Patent Application
20210071159
Current antimicrobial paradigms, such as antibiotics, broadly target microbial processes and cannot be specifically targeted against virulence factors or antibiotic-resistance genes, resulting in selection and dissemination of resistance among virulent and non-virulent organisms. Thus, this paradigm is always playing catch-up, as microbial consortia constantly trade and evolve traits to resist sporadic broad-spectrum treatments. As antibiotic resistance continues to spread and multidrug resistant strains continue to emerge, the production of novel antibiotic compounds remains behind.
Recent efforts to compensate for the lag in antibiotic production are directed to synthetic bacterial systems that rely on signal detection of pathogens and respond by producing antimicrobial agents (e.g., quorum-signaling molecules or pyocins). These current synthetic systems cannot directly target existing and emerging virulence factors or antibiotic resistance genes and, thus, are not useful for combating, for example, recombinant biothreat genes (e.g., genes that, when expressed, may be harmful), which may be engineered to be expressed by otherwise innocuous organisms such as probiotic strains of bacteria.
Various aspects and embodiments of the present disclosure relate to the discovery that engineered autonomously distributed circuits containing programmable nucleases (i.e., “programmable nuclease circuits”) can be delivered to microbial organisms in vivo to, for example, mediate sequence-specific elimination of microbial organisms that contain a target gene of interest (e.g., a gene that is harmful to humans). Some embodiments of the present disclosure relate to engineered variants of the Type II CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) system of Streptococcus pyogenes, which can be used in accordance with the present disclosure to reverse antibiotic resistance in, or specifically destroy, a wide range of microbial organisms. Other programmable nucleases that can be used in accordance with the present disclosure include other CRISPR-Cas systems, engineered TALEN (Transcription Activator-Like Effector Nuclease) variants and engineered zinc finger nuclease (ZFN) variants. Thus, the methods and engineered autonomously distributed circuits provided herein, in some embodiments, may be used to weaken resistance of microbial pathogens to existing antibiotics.
These programmable nuclease circuits may be delivered to microbial organisms using a variety of delivery methods. For example, in some embodiments, programmable nuclease circuits may be delivered to microbial organisms using recombinant bacteriophage and/or phagemids (also referred to as phagemid particles), which may specifically target select microbial cell populations in vivo without infecting, for example, neighboring cells (e.g., non-microbial cells). In other embodiments, broad-host-range bacteriophage and/or phagemid particles may be used to deliver programmable nuclease circuits to microbial organisms, whereby specificity of activity is conferred within the design of the nuclease circuits. In yet other embodiments, programmable nuclease circuits may be delivered to microbial organisms via conjugation using probiotic bacteria, wherein the vectors may be mobilizable such that they can be delivered from a donor to a recipient cell or self-transmissible such that they broadly propagate through a population. It should also be appreciated that programmable nuclease circuits may be delivered to microbial organisms using chemical-based delivery vehicles (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes) or nanoparticle-based delivery vehicles (or platforms). In still other embodiments, a combination approach may be taken for delivery, such as, but not limited to, using bacteriophage or nanoparticles to deliver self-transmissible plasmids to a recipient population. Other delivery vehicles/platforms are contemplated herein, including, without limitation, non-chemical-based delivery vehicles (e.g., electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment), and viral-based delivery vehicles (e.g., use of viruses as carriers). The choice of delivery vehicle/platform will depend, in part, on the type of cell to which the programmable nuclease circuits are delivered as well as the environment of the cells (e.g., in vivo v. in vitro).
Provided herein, in some aspects, is a transformative platform for targeted biothreat (e.g., antibiotic resistance) neutralization with engineered autonomously distributed circuits, or programmable nuclease circuits. The programmable nuclease circuits of the present disclosure enable, in some embodiments, sequence-specific destruction of any gene or nucleotide sequence of interest, including biothreat-causing virulence genes and antibiotic-resistance genes. The circuits can be engineered to specifically attenuate undesirable genes and/or to kill the target host pathogen. In other embodiments, the targeted gene or nucleotide sequence may be used to identify and/or eliminate a particular strain or broader grouping of bacteria (e.g., housekeeping genes, 16S rDNA, and other conserved DNA sequences). This platform can be efficiently and rapidly encoded into synthetic constructs for broad delivery and dissemination in microbial populations via the same mechanisms leveraged by biothreat pathways (e.g., conjugation and bacteriophages) and novel carriers (e.g., artificial gene delivery agents). Thus, the present disclosure is a paradigm shift in combating pathogens because it enables, inter alia, autonomous and distributed neutralization of biothreats at the gene level.
Various aspects of the present disclosure provide methods that comprise delivering to microbial cells (e.g., bacterial cells) in vivo a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit, which contains a programmable nuclease that targets a gene or nucleotide sequence of interest. In some embodiments, a nucleic acid encoding an engineered autonomously distributed circuit is stably expressed, while in other embodiments, a nucleic acid encoding an engineered autonomously distributed circuit is transiently expressed. In some embodiments, expression of a nucleic acid encoding an engineered autonomously distributed circuit is inducible.
Some aspects of the present disclosure provide phagemids that comprise an engineered autonomously distributed circuit, which contains at least one nucleic acid encoding a programmable nuclease that targets a gene or nucleotide sequence of interest.
Other aspects of the present disclosure provide bacteriophages that comprise an engineered autonomously distributed circuit, which contains at least one nucleic acid encoding a programmable nuclease that targets a gene or nucleotide sequence of interest.
Also provided in various aspects of the present disclosure are donor bacterial cells (e.g., probiotic bacterial cells and/or commensal bacterial cells) that comprise an engineered autonomously distributed circuit, which contains at least one nucleic acid encoding a programmable nuclease that targets a gene or nucleotide sequence of interest.
Additional aspects of the present disclosure provide nucleic acids encoding an engineered autonomously distributed circuit, which contains a programmable nuclease that targets a virulence factor or an antibiotic resistance gene. Also provided herein are phagemids and bacteriophages that comprise nucleic acids encoding engineered autonomously distributed circuits.
Yet other aspects of the present disclosure provide libraries that comprise a plurality of nucleic acids, each encoding an engineered autonomously distributed circuit, which contains a programmable nuclease that targets a virulence factor or an antibiotic resistance gene.
Various aspects of the present disclosure also provide methods that comprise culturing the microbial cells (e.g., bacterial cells) from a sample, delivering to the cultured microbial cells a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit, which contains a programmable nuclease that targets a gene or nucleotide sequence of interest, and determining whether or not the microbial cells that contain the programmable nuclease survive, wherein death of the microbial cells indicates that the sample comprises microbial cells with the gene of interest. In some embodiments, the sample is of a subject (e.g., a patient such as a human patient). In some embodiments, the sample is biological fluid sample (e.g., urine, blood (plasma or serum), mucosa), a tissue sample or a cell sample.
In some embodiments, the methods further comprise diagnosing the subject (e.g., a human). In some embodiments, the methods further comprise treating the subject (e.g., a human).
In some embodiments, the programmable nucleases provided herein are RNA-guided nucleases. Thus, in some embodiments, the engineered autonomously distributed circuits provided herein contain a Cas9 nuclease, a guide RNA (gRNA), and/or a transactivating small RNA (tracrRNA), with some or all regions derived from a CRISPR-Cas system (e.g., Type II CRISPR-Cas system of Streptococcus pyogenes). In some embodiments, the gRNA and the tracrRNA are each encoded separately (either on the same nucleic acid or different nucleic acids) to produce separate molecules. In some embodiments, the gRNA and the tracrRNA are encoded by a single nucleic acid to produce a chimeric (e.g., hybrid) single-guide RNA molecule (Jinek, M., et al. Science 337, 816-821 (2012)).
In other embodiments, the programmable nucleases are transcriptional activator like (TAL)-effector nucleases (TALENs).
In yet other embodiments, the programmable nucleases are zinc finger nucleases (ZFNs).
In some embodiments, the microbial cells are Gram-negative bacterial cells, Gram-positive bacterial cells, or a combination thereof.
In some embodiments, the microbial cells are pathogenic bacterial cells. For example, the pathogenic microbial cells may be extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumonia, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii, MDR Enterobacter spp. bacterial cells or a combination thereof. Non-ESBL microbial cells are also contemplated herein. In some embodiments, the microbial cells are non-pathogenic bacterial cells (e.g., probiotic and/or commensal bacterial cells). In some embodiments, the microbial cells form microbial flora (e.g., natural human microbial flora). Thus, the present disclosure contemplates microbial cells that are members of the phyla Actinobacteria, Bacteroidetes, Proteobacteria, Firmicutes, or others, or a combination thereof. In some embodiments, the microbial cells are used in industrial or environmental bioprocesses (e.g., bioremediation).
In some embodiments, the gene of interest is located episomally, while in other embodiments, the gene of interest is located chromosomally. In yet other embodiments, the gene of interest is located extracellularly (e.g., in the environment, in a neighboring cell, or in a bacteriophage) and is targeted upon receipt via horizontal gene transfer.
In some embodiments, the gene of interest is a virulence factor gene or a toxin gene. In other embodiments, the gene of interest is an antibiotic resistance gene. In yet other embodiments, the gene of interest is a remodeling gene. In still other embodiments, the gene of interest is a modulatory gene.
In some embodiments, the delivery vehicles are bacteriophages or engineered particles derived from bacteriophages. For example, in some embodiments, the delivery vehicles are phagemids.
In other embodiments, the delivery vehicles are probiotic bacterial cells comprising a conjugative plasmid.
