterial species with a segmented genome. Shuttle vectors are replicating DNA plasmids that can express both the RNA-guided nuclease and gRNA in the host bacterial strain.
In another aspect, an inducible expression cassette for expressing the RNA-guided nuclease and an expression cassette for the gRNA are encoded in the DNA of a bacteriophage. The term “phage” is interchangeable with the term “bacteriophage” and refers to a virus that infects bacterial cells. Phages include an outer protein capsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA, in either linear or circular form. Phages and phage vectors are well known to those of skill in the art and non-limiting examples of phages are λ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174, N4, Φ6, and Φ29. Bacteriophages allow efficient delivery of DNA sequence-specific RNA-guided nuclease antimicrobials into bacteria in vivo as a therapeutic option to treat bacterial infections, for example.
In another aspect, the RNA-guided nuclease and gRNA can be packaged and delivered in a liposome or nanoparticle, such as a lipid nanoparticle (LNP), in the form of plasmid DNA or in vitro transcribed RNA.
In another aspect, the pre-assembled RNA-guided nuclease and gRNA are formulated in a liposome or nanoparticle such as a LNP.
In another aspect, the RNA-guided nuclease and gRNA can be packaged and delivered in extracellular vesicles (EVs).
“Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Polynucleotides associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
A LNP is a non-viral delivery system that safely and effectively delivers nucleic acids to a target. The term “lipid nanoparticle” refers to a nanoscopic particle composed of lipids having a size measured in nanometers (e.g., 1-5,000 nm). In some embodiments, the lipids comprised in the lipid nanoparticles comprise cationic lipids and/or ionizable lipids. Cationic lipids and/or ionizable lipids known in the art can be used to formulate LNPs for delivery of gRNA and RNA-guided nuclease to the cells. Exemplary cationic lipids include one or more amine group(s) bearing positive charge. In some embodiments, the cationic lipids are ionizable such that they can exist in a positively charged or neutral from depending on pH. In some embodiments, the cationic lipid of the lipid nanoparticle comprises a protonatable tertiary amine head group that shows positive charge at low pH. The lipid nanoparticles can further comprise one or more neutral lipids (e.g., Di stearoylphosphatidylcholine (DSPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) etc. as a helper lipid), charged lipids, steroids, and polymers conjugated lipids. In some embodiments, the LNP can comprise cholesterol. In some embodiments, the LNP can comprise a polyethylene glycol (PEG) lipid.
In some embodiments, the molar percent of an ionizable lipid in the total lipid of a lipid nanoparticle is about, at least, at least about, at most or at most about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or a number or range between any two of these values. In some embodiments, the molar percent of an ionizable lipid in a lipid nanoparticle is in a range between about 40-70% (e.g., about 60%). In some embodiments, the lipid nanoparticle can further comprise a helper lipid (e.g., DSPC), a sterol lipid (e.g., cholesterol), and PEG lipid or a phospholipid PEG conjugate. In some embodiments, the molar percent of a helper lipid in a lipid nanoparticle is about 5%-20% (e.g., about 10.5%), the molar percent of a sterol lipid is about 10%-40% (e.g., about 21%), and the molar percent of a PEG lipid is about 0.5%-10% (e.g., about 8.5%).
EVs can be generated by organisms and function in cell-to-cell communication by transferring biomacromolecules (e.g. nucleic acids, proteins, lipids, glycoproteins). Borrelia sp. generate EVs that contains biomacromolecules and methods for EV isolation from Borrelia have been documented.
In an aspect, exposing the bacterial genome comprises administering the RNA-guided nuclease and gRNA to a mammalian subject. In an aspect, the mammal is a human, or a rodent species, for example.
Advantageously, the treatment method described herein is expected to be effective in all stages of infection. For example, in Lyme disease, stage one is localized Lyme disease in which the bacteria have not yet spread throughout the body. Stage 2 is early disseminated Lyme disease in which the bacteria have begun to spread through the body. And stage 3 is late disseminated Lyme disease in which the bacteria have spread to more distant sites such as joints and nerves.
The treatment described herein can be combined with administration of an antibiotic such as doxycycline, amoxicillin, cefuroxime, ceftriaxone, hygromycin A, and the like, and combinations thereof.