In some embodiments, the programmable nucleases are operably linked to an inducible promoter.
Also provided herein are methods that comprise delivering to microbial cells in vivo (a) a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest, and (b) a nucleic acid encoding an SOS-responsive reporter construct.
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing.
Antimicrobial resistance is a major global problem exacerbated by global travel, antibiotic overuse, horizontal gene transfer, bacterial evolution and a dearth of new antimicrobial agents (Boucher, H. W., et al. Clinical Infectious Diseases 48, 1-12 (2009)). Troublesome pathogens include vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli and Klebsiella pneumoniae, and multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. New classes of antimicrobials are needed, but few are being developed (Walsh, C. Nat Rev Microbiol 1, 65-70 (2003)). The antimicrobial pipeline is especially dry for MDR Gram-negative bacteria (Boucherm, H. W., et al. (2009)), resulting in sicker patients, longer hospitalizations, increased morbidity and mortality, and high economic costs (Hall-Stoodley, L., et al. Nat Rev Microbiol 2, 95-108 (2004); Wise, R. J. Antimicrob Chemother. 54, 306-310 (2004); Hall, B. G. Nat Rev Microbiol 2, 430-435 (2004)). Thus, there is a large unmet need for new therapeutic agents to treat MDR bacterial infections.
In addition to natural evolutionary processes, exponential improvements in DNA synthesis technologies enable the accelerated capability to engineer new pathogens or revive old pathogens. A fundamental challenge of detecting and neutralizing engineered biothreats is that they are linked to underlying genes (e.g., toxin genes, virulence genes and antibiotic-resistance genes), which can be readily mobilized into new cellular backgrounds.
Engineered autonomously distributed circuits containing programmable nucleases (i.e., “programmable nuclease circuits”) provide a platform for protecting against, inter alia, toxin genes, virulence genes and antibiotic-resistance genes.
In some embodiments, the programmable nuclease circuits of the invention use components of the clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene. In some embodiments, the programmable nuclease circuits of the present disclosure use components of the Type II CRISPR-Cas system to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene. CRISPR-Cas systems are used by various bacteria and archaea to mediate defense against viruses and other foreign nucleic acid. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats and serve as a ‘memory’ of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. Recent work has shown that Type II CRISPR-Cas systems can be engineered to direct targeted double-stranded DNA breaks in vitro to specific sequences. Programmable RNA-guided nucleases (RGNs) consist of two components: a short ˜100 nucleotide single guide RNA (gRNA), containing 20 variable nucleotides at the 5′ end involved in base pairing with a target DNA sequence, and the Cas9 nuclease, which cleaves the target DNA (Jinek, M., et al. Science 337, 816-821 (2012)).
The CRISPR-Cas system of Streptococcus pyogenes enables effective, programmable RGNs for genome editing in human, zebrafish, and microbes (Mali, P., et al. Science 339, 823-826 (2013); Jinek, M., et al. Science 337, 816-821 (2012); Jiang, W., et al. Nat Biotechnol 31, 233-239 (2013); Hwang, W. Y., et al. Nat Biotechnol 31, 227-229 (2013); Deltcheva, E., et al. Nature 471, 602-607 (2011); Cong, L., et al. Science 339, 819-823 (2013); Cho, S. W., et al. Nat Biotechnol 31, 230-232 (2013); Bikard, D., et al. Cell Host & Microbe 12, 177-186 (2012)). The specificity of CRISPR-Cas is dictated by the identity of spacer sequences flanked by direct repeats encoded in the CRISPR locus, which are transcribed and processed into mature guide RNAs (gRNA) (Jinek, M. et al. (2012)). With the aid of a trans-activating small RNA (tracrRNA), gRNAs license the Cas9 endonuclease to introduce double-stranded breaks in target DNA sequences (protospacers) (Jinek, M. et al. (2012); Bikard, D., et al. (2012)). Thus, through simple modifications of spacers in the CRISPR loci, an RGN can direct cleavage of almost any DNA sequence with the only design restriction being a NGG motif immediately 3′ of the protospacer (Jinek, M. et al. (2012)). Natural CRISPR loci have been discovered with up to 58 spacers to simultaneously license Cas9 to induce double stranded breaks at 58 different DNA sequences (Gunderson, F. F., et al. mBio 4(2013)). Thus, a single programmable nuclease circuit may be engineered to target at least 58 different DNA sequences, as described elsewhere herein.
In some embodiments of the present disclosure, the engineered autonomously distributed circuits of the present disclosure contain components of the CRISPR-Cas system, including a Cas9 nuclease, a guide RNA (gRNA) (also referred to herein as CRISPR RNA (crRNA)), and a trans-activating small RNA (tracrRNA) (e.g., dual-guide system). In some embodiments, this system is employed using a Cas9 nuclease and a chimeric single-guide RNA (sgRNA) that combines the crRNA and tracrRNA into a single transcript (referred to herein as a “single-guide system”) (Jinek, M., et al. Science 337, 816-821 (2012)).
In some embodiments, the programmable nuclease circuits of the present disclosure use components of the TAL/TALEN system to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene. Transcriptional activator-like (TAL) effectors are proteins that can be engineered to target specific gene sequences. The genes were originally discovered in a bacterial plant pathogen that uses the TAL effectors to alter host gene expression. TAL effector nucleases (TALENs) are novel classes of sequence-specific nucleases, created by the fusion of a TAL effector (TALE) DNA binding domain to the catalytic head of an endonuclease (N). For example, when a TALE is fused to a nuclease known as FokI, the resulting TALEN can dimerize with a second TALEN and cleave DNA flanked by left and right target sites (Christian, M. et al. Genetics 186, 757-61 (2010)).
The first component of a TALEN is the TALE domain. In the nucleus they bind specific sequences within gene promoters to modulate host resistance mechanisms. Each TALE contains a highly conserved central region consisting of varying numbers of repeat units of typically 33 to 35 amino acids that confer specific DNA sequence recognition to the TALE protein. These repeat units contain high levels of identity with the exception of two variable amino acids at positions 12 and 13. These residues, termed the Repeat-Variable Di-residues (RVDs), confer DNA-binding specificity to the repeat region. The mechanism of DNA recognition of a TALE is therefore based on a simple code whereby one RVD recognizes one nucleotide of DNA sequence and ensures that the DNA binding domain of each TALE is capable of targeting large recognition sites with high precision (15-30 nucleotides).
The second component of a TALEN is the catalytic domain of an endonuclease that introduces DNA double strand breaks (DSBs) in vitro and in vivo, and which is fused to the TALE DNA-binding domain.
Thus, in some embodiments of the present disclosure, the engineered autonomously distributed circuits of the present disclosure contain components of the TAL/TALEN system, including a transcriptional activator like effector (TALE) fused to an endonuclease.
In some embodiments, the programmable nuclease circuits of the present disclosure use zinc finger nucleases (ZFNs) to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene. ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain (Carroll, et al. Nat Protoc, 1(3), 1329-41 (2006)). Zinc finger domains can be engineered to target unique DNA sequences. The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 base pairs. The non-specific cleavage domain from the type II restriction endonuclease FokI is typically used as the cleavage domain in ZFNs (Kim, et al., Proc Natl Acad Sci USA, 93(3), 1156-60 (1996)). This cleavage domain dimerizes to cleave DNA (Bitinaite, et al. Proc Natl Acad Sci USA 95(18), 10570-5 (1998)) and thus a pair of ZFNs target non-palindromic DNA sites. In some embodiments, ZFNs are engineered by fusing the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs bind opposite strands of DNA with their C-termini a certain distance apart. In some embodiments, the linker sequences between the zinc finger domain and the cleavage domain requires the 5′ edge of each binding site to be separated by 5 to 7 base pairs (bp) (Cathomen, et al. Mol. Ther. 16(7), 1200-7 (2008)).
Programmable nuclease circuits may be used to target most genes including, without limitation, genes or single nucleotide polymorphisms (SNPs) that confer resistance to traditional small molecule antimicrobials, genes that confer virulence traits to pathogens (e.g., Escherichia coli, Shigella dysenteriae, Yersinia pestis, Francisella tularensis, Bacillus anthraces, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, and Salmonella enterica Typhi), highly conserved genes that can typify microbial species, genera or phyla for the purposes of, e.g., remodeling complex microbial communities, and genes that encode enzymes involved in biochemical pathways with products that can modulate host physiology.
Examples of genes that confer aminoglycoside resistance include, without limitation, aph, aac and aad variants and other genes that encode aminoglycoside-modifying enzymes. Examples of genes with SNPs that confer aminoglycoside resistance include, without limitation, rpsL, rrnA and rrnB.
Examples of genes that confer beta-lactam resistance include, without limitation, genes that encode beta-lactamase (bla) (e.g., TEM, SHV, CTX-M, OXA, AmpC, IMP, VIM, KPC, NDM-1, family beta-lactamases) and mecA.
Examples of genes with SNPs that confer daptomycin resistance include, without limitation, mprF, yycFG, rpoB and rpoC.
Examples of genes that confer macrolide-lincosamide-streptogramin B resistance include, without limitation, ermA, ermB and ermC.
Examples of genes that confer quinolone resistance include, without limitation, qnrA, qnrS, qnrB, qnrC and qnrD. Examples of genes with SNPs that confer quinolone resistance include, without limitation, gyrA and parC.
Examples of genes with SNPs that confer trimethoprim/sulfonamide resistance include, without limitation, the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes.
Examples of genes that confer vancomycin resistance include, without limitation, vanA (e.g., vanRS and vanHAX), vanB and vanC operons.
In some embodiments, the engineered autonomously distributed circuits may be used to target SNPs, which cause overexpression of genes that encode multi-drug efflux pumps, such as acrAB, mexAB, mexXY, mexCD, mefA, msrA and tetL.
Examples of genes that confer virulence traits to Escherichia coli (e.g., O157:H7) include, without limitation, stx1 and stx2 (encode Shiga-like toxins) and espA (responsible for induction of enterocyte effacement (LEE) A/E lesions). Other examples of genes that confer virulence traits to Escherichia coli include fimA (fimbriae major subunit), csgD (curli regulator) and csgA.
Examples of genes that confer virulence traits to Shigella dysenteriae include, without limitation, stx1 and stx2.
An example of a gene that confers virulence traits to Yersinia pestis is yscF (plasmid-borne (pCD1) T3SS external needle subunit).
An example of a gene that confers virulence traits to Francisella tularensis is fslA.
An example of a gene that confers virulence traits to Bacillus anthraces is pag (Anthrax toxin, cell-binding protective antigen).
Examples of genes that confer virulence traits to Vibrio cholerae include, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator).
Examples of genes that confer virulence traits to Pseudomonas aeruginosa include, without limitation, genes that encode for the production of siderophore pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdI, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT and opmQ), genes that encode for the production of siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and genes that encode for toxins (e.g., exoU, exoS and exoT).
Examples of genes that confer virulence traits to Klebsiella pneumoniae include, without limitation, fimA (adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
Examples of genes that confer virulence traits to Acinetobacter baumannii include, without limitation, ptk (capsule polymerization) and epsA (assembly).
Examples of genes that confer virulence traits to Salmonella enterica Typhi include, without limitation, hilA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile tolerance, including efflux pump genes acrA, acrB and tolC.
Examples of highly conserved genes that can typify microbial species, genera or phyla for the purposes of remodeling complex microbial communities (referred to herein as “remodeling” genes) include, without limitation, ribosomal components rrnA, rpsL, rpsJ, rplO, rpsM, rplC, rpsH, rplP, and rpsK, transcription initiation factor infB, and tRNA synthetase pheS.
Examples of genes encoding enzymes involved in biochemical pathways with products that can modulate host physiology (referred to herein as “modulatory” genes include, without limitation, (1) genes encoding enzymes involved in deoxycholate production linked to hepatocellular carcinoma, (2) genes encoding enzymes involved in polysaccharide A production by Bacteroides fragilis, leading to development of regulatory T cells (TREG), IL-10 response and increased TH1 cell numbers, (3) genes encoding enzymes involved in butyrate production leading to secretion of inducible antimicrobial peptides, (4) genes encoding enzymes involved in short-chain fatty acid production leading to increased energy harvest, obesity, inflammatory modulation and gastrointestinal wound healing, (5) genes encoding enzymes involved in transformation of choline into methylamines, which can disrupt glucose homeostasis, leading to non-alcoholic fatty liver disease and cardiovascular disease, (6) genes encoding enzymes involved in the generation of neuromodulatory compounds such as γ-aminobutyric acid, noradrenaline, 5-HT, dopamine and acetylcholine, and (7) genes encoding enzymes involved in the formation of lactic acid and propionic acid linked to anxiety.
Any of the foregoing genes or any other gene (e.g., biothreat gene) may be targeted by the programmable nuclease circuits of the present disclosure.
Programmable nuclease circuits may be loaded into vehicles for target, or broad, delivery and dissemination including, without limitation, engineered phagemids (Lu, T. K., et al. Current Opinion in Microbiology 14, 524-531 (2011)), bacteriophages and broad-host-range and plasmids (e.g., conjugative plasmids) delivered via transfer-competent probiotic bacteria. Other delivery vehicles contemplated herein include, without limitation, chemical-based delivery vehicles (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes), nanoparticle-based delivery vehicles (or platforms), non-chemical-based delivery vehicles (e.g., electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment, cell-penetrating peptides), and viral-based delivery vehicles (e.g., use of viruses as carriers). The choice of delivery vehicle/platform will depend, in part, on the type of cell to which the programmable nuclease circuits are delivered as well as the environment of the cells (e.g., in vivo v. in vitro). In some embodiments, microbial cells may be naturally competent and take up DNA from the environment on their own. In some embodiments, two or more different bacteriophages or phagemids or other combination of delivery vehicles may be used together as a delivery cocktail (Goodridge, L. D. Current Pharmaceutical Biotechnology 11, 15-27 (2010)). These delivery platforms enable the introduction and distribution of programmable nuclease circuits in microbial populations, where they can, in some instances, autonomously spread.
Phagemids
In some embodiments of the present disclosure, programmable nuclease circuits may be loaded into engineered phagemids, or phasmids, for delivery to microbial cell populations. As used herein, a phagemid refers to a phage-derived vector containing the replication origin of a plasmid and the packaging site of a bacteriophage.
Examples of phagemids that may be used in accordance with the invention include, without limitation, M13-derived phagemids containing the fl origin for filamentous phage packaging such as, for example, pBluescript II SK (+/−) and KS (+/−) phagemids, pBC SK and KS phagemids, pADL and P1-based phagemids (see, e.g., Westwater C A et al., Microbiology 148, 943-50 (2002); Kittleson J T et al., ACS Synthetoc Biology 1, 583-89 (2012); Mead D A et al., Biotechnology 10, 85-102 (1988)). Other phagemids may be used in accordance with the present disclosure and, for example, can be made to work with packaging systems from natural, engineered or evolved bacteriophage.
Bacteriophages
In some embodiments of the present disclosure, programmable nuclease circuits, or phagemids containing programmable nuclease circuits, may be loaded into naturally-occurring, engineered (e.g., rationally engineered), or adaptively evolved bacteriophage for delivery to microbial cell populations, e.g., endogenous microbial cells. A bacteriophage, or phage, is a virus that infects and replicates in bacteria. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Bacteriophages replicate within bacteria following the injection of their genome into the cytoplasm and do so using either a lytic cycle, which results in bacterial cell lysis, or a lysogenic (non-lytic) cycle, which leaves the bacterial cell intact. The bacteriophages of the present disclosure are, in some embodiments, non-lytic (also referred to as lysogenic or temperate). Non-lytic phage may also include those that are actively secreted from infected cells in the absence of lysis, including, without limitation, filamentous phage such as, for example, M13, fd, IKe, CTX-φ, Pf1, Pf2 and Pf3. Thus, after phage delivery of a programmable nuclease circuit to a bacterial cell, the bacterial cell may remain viable and able to stably maintain expression of the circuit. In some embodiments, lytic bacteriophage may be used as delivery vehicles. When used with phagemid systems, naturally lytic phage serve as cargo shuttles and do not inherently lyse target cells.
Examples of non-lytic bacteriophage for use in accordance with the present disclosure include, without limitation, Myoviridae (P1-like viruses; P2-like viruses; Mu-like viruses; SPO1-like viruses; phiH-like viruses); Siphoviridae (λ-like viruses, γ-like viruses, T1-like viruses; T5-like viruses; c2-like viruses; L5-like viruses; psiM1-like viruses; phiC31-like viruses; N15-like viruses); Podoviridae (phi29-like viruses; P22-like viruses; N4-like viruses); Tectiviridae (Tectivirus); Corticoviridae (Corticovirus); Lipothrixviridae (Alphalipothrixvirus, Betalipothrixvirus, Gammalipothrixvirus, Deltalipothrixvirus); Plasmaviridae (Plasmavirus); Rudiviridae (Rudivirus); Fuselloviridae (Fusellovirus); Inoviridae (Inovirus, Plectrovirus, M13-like viruses, fd-like viruses); Microviridae (Microvirus, Spiromicrovirus, Bdellomicrovirus, Chlamydiamicrovirus); Leviviridae (Levivirus, Allolevivirus) and Cystoviridae (Cystovirus). Such phages may be naturally occurring or engineered phages. In some embodiments, the bacteriophage is a coliphage (e.g., infects Escherichia coli). In some embodiments, the bacteriophage of the present disclosure target bacteria other than Escherichia coli, including, without limitation, Bacteroides thetaiotamicron (e.g., B1), B. fragilis (e.g., ATCC 51477-B1, B40-8, Bf-1), B. caccae (e.g., phiHSC01), B. ovatus (e.g., phiHSC02), Clostridium difficile (e.g., phiC2, phiC5, phiC6, phiC8, phiCD119, phiCD27), Klebsiella pneumoniae (e.g., KPO1K2, K11, Kpn5, KP34, JD001), Staphylococcus aureus (e.g., phiNM1, 80alpha), Enterococcus faecalis (e.g., IME-EF1), Enterococcus faecium (e.g., ENB6, C33), and Pseudomonas aeruginosa (e.g., phiKMV, PAK-P1, LKD16, LKA1, delta, sigma-1, J-1). Other bacteriophage may be used in accordance with the present disclosure.
In some embodiments, the bacteriophage of the present disclosure is an M13 bacteriophage. M13 is a filamentous bacteriophage of the family Inoviridae and is composed of circular single-stranded DNA. M13 phages are about 900 nm long and 6-7 nm in diameter with 5 proteins. The minor coat protein, P3, attaches to the receptor at the tip of the F pilus of an Escherichia coli host cell. Thus, in some embodiments, the methods comprise delivering to bacterial cells a recombinant M13 bacteriophage that is engineered to contain at least one programmable nuclease circuit.