The invention is further illustrated by the following non-limiting examples.
E. coli strains and growth conditions: E. coli host strain 5-alpha F′ 1q (New England Biolabs) was exclusively used to generate, store, and amplify the E. coli/B. burgdorferi shuttle vectors listed in Table 2. The resulting strains were grown on LB agar plates or in Super Broth (35 g/L bacto-tryptone, 20 g/L yeast extract, 5 g/L NaCl, and 6 mM NaOH) liquid medium with shaking at 30° C. Transformation was achieved by heat shock followed by recovery in SOC medium (New England Biolabs) for 1h at 30° C. with shaking. Antibiotic selection was achieved using spectinomycin at 50 μg/mL or rifampin at 25 μg/mL in liquid culture or 50 μg/mL in plates.
B. burgdorferi strains and growth conditions: Previously described B. burgdorferi strain B31-A3-68-Δbbe02::PflgB-aphI, also known as K2, is an infectious, highly transformable derivative of the type strain B31. To derive strain CJW_Bb471 from K2, pseudogene bbf29 of plasmid lp28-1 was disrupted by insertion of a gentamicin resistance cassette. Strains K2 and CJW_Bb471 contain 18 of the 21 endogenous plasmids of parental strain B31; they both lack endogenous plasmids cp9, lp5, and lp56. To generate strain CJW_Bb471, 75 μg of plasmid p28-1::flgBp-aacC1 were digested with AgeI-HF (New England Biolabs), ethanol precipitated, resuspended in 25 μL water, and electroporated into a 100 μL aliquot of K2 electrocompetent cells. Electroporated cells were immediately transferred to 6 mL complete Barbour-Stoenner-Kelly (BSK)-II medium and allowed to recover overnight. The following day, cells were plated in semisolid BSK-agarose medium under kanamycin and gentamicin selection. A clone was grown and confirmed to have correct insertion of the gentamicin resistance cassette into lp28-1 and to contain all the endogenous plasmids of the parental strain.
B. burgdorferi strains were grown in complete BSK-II medium at 34° C. in a humidified 5% CO2 incubator. BSK-II medium contained 50 g/L Bovine Serum Albumin, Universal Grade (Millipore), 9.7 g/L CMRL-1066 (US Biological), 5 g/L Neopeptone (Difco), 2 g/L Yeastolate (Difco) 6 g/L HEPES (Millipore), 5 g/L glucose (Sigma-Aldrich), 2.2 g/L sodium bicarbonate (Sigma-Aldrich), 0.8 g/L sodium pyruvate (Sigma-Aldrich), 0.7 g/L sodium citrate (Fisher Scientific), 0.4 g/L N-acetylglucosamine (Sigma-Aldrich), 60 mL/L heat-inactivated rabbit serum (Gibco), and had a pH of 7.6. For plating in semisolid BSK-agarose medium, each 10-cm plate was seeded with up to 1 mL B. burgdorferi culture. BSK-agarose plating medium was made by mixing two volumes of 1.7% agarose in water, melted and pre-equilibrated at 55° C. with three volumes of BSK-1.5 medium, also briefly (for less than 5 min) pre-equilibrated at 55° C. and containing appropriate amounts of antibiotics. Then, 25 mL of the BSK-agarose mix was added to each seeded plate, which was then gently swirled and allowed to solidify for approximately 30 min at room temperature in a biosafety cabinet. The plates were then transferred to a humidified 5% CO2 incubator kept at 34° C. BSK-1.5 medium contained 69.4 g/L BSA, 12.7 g/L CMRL-1066, 6.9 g/L Neopeptone, 3.5 g/L Yeastolate, 8.3 g/L HEPES, 6.9 g/L glucose, 6.4 g/L sodium bicarbonate, 1.1 g/L sodium pyruvate, 1.0 g/L sodium citrate, 0.6 g/L N-acetylglucosamine, 40 mL/L heat-inactivated rabbit serum, and had a pH of 7.5. Antibiotics were used at the following concentrations: streptomycin at 100m/mL, gentamicin at 40 μg/mL, and kanamycin at 200m/mL. Unless otherwise indicated, B. burgdorferi cultures were maintained in exponential growth by diluting cultures into fresh medium before cultures densities reached approximately 5×10 7 cells/mL. Cell density of cultures was determined by direct counting under darkfield illumination using disposable hemocytometers, as previously described in the art.
B. burgdorferi transformation, clone isolation, and characterization. Electrocompetent cells were generated as previously described and stored as single use 50 or 100 μL aliquots at −80° C. For shuttle vector transformations, 30 or 50 μg of plasmid eluted in water were electroporated (2.5 kV, 25 1.4F, 200 Ω, 2 mm gap cuvette) into 50 μL aliquots of competent cells. Electroporated cells were immediately transferred to 6 mL BSK-II and allowed to recover overnight. The next day, 100, 300, and 900 μL aliquots of the culture were each plated in semisolid BSK-agarose under selection. The remaining culture was diluted 6-fold in BSK-II and selected in liquid culture with appropriate antibiotics. Once transformants were observed as motile spirochetes, the liquid cultures were plated for clone isolation. Agarose plugs containing individual colonies were used to inoculate 6 mL BSK-II cultures. After 3 days, 500 to 1000 μL of each clonal culture was removed and pelleted at 10,000×g for 10 min, the cells were resuspended and lysed in 50-100 μL water, and the resulting solution was used to perform multiplex PCR using primer pairs specific for each endogenous plasmid of strain B31 and the DreamTaq™ Green DNA Polymerase (Thermo Scientific). For genomic DNA extraction, approximately 14 mL cultures were grown to approximately 10 8 cells/mL and then pelleted at 4,300×g for 10 min at room temperature in a Beckman Coulter X-14R centrifuge equipped with a swinging bucket rotor. The media was removed and the pellet was processed for DNA extraction using QIAGEN's DNeasy® Blood & Tissue Kit protocol for Gram-negative bacteria. Final elution was carried out in 10 mM Tris-HCl, 0.1 mM EDTA, pH 9.0.
Generation of E. coli/B. burgdorferi shuttle vectors for Cas9 and sgRNA expression: Table 2 lists the E. coli/B. burgdorferi shuttle vectors used or generated in this study. They were based on the previously described B. burgdorferi CRISPR interference platform. The shuttle vectors express one of the following Cas9 versions: wild-type Cas9, the nickases Cas9D10A or Cas9H840A, or the catalytically inactive dCas9 that carries both the D10A and H840A mutations. To revert the D10A mutation, site-directed mutagenesis was performed on appropriate template plasmids using Agilent's QuickChange® Lightning Site-Directed Mutagenesis kit and primers NT651 and NT652. To revert the H840A mutation, site-directed mutagenesis was performed on appropriate template plasmids using primers NT749 and NT750. To generate plasmids with decreased basal expression of Cas9 proteins, site-directed mutagenesis was performed on appropriate plasmid templates using primers NT669 and NT670, which generated a weakened ribosomal binding site (“RB Smut” constructs), or primers NT677 and NT678, which introduced a mutation in the −10 region of the Cas9 promoter (“-10TC” constructs). Expression cassettes for the sgRNAs were moved among plasmids using restriction endonucleases AscI and EagI. To generate sgRNA expression cassettes, SapI-digested Psyn-sgRNA500-containing plasmids were ligated with annealed primer pairs, as follows: primers NT657 and NT658 generated sgRNAvlsE1; NT660 and NT661 generated sgRNAvlsE2; NT721 and NT722 generated sgRNAvls11; NT723 and NT724 generated sgRNAbbe10; NT725 and NT726 generated sgRNAbbe17; and NT727 and NT728 generated sgRNAbbf03. Primer annealing was achieved by mixing 10 μL volumes of each primer at 5 μM concentration, then cycling the mix five times between 30 s at 95° C. and 30 s at 55° C., followed by cooling to room temperature. Nucleotide sequences of primers used to generate the E. coli/B. burgdorferi shuttle vectors in this study are given in Table 6.