It should be appreciated that the bacteriophages of the present disclosure can be isolated from any environment where bacteria exist. In some embodiments, the bacteriophages of the invention are isolated from (e.g., collected from, obtained from) stool or sewage. In other embodiments, the bacteriophages of the present disclosure are isolated from terrestrial or marine environments. Isolated bacteriophages may be further modified by rational design including, without limitation, alterations in host range determination factors such as the addition, deletion, or swapping of components of tail fibers, capsule binding and/or degrading enzymes, phage counter defense genes (immunity to restriction, anti-CRISPR genes, antitoxins or other products to subvert programmed cell death), biocontainment modules, and modifications involved in the generation of phagemid packaging systems (e.g. modification or deletion of packaging site from phage genome). These components and unknown components may also be modified through adaptive evolution designed to improve efficiency of delivery or expand the host range of the phages.
Broad-Host-Range and Conjugative Plasmids
In some embodiments of the present disclosure, programmable nuclease circuits may be loaded into broad-host-range and conjugative plasmids for delivery to microbial cell populations, e.g., endogenous microbial cells. Conjugative transfer of bacterial plasmids is the primary route of broad host range DNA transfer between different genera of bacteria and is the most efficient way of horizontal gene spread. Conjugative transfer is thus considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Various aspects of the present disclosure are directed to exploiting the natural phenomena of conjugative transfer of plasmids to deliver programmable nuclease circuits to microbial cells in vivo, which prevents and/or reverses, for example, antibiotic resistance in the cells.
To promote propagation of programmable nuclease circuits, the present disclosure contemplates the use of plasmids with broad-host-range origins of replication. Such plasmid origins include, without limitation, those derived from major broad-host-range incompatibility groups IncQ (e.g., RSF1010, R300B, R1162), IncW (e.g., pSa, pR388), IncP (e.g., R18, R68, RK2, RP1, RP4), IncN and IncU (e.g., RA3), and pBBR1 (Lale, R., et al. Strain Engineering, Vol. 765 (ed. Williams, J. A.) 327-343 (Humana Press, 2011)). Other broad-host-range and conjugative plasmids may be used in accordance with the invention.
Mobile Genetic Elements
In some embodiments of the invention, mobile genetic elements other than conjugative vectors can be used to facilitate transfer into target strains. Such elements may include, without limitation, integrative conjugative elements (e.g., SXT, PAPI-1), integrative and mobilizable elements (e.g., SGI1), cis-mobilizable elements, conjugative transposons (e.g., Tn916, Tn1545, Tn5276, Tn5397, CTnDOT), mobile pathogenicity islands (e.g., SaPI1), and prophages (e.g., P1, Lambda, 933W, phiNM1, 80alpha), among others.
Donor Bacteria
In some embodiments of the present disclosure, programmable nuclease circuits, or broad-host-range and conjugative plasmids, may be loaded into donor bacteria (e.g., probiotic bacteria and/or commensal bacteria) for delivery to microbial cell populations, e.g., endogenous microbial cells. Probiotic bacteria, for example, are live bacteria that may confer a health benefit on the host and/or, at the very least, are not harmful (e.g., not pathogenic) to the host. Commensal bacteria (also referred to as commensal microflora), as another example, include bacteria present on body surfaces covered by epithelial cells that are exposed to the external environment (e.g., gastrointestinal and respiratory tract, vaginal, skin). Commensal bacteria are found, for example, in normal microflora and indigenous microbiota. As discussed above, bacterial plasmids are transferred from bacteria to bacteria through conjugation. Thus, aspects of the invention contemplate the use of donor bacteria (e.g., probiotic bacteria and/or commensal bacteria) loaded with plasmids containing programmable nuclease circuits to deliver the circuits to microbial cells (e.g., pathogenic microbial cells) in vivo. In other embodiments of the present disclosure, probiotic bacteria may be loaded with non-lytic or inducible bacteriophage that can express and generate bacteriophage-based delivery particles in situ.
Examples of probiotic bacteria for use in accordance with the present disclosure include, without limitation, Bacillus coagulans GBI-30, 6086, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis, Escherichia coli Nissle 1917, Lactobacillus acidophilus NCFM, Lactobacillus paracasei St11 (or NCC2461), Lactobacillus johnsonii La1 (also referred to as Lactobacillus LC1, Lactobacillus johnsonii NCC533), Lactobacillus plantarum 299v, Lactobacillus reuteri ATCC 55730 (Lactobacillus reuteri SD2112), Lactobacillus reuteri Protectis (DSM 17938, daughter strain of ATCC 55730), Lactobacillus reuteri Prodentis (DSM 17938/ATCC 55730 and ATCC PTA 5289 in combination), Lactobacillus rhamnosus GG, Saccharomyces boulardii, mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, a mixture of Lactobacillus acidophilus NCFM and Bifidobacterium bifidum BB-12, a mixture of Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC8OR, a mixture of Lactobacillus plantarum HEAL 9 and Lactobacillus paracasei 8700:2, Lactobacillus bulgaricus, Lactococcus thermophiles and/or Lactobacillus bifidus. In some embodiments, probiotic bacteria for use in accordance with the present disclosure may be a mixture of any two or more of the foregoing strains.
In some embodiments, live probiotic bacterial cells may be derived/obtained/isolated from, and/or delivered in vivo in, fermented dairy products and probiotic fortified foods (e.g., pickled vegetables, fermented bean paste (e.g., tempeh, miso, doenjang), kefir, buttermilk or karnemelk, kimchi, pao cai, sauerkraut, soy sauce, zha cai). In other embodiments, the probiotic bacteria may be delivered in vivo as tablets, capsules, powders and/or sachets containing the bacteria in freeze dried form. Alternatively, in some embodiments, probiotic bacteria may be delivered by means of a fecal microbiota transplant.
Bacteria are small (typical linear dimensions of around 1 micron), non-compartmentalized organisms, with at least one circular DNA chromosomes and ribosomes of 70S. As used herein, the term “bacteria” encompasses all variants of bacteria (e.g., endogenous bacteria, which naturally reside in a closed system, environmental bacteria or bacteria released for bioremediation or other efforts). Bacterial cells of the present disclosure include bacterial subdivisions of Eubacteria and Archaebacteria. Eubacteria can be further subdivided into Gram-positive and Gram-negative Eubacteria, which depend upon a difference in cell wall structure. Also included herein are those classified based on gross morphology alone (e.g., cocci, bacilli). In some embodiments, the bacterial cells are Gram-negative cells, and in some embodiments, the bacterial cells are Gram-positive cells. Examples of bacterial cells of the present disclosure include, without limitation, cells from Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., or Lactobacillus spp. In some embodiments, the bacterial cells are from Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphlococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Streptococcus spp., Enterococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, or Streptomyces ghanaenis. Thus, the bacteriophage of the present disclosure may target (e.g., specifically target) a bacterial cell from any one or more of the foregoing genus and/or species of bacteria. Other bacterial cells and microbes may also be targeted. As used herein, “endogenous” bacterial cells may refer to non-pathogenic bacteria that are part of a normal internal ecosystem such as bacterial flora.
In some embodiments, bacterial cells of the present disclosure are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells such as, for example, Escherichia coli, Shewanella oneidensis and Listeria monocytogenes. Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species. In humans, for example, anaerobic bacterial cells are most commonly found in the gastrointestinal tract. Thus, the bacteriophage of the present disclosure may target (e.g., specifically target) anaerobic bacterial cells.
The programmable nuclease circuits of the present disclosure contain at least one genetic element that can regulate gene/protein expression. A “genetic element,” as used herein, refers to a nucleotide sequence that has a role in gene expression. For example, nucleic acids (e.g., recombinant nucleic acids) encoding proteins, promoters, enhancers and terminators are considered to be genetic elements. The programmable nuclease circuits of the invention may be engineered using, for example, standard molecular cloning methods (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. M., et al., New York: John Wiley & Sons, 2006; Molecular Cloning: A Laboratory Manual, Green, M. R. and Sambrook J., New York: Cold Spring Harbor Laboratory Press, 2012; Gibson, D. G., et al., Nature Methods 6(5):343-345 (2009), the teachings of which relating to molecular cloning are herein incorporated by reference).
Nucleic Acids
As used herein, the term “nucleic acid” refers to at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds (e.g., a phosphodiester “backbone”). In some embodiments, a nucleic acid of the present disclosure may be considered to be a nucleic acid analog, which may contain other backbones comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, and/or peptide nucleic acids. Nucleic acids (e.g., components, or portions, of the nucleic acids) of the present disclosure may be naturally occurring or engineered. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. “Recombinant nucleic acids” may refer to molecules that are constructed by joining nucleic acid molecules and, in some embodiments, can replicate in a living cell. “Synthetic nucleic acids” may refer to molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
The nucleic acids may be single-stranded (ss) or double-stranded (ds), as specified, or may contain portions of both single-stranded and double-stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid/chimeric, where the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.
Some aspects of the present disclosure provide methods that include delivering to microbial cells in vivo a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest. It should be understood that components of the circuit, including the programmable nuclease, may be encoded by a single nucleic acid (e.g., on the same plasmid or other vector) or by multiple different (e.g., independently-replicating) nucleic acids.
Promoters
The programmable nuclease circuits of the present disclosure may contain nucleic acids with promoter sequences, or promoters, operably linked to a nucleotide sequence encoding a gene product of interest. As used herein, a “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, activatable, repressible, tissue-specific or any combination thereof.
A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.