DNA sequence analysis: To determine the sequence of the vls locus of B. burgdorferi strain K2, the 10910 base pair region encompassing vlsE and silent cassettes vls2-vls16 was amplified using Platinum™ SuperFi™ DNA Polymerase (Thermo Fisher Scientific) and primers YN-LI_266 and YN-LI_267 (Table 6) and then sequenced with a SMRT Cell™ using 10-h data collection (Pacific Biosciences). The resulting reads were subjected to read-of-insert (ROI) analysis using SMRT Link v6.0.0 (Pacific Biosciences), followed by multiple sequence alignment, to obtain the final consensus sequence.
The previous report establishing CRISPRi in B. burgdorferi relied in part on all-in-one E. coli 1 B. burgdorferi shuttle vectors that carry a constitutive sgRNA expression cassette as well as an isopropyl β-D-1-thioglactopyranoside (IPTG)-inducible dCas9 expression cassette. Using these CRISPRi shuttle vectors or control vectors that lack the sgRNA as background, we generated vectors (
In separate cultures, Cas9WT or its nickase versions were targeted to two endogenous B. burgdorferi plasmids, lp25 and lp28-1 (
B.
burgdorferi
For these experiments, strain B31-A3-68 Δbbe02::PflgB-aphI, also known as K2, a transformable, clonal, infectious derivative of the type strain B31, was used. A mouse passage occurred during the derivation of strain K2 from the parental, sequenced B31 strain. During that mouse passage, gene conversion events likely changed the vlsE sequence. The entire vls locus of strain K2 was sequenced using long read single-molecule, real-time (SMRT) sequencing to obtain an accurate sequence encompassing the expressed vlsE gene and the repetitive silent vls cassettes. The sequence of the silent vls cassette region was identical to the B. burgdorferi B31 reference sequence (GenBank accession number AE000794.2). In contrast, the sequence of the vlsE gene of strain K2 had indeed diverged from the parental B31 vlsE, as expected. Five clusters of changes were detected that could be attributed to segmental gene conversion events in which the original sequence was replaced by segments copied from the vls2-vls16 silent cassette sequences (
E. coli/B. burgdorferi shuttle vectorsa used in this study
aNaming of the E. coli/B. burgdorferi shuttle vectors follows the nomenclature established and described in detail in Takas 2012. Of note, Cas9 variant expression is driven either by the IPTG-inducible PpQE30 promoter or by its mutant versions in which the −10 region of the promoter (−10 TC) or the ribosome binding site (RBSmut) were mutated to reduce basal Cas9 expression;
bWhen requesting a plasmid from the Jacobs- Wagner lab, please include the CJW strain number alongside the plasmid name. For constructs previously published in Takas 2012, a CJW strain number is not provided, as the plasmids are available from Addgene. Refer to the original publication for the Addgene catalog numbers;
cSm/Sp, streptomycin/spectinomycin resistance conferred by the aadA gene; Rf, rifampin resistance conferred by the arr2 gene; Gm, gentamicin resistance conferred by the aacC1 gene.
The shuttle vectors described above were electroporated into strain K2. As controls, shuttle vectors lacking the sgRNA cassette and shuttle vectors expressing dCas9 rather than Cas9WT were used (Table 2). For each construct, the electroporated cells were plated after about three generations, a small number of the resulting clones were grown, and their endogenous plasmid content was determined by multiplex PCR, as described in the art. All clones that had received a shuttle vector expressing Cas9WT and the vlsE-targeting sgRNAvlsE1 had lost the vlsE-carrying plasmid lp28-1 (Table 3,
B. burgdorferi
bData was aggregated based on the Cas9 version and the sgRNA expressed by the shuttle vector. Transformed strains carrying the same sgRNA but expressing different basal levels of the Cas9 variant were analyzed together. Plasmid detection was achieved by multiplex PCR;
bData compares the number of endogenous plasmids detected in the analyzed clones with the expected number of endogenous plasmids if they had all been retained. All plasmid counts are combined for the non- targeted plasmids. A total of 18 non-targeted plasmids were assayed for each clone obtained by transformation with a shuttle vector lacking a sgRNA. A total of 17 non-targeted plasmids wereassayed for each clone obtained by transformation with a shuttle vector expressing a sgRNA;
cN/A, not applicable.
Performing multiplex PCR assays on individual clones is relatively labor-intensive. Additionally, if Cas9WT-mediated plasmid loss is not 100% effective, the fraction of cells that still retain the targeted plasmid might be below detection. To avoid these drawbacks, endogenous plasmid retention was quantified by plating electroporated B. burgdorferi populations under differential antibiotic selection. In these plating assays, strain K2 was used, in which retention of plasmid lp25 allows colony formation in the presence of kanamycin. Additionally, strain CJW Bb471 was derived from strain K2 by inserting a gentamicin resistance cassette in its lp28-1 plasmid. This genetic modification does not interfere with B. burgdorferi's ability to infect mice or be acquired by ticks. Plating CJW Bb471 transformants in the presence of kanamycin assays retention of lp25, while plating in the presence of gentamicin examines retention of lp28-1. In both cases, acquisition of streptomycin resistance indicates successful delivery of the Cas9-expressing shuttle vector. The number of streptomycin-resistant transformants detected in these experiments varied significantly both within an experiment and between experiments (
dDifferent volumes of transformant cultures were plated under streptomycin selection (which selects for the shuttle vector), or streptomycin + kanamycin selection (which selects for lp25). Colonies were counted after 2-3 weeks and the resulting count was used to calculate the concentration of selectable cells in the parental population of transformants, expressed as colony forming units (CFU) per mL;
eRetention of both lp25 and lp28-1 was assayed in experiment 3 following electroporation of the indicated constructs. For this reason, results from this experiment are presented in both Tables 4 and 5.
fDifferent volumes of transformant cultures were plated under streptomycin selection (which selects for the shuttle vector), or streptomycin + gentamicin (which selects for lp28-1). Colonies were counted and the resulting count was used to calculate the concentration of selectable cells in the parental population of transformants, expressed as colony forming units (CFU) per mL;
gRetention of both lp25 and lp28-1 was assayed in experiment 3 following electroporation of the indicated constructs. For this reason, results from this experiment are presented in both Tables 4 and 5.
While Cas9WT robustly and specifically induced plasmid loss when targeted to lp25 or lp28-1 (Tables 3-5,
Due to constitutive Cas9-targeting, the use of the original Cas9 construct (Table 2,
Targeting dCas9 to selected B. burgdorferi genes causes specific and efficient downregulation of gene expression, allowing for relatively easy and fast strain generation and phenotypic investigation. As shown herein, targeting Cas9WT or its nickase variants to plasmid-encoded loci results in plasmid loss, though to a varying degree (Tables 3-5 and
Importantly, targeting Cas9WT to an endogenous B. burgdorferi plasmid is an easy and efficient method to displace the plasmid. The Cas9 nickases can also be used to achieve this outcome, but they are less effective. The Cas9-based approach provides an alternative to the previously developed method that displaces endogenous plasmids through introduction of shuttle vectors belonging to the same plasmid compatibility class. Both methods yield clones in which the targeted endogenous plasmid is replaced by a shuttle vector that carries an antibiotic resistance marker. The Cas9-based method, however, does not require prior knowledge of the targeted plasmid's replication and segregation locus, and involves only an easy cloning step to insert the sgRNA sequence into the Cas9 shuttle vector. Additionally, as Cas9 activity can be simultaneously targeted to multiple locations in the genome by co-expression of relevant sgRNAs, simultaneous removal of multiple plasmids from a B. burgdorferi strain should be achievable via a single transformation.
While the degree of genome segmentation in Borreliaceae is the highest among the known bacteria, other bacteria have segmented genomes that can include circular and linear chromosomes, chromids, megaplasmids, as well as smaller plasmids. Plasmids often encode virulence factors or antibiotic resistance genes and are stably maintained by highly effective plasmid segregation mechanisms that ensure faithful inheritance by daughter cells over generations. The study of plasmid-encoded functions in bacteria other than the Lyme disease spirochetes can therefore be facilitated by implementation of a Cas9-mediated plasmid curation protocol. Translation of this approach across bacterial phyla is likely feasible, as demonstrated by the successful broad implementation of CRISPR-based methods of gene regulation.
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims priority to U.S. Provisional Application 63/407,955 filed on Sep. 19, 2022, which is incorporated herein by reference in its entirety.
This invention was made with government support under GM127029 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63407955 | Sep 2022 | US |