A promoter may be classified as strong or weak according to its affinity for RNA polymerase (and/or sigma factor); this is related to how closely the promoter sequence resembles the ideal consensus sequence for the polymerase. The strength of a promoter may depend on whether initiation of transcription occurs at that promoter with high or low frequency. Different promoters with different strengths may be used to construct programmable nuclease circuits with different levels of gene/protein expression (e.g., the level of expression initiated from a weak promoter is lower than the level of expression initiated from a strong promoter).
A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.” Similarly, an activator/enhancer may be one naturally associated with a nucleic acid sequence, located either within or downstream or upstream of that sequence.
In some embodiments, a coding nucleic acid segment may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).
Inducible Promoters
As used herein, an “inducible promoter” is one that is characterized by initiating or enhancing transcriptional activity when in the presence of, influenced by or contacted by an inducer or inducing agent. An “inducer” or “inducing agent” may be endogenous or a normally exogenous condition, compound or protein that contacts a programmable nuclease circuit in such a way as to be active in inducing transcriptional activity from the inducible promoter.
Inducible promoters for use in accordance with the present disclosure function in a microbial cell such as a bacterial cell. Examples of inducible promoters for use herein include, without limitation, bacteriophage promoters (e.g. Plslcon, T3, T7, SP6, PL) and bacterial promoters (e.g. Pbad, PmgrB, Ptrc2, Plac/ara, Ptac, Pm), or hybrids thereof (e.g. PLlacO, PLtetO). Examples of bacterial promoters for use in accordance with the present disclosure include, without limitation, positively regulated E. coli promoters such as positively regulated σ70 promoters (e.g., inducible pBad/araC promoter, Lux cassette right promoter, modified lamdba Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), σS promoters (e.g., Pdps), σ32 promoters (e.g., heat shock) and σ54 promoters (e.g., glnAp2); negatively regulated E. coli promoters such as negatively regulated σ70 promoters (e.g., Promoter (PRM+), modified lamdba Prm promoter, TetR-TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLacO1, dapAp, FecA, Pspac-hy, pcI, plux-cI, plux-lac, CinR, CinL, glucose controlled, modifed Pr, modifed Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR_regulated, BetI_regulated, pLac_lux, pTet_Lac, pLac/Mnt, pTet/Mnt, LsrA/cI, pLux/cI, LacIQ, pLacIQ1, pLas/cI, pLas/Lux, pLux/Las, pRecA with LexA binding site, reverse BBa_R0011, pLacI/ara-1, pLacIq, rrnB P1, cadC, hns, PfhuA, pBad/araC, nhaA, OmpF, RcnR), σS promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ38), σ32 promoters (e.g., Lutz-Bujard LacO with alternative sigma factor σ32), and σ54 promoters (e.g., glnAp2); negatively regulated B. subtilis promoters such as repressible B. subtilis σA promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank) and σB promoters. Other inducible microbial promoters and/or bacterial promoters may be used in accordance with the present disclosure.
The administration or removal of an inducer results in a switch between activation and inactivation of the transcription of the operably linked nucleic acid sequence (e.g., nucleic acid encoding a gene product of interest). Thus, as used herein, the active state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is expressed). Conversely, the inactive state of a promoter operably linked to a nucleic acid sequence refers to the state when the promoter is not actively driving transcription of the nucleic acid sequence (i.e., the linked nucleic acid sequence is not expressed).
An inducible promoter for use in accordance with the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s). The extrinsic inducer or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations thereof.
Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).
In some embodiments, the inducer used in accordance with the present disclosure is an N-acyl homoserine lactone (AHL), which is a class of signaling molecules involved in bacterial quorum sensing. Quorum sensing is a method of communication between bacteria that enables the coordination of group based behavior based on population density. AHL can diffuse across cell membranes and is stable in growth media over a range of pH values. AHL can bind to transcriptional activators such as LuxR and stimulate transcription from cognate promoters. In some embodiments, the inducer used in accordance with the present disclosure is anhydrotetracycline (aTc), which is a derivative of tetracycline that exhibits no antibiotic activity and is designed for use with tetracycline-controlled gene expression systems, for example, in bacteria.
Other inducible promoter systems are known in the art and may be used in accordance with the present disclosure.
Enhancers
In some embodiments of the present disclosure, a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter. The enhancer may be located at any functional location before or after the promoter.
Terminators
In some embodiments, a programmable nuclease circuit may contain a terminator sequence, or terminator. A “terminator,” as used herein, is a nucleic acid sequence that causes transcription to stop. A terminator may be unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters. Thus, in certain embodiments, a terminator that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable gene/protein expression levels.
The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid sequence that is usually transcribed, a forward transcriptional terminator will cause transcription to abort. In some embodiments, bidirectional transcriptional terminators are provided, which usually cause transcription to terminate on both the forward and reverse strand. In some embodiments, reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only.
In prokaryotic systems, terminators usually fall into two categories (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators are generally composed of palindromic sequence that forms a stem loop rich in G-C base pairs followed by a string of uracil bases.
Terminators for use in accordance with the present disclosure include any terminator of transcription described herein or known to one of ordinary skill in the art. Examples of terminators include, without limitation, the termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the TO terminator, the TE terminator, Lambda T1 and the T1T2 terminator found in bacterial systems. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation.
Other genetic elements are known in the art and may be used in accordance with the present disclosure.
In some embodiments of the present disclosure, the microbial cells (e.g., bacterial cells) may be functionalized with recombinase-based programmable nuclease circuits. A “recombinase,” as used herein, is a site-specific enzyme that recognizes short DNA sequence(s), which sequence(s) are typically between about 30 base pairs (bp) and 40 bp, and that mediates the recombination between these recombinase recognition sequences, which results in the excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. For example, in some embodiments, recombinant bacteriophages of the present disclosure may be engineered to deliver at least two programmable nuclease circuits, one containing a nucleic acid with an inducible promoter operably linked to a nucleic acid encoding a recombinase, and the other containing a nucleic acid with a promoter operably linked to a nucleotide sequence encoding a gene product of interest and optionally containing a terminator, wherein at least one of the promoter and terminator is flanked by a forward and a reverse recognition site of the recombinase. In such embodiments, expression of the gene product of interest of one circuit is regulated by recombinase activity, or inactivity, of the other circuit.
Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases), based on distinct biochemical properties. Serine recombinases and tyrosine recombinases are further divided into bidirectional recombinases and unidirectional recombinases. Examples of bidirectional serine recombinases for use herein include, without limitation, β-six, CinH, ParA and γδ; and examples of unidirectional serine recombinases include, without limitation, Bxb1, φC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118, U153 and gp29. Examples of bidirectional tyrosine recombinases for use herein include, without limitation, Cre, FLP, and R; and unidirectional tyrosine recombinases include, without limitation, Lambda, HK101, HK022 and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA and which becomes covalently linked to the DNA during strand exchange.
The outcome of recombination depends, in part, on the location and orientation of two short repeated DNA sequences that are to be recombined, typically less than 30 bp long. Recombinases bind to these repeated sequences, which are specific to each recombinase, and are herein referred to as “recombinase recognition sequences” or “recombinase recognition sites.” Thus, as used herein, a recombinase is “specific for” a recombinase recognition site when the recombinase can mediate inversion or excision between the repeat nucleotide sequences. As used herein, a recombinase may also be said to recognize its “cognate recombinase recognition sites,” which flank an intervening genetic element (e.g., promoter, terminator, or output nucleic acid sequence). A genetic element is said to be “flanked” by recombinase recognition sites when the element is located between and immediately adjacent to two repeated nucleotide sequences.
Recombinases can also be classified as irreversible or reversible. As used herein, an “irreversible recombinase” refers to a recombinase that can catalyze recombination between two complementary recombination sites, but cannot catalyze recombination between the hybrid sites that are formed by this recombination without the assistance of an additional factor. Thus, an “irreversible recognition site” refers to a recombinase recognition site that can serve as the first of two nucleotide recognition sequences for an irreversible recombinase and that is modified to a hybrid recognition site following recombination at that site. A “complementary irreversible recognition site” refers to a recombinase recognition site that can serve as the second of two nucleotide recognition sequences for an irreversible recombinase and that is modified to a hybrid recombination site following homologous recombination at that site.
Irreversible recombinases, and nucleic acids that encode the irreversible recombinases, are described in the art and can be obtained using routine methods. Examples of irreversible recombinases include, without limitation, phiC31 (φC31) recombinase, coliphage P4 recombinase (Ow & Ausubel, J. Bacteriol. 155, 704-713 (1983)), coliphage lambda integrase (Lorbach et al., J. Mol. Biol., 296, 1175-81 (2000)), Listeria A118 phage recombinase (Loessner et al., Mol. Micro. 35, 324-340 (2000)), and actinophage R4 Sre recombinase (Matsuura et al., J. Bacteriol. 178, 3374-3376 (1996)), HK101, HK022, pSAM2, Bxb1, TP901, TG1, φBT1, φRV1, φFC1, MR11, U153 and gp29.
Conversely, a “reversible recombinase” refers to a recombinase that can catalyze recombination between two complementary recombinase recognition sites and, without the assistance of an additional factor, can catalyze recombination between the sites that are formed by the initial recombination event, thereby reversing it. The product-sites generated by recombination are themselves substrates for subsequent recombination. Examples of reversible recombinase systems include, without limitation, the Cre-lox and the Flp-frt systems, R, β-six, CinH, ParA and γδ.
In some embodiments, the recombinase is serine recombinase. Thus, in some embodiments, the recombinase is considered to be irreversible. In some embodiments, the recombinase is a tyrosine recombinase. Thus, in some embodiments, the recombinase is considered to be reversible.
The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the present disclosure. The complexity of the programmable nuclease circuits of the present disclosure can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (Groth, A. C. & Calos, M. P. J. Mol Biol 335, 667-678, (2004); Gordley, R. M., et al. Proc Natl Acad Sci USA 106, 5053-5058 (2009)). Other examples of recombinases that are useful in the programmable nuclease circuits described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the invention.
Provided herein are libraries of (e.g., a plurality of) programmable nuclease circuits, which are engineered to target a myriad of plasmid and chromosomal genes of interest (e.g., biothreat genes, remodeling genes, modulatory genes). The libraries of the present disclosure may contain any number of programmable nuclease circuits, each engineered to destroy/attenuate a target gene of interest. For example, the present disclosure contemplates “biothreat control” libraries of programmable nuclease circuits that target genes or single nucleotide polymorphisms (SNPs) that confer resistance to traditional small molecule antimicrobials, genes that confer virulence traits to pathogens, highly conserved genes that can typify microbial species, genera or phyla for the purposes of, e.g., remodeling complex microbial communities, and genes that encode enzymes involved in biochemical pathways with products that can modulate host physiology. Such pre-engineered libraries are particularly useful if the need to address a biothreat is immediate. A library of the present disclosure may contain, for each gene target, at least two (e.g., at least 3, at least 4, at least 5, at least 10, at least 25) programmable nuclease circuits targeting different loci of the gene target, thereby increasing efficiency and efficacy of gene target destruction/attenuation.
The present disclosure also contemplates multiplexed programmable nuclease circuit libraries wherein multiple guide RNA sequences are encoded into a single circuit to enable simultaneous targeting of several genes of interest. Natural CRISPR loci have been found to contain 58 spacers to simultaneously license Cas9 to induce double stranded breaks at 58 different DNA sequences (Gunderson, F. F., et al. mBio 4(2013)). Thus, in some embodiments, a programmable nuclease circuit may be engineered to target 2 to 60 different loci within the same gene target or across multiple gene targets. For example, a programmable nuclease circuit may be engineered to target 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 different DNA sequences. In some embodiments, a programmable nuclease circuit may be engineered to target more than 60 different loci within the same gene target or across multiple gene targets. For example, a programmable nuclease circuit may be engineered to target 65, 70, 75, 80, 85, 90, 95, 100 or more different DNA sequences.
Various aspects and embodiments of the present disclosure are directed to the use of programmable nucleases circuits to specifically target existing or new biothreat genes (e.g., harmful genes such as toxin genes, virulence genes, antibiotic resistance genes) or other genes or nucleotide sequences of interest in microbial/bacterial pathogens in vivo, as described elsewhere herein. The programmable nuclease circuits may be used, in some embodiments, to eliminate plasmid-borne biothreat genes (or other genes or nucleotide sequences of interest) or destroy, or put substantial selective pressure against, cells with chromosomally integrated biothreat genes (or other genes or nucleotide sequences of interest).
The programmable nuclease circuits may be delivered to microbial/bacterial cells alone or together with an antibiotic or other antimicrobial agent. Examples of antibiotics and antimicrobial agents that may be used in accordance with the present disclosure include, without limitation, aminoglycosides, ansamycins, carbapenems, cephalosporins (1st-5th generation), glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins (and combinations with penecillins), polypeptides, quinolones, sulfonamides, tetracyclines, drugs against mycobacteria, and others.
Examples of aminoglycosides include, without limitation, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin and Spectinomycin.
Examples of ansamycins include, without limitation, Geldanamycin, Herbimycin and Rifaximin (streptomycin).
Examples of carbapenems include, without limitation, Ertapenem, Doripenem, Imipenem/Cilastatina and Meropenem.
Examples of cephalosporins include, without limitation, Cefadroxil, Cefazolin, Cefalotin or Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil and Ceftobiprole.
Examples of glycopeptides include, without limitation, Teicoplanin, Vancomycin and Telavancin.
Examples of lincosamides include, without limitation, Clindamycin and Lincomycin.
An example of a lipopeptide includes, without limitation, Daptomycin.
Examples of macrolides include, without limitation, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spiramycin.
An example of a monobactams includes, without limitation, Aztreonam.
Examples of nitrofurans include, without limitation, Furazolidone and Nitrofurantoin.
Examples of oxazolidonones include, without limitation, Linezolid, Posizolid, Radezolid and Torezolid.
Examples of penicillins include, without limitation, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin and penicillin combinations (e.g., amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate).
Examples of polypeptides include, without limitation, bacitracin, colistin and polymyxin B.
Examples of quinolones include, without limitation, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin.
Examples of sulfonamides include, without limitation, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide (archaic), Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole(Co-trimoxazole) (TMP-SMX) and Sulfonamidochrysoidine(archaic).
Examples of tetracyclines include, without limitation, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline and Tetracycline.
Examples of drugs against mycobacteria include, without limitation, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin (Rifampin in US), Rifabutin, Rifapentine and Streptomycin.
Examples of other antibiotic and antimicrobial agents include, without limitation, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin,
Platensimycin, Quinupristin/Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole and Trimethoprim.
In some embodiments, programmable nuclease circuits of the present disclosure may be used to target components of the CRISPR-Cas system in bacteria. Bacteria have evolved the CRISPR-Cas system defense mechanism to cope with various environmental stressors, including virus (e.g., bacteriophage) attack. By targeting components of the CRISPR-Cas system using the programmable nuclease circuits of the present disclosure, bacterial defenses, for example, to bacteriophage infection can be weakened. Thus, in some embodiments, provided herein are methods that comprise culturing bacterial cells and delivering to the cultured bacterial cells a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a component of the CRISPR-Cas system (e.g., Cas9 nuclease, a guide RNA (gRNA), and/or a transactivating small RNA (tracrRNA) derived from the Type II CRISPR-Cas system of Streptococcus pyogenes, or derived from other CRISPR-Cas systems).
Also contemplated herein, in some embodiments, is the use of programmable nuclease circuits to identify bacteria that contain a gene or SNP of interest (e.g., an antibiotic resistance gene). Thus, in some embodiments, provided herein are methods that comprise culturing the bacterial cells of a sample, delivering to the cultured bacterial cells a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest, and determining whether or not the bacterial cell survives, wherein bacterial cell death indicates the sample comprises bacterial cells with the gene of interest. Such methods may be useful in clinical applications to identify samples (e.g., biological fluid or tissue samples) from a subject that may contain (e.g., are suspected of containing), for example, antibiotic resistant bacterial cells. In some embodiments, the methods further comprise diagnosing the subject, or deciding on a course of therapy for the subject, based on whether or not the sample from the subject contains, for example, antibiotic resistant bacterial cells (or bacterial cells containing other genes of interest). Such methods of the present disclosure are particularly useful if, for example, a subject undergoing treatment for a bacterial infection is non-responsive to a particular antibiotic. In such cases, the methods of the present disclosure may be used to determine whether or not the subject is infected with pathogenic bacterial cells (e.g., MDR bacterial cells) that are resistant to the particular antibiotic being used to treat the subject (or to other antibiotics that may be considered as an alternative treatment).
In some embodiments, programmable nuclease circuits of the present disclosure may be used as a diagnostic. As indicated above and shown below in the Examples, programmable nuclease circuits can be used to detect nucleic acid mutations, including single nucleotide mutations. The programmable nuclease circuits can also be used to detect the presence or absence of genes (e.g., antibiotic resistance genes) and/or gene copy number.
This may be achieved, for example, by coupling a programmable nuclease circuit to an SOS-responsive reporter construct, which includes, for example, an SOS-responsive promoter operably linked to a gene encoding a reporter molecule, such as green fluorescent protein (GFP). Other reporter molecules (e.g., other fluorescent reporter molecules) are contemplated herein. The SOS response is a response to DNA damage in which the cell cycle is arrested, and DNA repair and mutagenesis are induced. The response involves the RecA protein, which when stimulated by single-stranded DNA, is involved in the inactivation of the LexA repressor, thereby inducing the SOS response. Upon induction of DNA damage by a programmable nuclease reaching its target sequence (e.g., a mutated sequence or a sequence of a gene of interest), the cellular SOS response is activated. When the SOS response is activated in the presence of an SOS-responsive reporter construct, transcription of the reporter molecule is concomitantly activated.
Thus, in some embodiments, programmable nuclease circuits may be coupled with, for example, an SOS-responsive reporter construct to detect nucleic acid mutations associated with, for example, antibiotic resistance genes and/or virulence genes in bacterial cells (e.g., bacterial cell niches). In some embodiments, programmable nuclease circuits may be coupled with a reporter construct that responds to a signal resulting from DNA cleavage to detect other genes of interest and/or gene copy number. For example, a programmable nuclease circuit may be coupled with an SOS-responsive reporter construct.
In some embodiments, provided herein are methods of diagnosing a subject (e.g., a human subject) having bacteria that contain virulance factor and/or and antibiotic resistance gene. In some embodiments, the methods comprise administering to a subject (a) a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest, and (b) a nucleic acid encoding an SOS-responsive reporter construct.
Also contemplated herein, in some embodiments, is the use of programmable nuclease circuits to sculpt the composition of bacterial populations (e.g., complex bacterial populations). For example, programmable nuclease circuits may be used to selectively remove bacteria with specific genomic contents, such as, for example, antibiotic resistance loci and/or virulence loci and/or metabolic pathways. Selective removal of such targeted loci can be achieved without affecting neighboring cells that do not contain the targeted loci. Thus, in some embodiments, programmable nuclease circuits are used to selectively knockdown bacteria that contain target DNA sequences while allowing the remaining non-target bacteria to dominate the population. By pairing RGNs with broad-host- range phages or conjugative plasmids (Westwater, C. et al. Microbiology 148, 943-50 (2002); Filutowicz, M. et al. Plasmid 60, 38-44 (2008)), targeted ‘bacterial knockdown,’ in some embodiments, may be used to selectively remove virulence genes and/or antibiotic resistance genes in pathogenic and/or commensal bacterial populations, enable functional studies of complex microbiota, and/or complement additive therapies, such as probiotics, for microbiome-associated disease by, for example, clearing specific niches and/or removing defined genes from bacterial populations.
Also provided herein are compositions and kits that comprise any one or more of the phagemids, bacteriophages, probiotic bacterial cells, nucleic acids and/or libraries of the present disclosure. The compositions and kits may further comprise additional reagents such as buffers, salts and the like. In some embodiments, the compositions are pharmaceutical compositions optionally comprising one or more pharmaceutical carriers and/or excipients.
As used herein, for simplicity, engineered autonomously distributed circuits that contain a programmable nuclease of the present disclosure will be referred to as “programmable nuclease circuits.” A programmable nuclease circuit of the present disclosure may express an RNA-guided nuclease (RGN) (“programmable RGN circuit”), a TAL effector nuclease (TALEN) (“programmable TALEN circuit”), or a zinc finger nuclease (ZFN) (“programmable ZFN circuit”).
By packaging programmable nuclease circuits into non-lytic bacteriophage particles or harnessing mobilizable plasmids for high-efficiency delivery, conditional-lethality devices with high specificity, modularity, and multiplexability against undesired DNA sequences can be implemented, in some embodiments (
To establish RNA-guided nuclease (RGN) functionality in mediating sequence-specific cytotoxicity, programmable nuclease circuits were designed to induce double-stranded breaks in blaSHV-18 or blaNDM-1, which encode extended spectrum and pan-resistance to β-lactam antibiotics, respectively (Table 1) (Rasheed, J. K. et al. Antimicrob. Agents Chemother. 44, 2382-8 (2000); Rasheed, J. K. et al. Emerg. Infect. Dis. 19, 870-8 (2013)). Transformation of plasmid-borne RGNs (pRGNs) into E. coli containing a chromosomal copy of these target genes resulted in nearly a thousand-fold decrease in transformation efficiency as compared to wild-type cells lacking the target (
Sequence analysis of 30 escape mutants, cells that received an RGN plasmid despite the presence of a target sequence, revealed that tolerance was exclusively due to a defective construct, frequently resulting from deletion of the targeting spacer within the crRNA locus (
Similarly, introduction of single-guide RGNs (ssRGN), utilizing a single hybrid/chimeric gRNA in place of the natural crRNA and required tracrRNA adaptor (Jinek, M. et al. Science 337, 816-21 (2012)), demonstrated similar reductions in transformation efficiency when introduced into cells containing the cognate target sequence (
Antibiotic resistance genes, such as blaSHV-18 and blaNDM-1, often reside on large, multi-copy plasmids capable of autonomous transfer in microbial populations, leading to horizontal dissemination of drug resistance (Nordmann, P. et al. Trends Mol. Med. 18, 263-72 (2012)). RGN activity against high-copy plasmids was verified with a GFP-expressing, ColE1-derived vector containing a standard β-lactamase selectable marker (pZE-blaz-gfp) (Lutz, R. & Bujard, H. Nucleic Acids Res. 25, 1203-10 (1997)) or blaNDM-1 (pZE-blaNDM-1-gfp). Vectors bearing this ColE1 origin are present at copy numbers of 50-70 per cell (Lutz, R. & Bujard, H., 1997). Transformation of pRGNndm-1, a plasmid-borne RGN targeting blaNDM-1, into cells containing pZE-blaNDM-1-gfp led to a three-log reduction in carbenicillin-resistant transformants, whereas transformation of pRGNndm-1 into cells containing target-free pZE-blaz-gfp did not lead to any significant reduction in resistant transformants (
Further, because many antibiotic resistance genes are encoded on episomes, whether programmable nuclease circuits can induce plasmid loss in Escherichia coli (E. coli) was explored. Wild-type EMG2 cells were first transformed with high copy plasmids (ColE1-derived) that contained either the bla or blaNDM-1 gene as the resistance marker (named pZE12-gfp and pZE-ndm1-gfp, respectively). These plasmid-containing cells were made chemically competent using a TSS protocol, and transformed with single (Cas9 and hybrid/chimeric gRNA) or dual-guide (Cas9, tracrRNA, and crRNA) RGNs targeting blaNDM-1 or blaSHV-18. Following a 90 minute recovery in SOC, cultures were diluted 1:100 in fresh LB chloramphenicol to select for transformants and incubated for 16 hours until saturation. The samples were plated on LB plates supplemented with chloramphenicol and carbenicillin or with chloramphenicol only. Introduction of a cognate RGN into cells bearing a blaNDM-1 containing plasmid induced potent loss of plasmid as measured by the ratio of carbenicillin resistant to total number of colonies (
The viability of programmable nuclease circuits as an antimicrobial therapy hinges, in some instances, on high-efficiency delivery of genetic constructs to bacterial cells. Bacteriophages are natural predators of prokaryotes and are highly adept at injecting DNA into host cells. To use phage particles for programmable nuclease circuit delivery, phagemid vectors were engineered by pairing the blaNDM-1 or blaSHV-18 RGN constructs with an f1 origin for packaging into M13 particles. Phage-packaged RGNndm-1 (“ΦRGNndm-1”) was capable of comprehensively transducing a population of E. coli EMG2 (
CRISPR-Cas antimicrobials can be multiplexed to target multiple different genetic loci. To mitigate the occurrence of variants that may arise due to modifications of gene target sequences or programmable nuclease circuits, for example, multiplexed programmable nuclease circuits may be designed to target genes at multiple protospacers such that target inactivation is mediated by more than one guide RNA (gRNA). Natural CRISPR loci can contain up to 58 spacers to simultaneously license Cas9 to induce double stranded breaks at 58 different DNA sequences (Gunderson, F. F., et al. mBio 4(2): 1-11 (2013)). Thus, engineered CRISPR loci can target multiple genes to create multi-functional, highly selective antimicrobial devices. Herein, an efficient one-step DNA assembly strategy was created to redirect the CRISPR-Cas system against multiple heterologous targets (e.g., Target A, Target B, etc.) with only short, inexpensive, DNA oligonucleotides (
In order to exact multiplexed targeting by a single construct, an programmable nuclease circuit was engineered to contain spacers from both the blaNDM-1- and blaSHV-18-targeting RGNs (ΦRGNndm-1/shv-18). ΦRGNndm-1/shv-18 generated 2-3-log reductions in viable cells counts of both EMG2 pNDM-1 and EMG2 pSHV-18, but not of wild-type EMG2 (
In addition to antibiotic-modifying enzymes, such as β-lactamases, alterations in host proteins constitute a major antibiotic resistance mechanism (Jacoby, G. A. Clin. Infect. Dis. 41 Suppl 2, S120-6 (2005)). CRISPR-Cas systems show exquisite sequence specificity, where single base pair mutation in, or proximal to, the protospacer-adjacent motif (PAM) can fully abrogate activity. Due to the specificity of the CRISPR-Cas system in prokaryotes, it was suspected that programmable nuclease circuits could discriminate between susceptible and resistant strains that differ by a single nucleotide mutation in DNA gyrase (gyrA), which confers resistance to quinolone antibiotics (Table 1) (Jacoby, G. A. (2005)). Indeed, ΦRGNgyrAD87G exhibited specific cytotoxicity only towards quinolone-resistant E. coli harboring the gyrAD87G mutation and not towards otherwise isogenic strains with the wild-type gyrA gene (
To further exploit this sensitivity to detect small nucleotide polymorphisms (SNPs) in the E. coli genome that confer resistance to quinolones, a programmable RGN circuit was designed to specifically target the gyrA gene in the quinolone-resistant RFS289 E. coli strain, which contains a gyrA111 mutation. RFS289 and a similar wild-type strain, MG1655pro, were transformed with either RGNØ or RGNgyrA111, and the transformation efficiency of each strain was determined by counting colony forming units (cfu) of transformants following plating on LB chloramphenicol plates. RGNgyrA111 was specifically toxic to RFS289, although the wild-type and mutant sequences differed by only a single base pair (
Based on these results and the selective toxicity of RGNs, the present disclosure contemplates the use of programmable nuclease circuits to identify SNPs in bacterial genomes.
Killing curves revealed that (DRGNs mediated rapid killing of target cells, with viable cell counts that decayed exponentially (t1/2˜13 minutes) and maximal bactericidal effect achieved by 2-4 hours (
It was intriguing to observe that targeted cleavage of blaNDM-1 in their native plasmids was lethal to host cells, whereas targeted cleavage of the same gene in a standard Lutz-based cloning vector (“pZA-ndm-1-gfp”) was not (
To further demonstrate the versatility of programmable nuclease circuits for specifically combating pathogens, a ΦRGN was designed to target intimin, a virulence factor of enterohemorrhagic E. coli O157:H7 (EHEC) necessary for intestinal colonization and pathology. Encoded by the eae gene, intimin is a cell-surface adhesin that mediates intimate attachment to the host epithelium, permitting subsequent disruption of intestinal tight junctions and effacement of microvilli (Kaper, J. B. et al. Nat. Rev. Microbiol. 2,123-40 (2004)). Treatment of EHEC with ΦRGNeae resulted in a 20-fold reduction in viable cell counts; this cytotoxicity was increased an additional 100-fold under selection for ΦRGNeae transductants (
Apart from their therapeutic potential, RGN activity can be coupled with a fluorescence reporter to assay the presence of target sequences in bacterial cultures. E. coli and other bacteria respond to chromosomal double-stranded breaks by inducing DNA repair through the activation of the SOS response (Pennington, J. M. & Rosenberg, S. M. Nat Genet 39, 797-802 (2007)). Using a SOS-responsive GFP reporter plasmid (Dwyer, D. J. et al. Mol Syst Biol 3, (2007)), a 8-fold and 4-fold increase in fluorescence was observed in cells containing a chromosomal (gyrAD87G) or plasmid-borne (blaNDM-1) target site, respectively, when treated with their cognate DRGNs relative to untreated cells (
To expand the utility of programmable nuclease circuits, R1162, a mobilizable broad-host-range IncQ-type plasmid (Meyer, R. Plasmid 62, 57-70 (2009)), was used as another platform to shuttle RGNs into target cells. This approach was used to selectively remove multidrug-resistant E. coli. High-efficiency mobilization of R1162 was achieved using E. coli S17-1, which has the conjugative machinery of plasmid RP4 integrated into its chromosome (Simon, R. et al. Nat. Biotechnol. 1, 784-791 (1983)). Although constrained by requirements for cell-cell contact, conjugative plasmids tend to possess the largest host ranges amongst all forms of horizontal gene transfer (Smillie, C. et al. Microbiol. Mol. Biol. Rev. 74, 434-52 (2010)) and there are no known recipient factors necessary for DNA uptake (Pérez-Mendoza, D. & de la Cruz, F. BMC Genomics 10, 71 (2009)). After 3-hour matings, transfer of a mobilizable RGN (mRGN) enabled a 40-60-fold reduction of carbenicillin-resistant recipient cells possessing target chromosomal antibiotic resistance determinants in the absence of any selection for the mRGN in the target cells (
In addition to implementing targeted antimicrobial therapies, programmable nuclease circuits can be used to sculpt the composition of complex bacterial populations (
Programmable nuclease circuits delivered on plasmids in E. coli can prevent the incoming conjugation of plasmids with blaNDM-1 and blasSHV-18 antibiotic-resistance genes from clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumonia) (
M13 bacteriophages (“M13 phages”) and phagemids that express programmable nuclease circuits are engineered for delivery into Escherichia coli (E. coli) containing chromosomally integrated and plasmid-borne genes of interest (e.g., biothreat genes). One advantage of using phages for delivery of the programmable nuclease circuits is that they can replicate within infected cells and amplify themselves to achieve near-complete infection of a target population. One advantage of using phagemids for delivery is that they do not produce new infectious particles after delivery and, thus, are self-limited. As a result, phagemids must be packaged into high-titer and efficient phage particles to ensure near-complete infection of a target population.
M13 bacteriophage delivery vehicles of the invention are engineered by cloning a programmable RGN circuit expressing the trans-activating small RNA, the Cas9 endonuclease, and gDNAs into the multiple-cloning site of M13mp18. This phage vector enables straightforward blue-white screening of recombinant phages. M13 phage DNA is isolated through standard miniprep protocols for cloning. Recombinant phages are amplified in E. coli by infecting mid-log cell populations, removing the cells through centrifugation and filtration, and storing the supernatants where the phage particles are present. The phage stocks are titered to determine the number of viable phage particles through serial dilution and enumeration of plaque-forming units (pfu) on lawns of susceptible E. coli.
M13 phagemids of the invention are engineered by cloning an M13 origin of replication (fl) into vectors containing the programmable RGN circuits and a selectable antibiotic-resistance marker to aid in cloning. These phagemid vectors are placed into special packaging strains of bacteria that produce phage particles containing the phagemid (Chasteen, L., et al. Nucleic acids research 34, e145 (2006)). After growth of and phagemid packaging in these bacteria, the phage particles are purified by removing the bacteria cells through centrifugation and filtration of the resulting supernatant fluid where the phage particles are present. The number of phage particles containing the engineered phagemid is determined by exposing serial dilutions of these supernatants to phage-susceptible cells and counting the number of cells that subsequently demonstrate resistance to the antibiotic marker on the phagemid containing the programmable RGN circuit.
Genotypic and phenotypic assays are used to determine the effectiveness of M13 phages and phagemids at targeting plasmid-borne and chromosomally integrated genes, including antibiotic-resistance genes and virulence factors. DNA sequencing is used to determine the effects of RGNs on the target genes at the sequence level. For antibiotic-resistance genes such as blaNDM-1, cells are plated on non-selective and selective plates to determine whether treatment with programmable RGN circuits reduces the number of bacteria that still display antibiotic resistance.
For virulence genes, specific phenotypic assays are performed. For example, assays for directly detecting the production of Stx have been described (Gould, L. H., et al. MMWR.
Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports/Centers for Disease Control 58, 1-14 (2009)). Two methods are used to analyze stx targeting: a cytotoxicity-based assay and direct detection of the toxin by a Stx-specific ELISA. Cytotoxicity of spent culture medium from Stx2-producing bacteria is assessed by applying sterile-filtered supernatants from these cultures onto a monolayer of Vero cells (African green monkey kidney epithelial cells) and quantifying cell viability using a methyl tetrazolium (MTT) assay (Fotakis, G., et al. Toxicology letters 160, 171-177 (2006)). Vero cells are often used in such assays owing to an increased prevalence of the surface receptor Gb3 and resulting heightened sensitivity to the toxin (Paton, J. C., et al. Clin Microbiol Rev 11, 450-479 (1998); Konowalchuk, J., et al. Infect Immun 18, 775-779 (1977)). Cytotoxicity due to Stx is further confirmed by demonstrating a loss of cytotoxicity in the presence of Stx-neutralizing antibodies (Karmali, M. A., et al. J Infect Dis 151, 775-782 (1985)). In addition to cytotoxicity assays, ELISA assays are used to titer the concentration of Stx and commercial kits such as the Premier™ EHEC Kit (Meridian Biosciences, Inc.) are used to evaluate sensitivity and specificity (Willford, J., et al. J Food Prot 72, 741-747 (2009); Staples, M., et al. Diagn Microbiol Infect Dis 73, 322-325 (2012)).
A urinary tract infection (UTI) model in mice is used to study the in vivo efficacy of phagemid and phage delivery of programmable nuclease circuits. The circuits are tested against E. coli with plasmid-borne and chromosomally integrated genes of interest (e.g., biothreat genes). Urinary catheters are inoculated with E. coli for 24 hours to establish infections. These urinary catheters are placed into mice and then exposed to intra-catheter treatments. The following delivery vehicles are used: phage carrying a programmable nuclease circuit, phagemids carrying a programmable nuclease circuit, control phages without a programmable nuclease circuit, and control phages with a non-cognate programmable nuclease circuit. Urine is collected at multiple time points and centrifuged to collect bacteria. Bacteria are plated on selective media to determine the proportion of cells that receive a programmable nuclease circuit (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits). Furthermore, genotypic assays, such as DNA sequencing, and phenotypic assays, such as antibiotic resistance and toxin expression are performed as described elsewhere herein.
Programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) targeting genes of interest in Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and vancomycin-resistant Enterococci (VRE) are engineered. Targets of interest in K. pneumoniae include blaSHV-18 and blaNDM-1, which are plasmid-borne genes that confer high-level resistance to beta-lactam antibiotics. Targets of interest in P. aeruginosa include gyrA mutations, which are chromosomal mutations leading to resistance to quinolone antibiotics, efflux pump mutations, which are chromosomal mutations in genes such as mexR and mexS that confer multi-drug resistance, porin loss mutations, which are chromosomal mutations in genes such as oprD that confer resistance to imipenem resistance, and virulence factors such as pyoverdin and exotoxin A. Targets of interested in VRE include the vanA and vanB mutations that lead to vancomycin resistance (Eliopoulos, G. M., et al. Clinical Infectious Diseases 33, 210-219 (2001)).
To permit programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) to propagate broadly in different bacterial strains, the circuits are loaded onto plasmids with broad-host-range origins of replication. Plasmid origins of interest include those derived from the major broad-host-range incompatibility groups IncQ (e.g., RSF1010, R300B, R1162), IncW (e.g., pSa, pR388), IncP (e.g., R18, R68, RK2, RP1, RP4), and pBBR141-based (Lale, R., et al. Broad-Host-Range Plasmid Vectors for Gene Expression in Bacteria in Strain Engineering, Vol. 765 (ed. Williams, J. A.) 327-343 (Humana Press, 2011)). For example, the origin derived from pBBR1, a small plasmid isolated from Bordetella bronchiseptica, confers stable maintenance in Pseudomonas, Klebsiella, Citrobacter, Shigella, Escherichia, Bordetella, Vibrio, Rhizobium and other species (Antoine, R., et al. Molecular microbiology 6, 1785-1799 (1992)). pBBR1 does not belong to the common broad host range incompatibility groups IncP, IncQ, or IncW and is compatible with ColE1 and p15A plasmids, an important feature to help avoid exclusion from strains harboring native plasmids (Antoine, R., et al. (1992)). Of note, pBBR1 has been used successfully in a phagemid system and can be mobilized via conjugation (Antoine, R., et al. (1992); Westwater, C., et al. Microbiology (Reading, England) 148, 943-950 (2002)).
To express the programmable nuclease circuits, promoters that are active in the target organism are used. Broad-activity promoters that have been shown to be active in these hosts to drive expression of RGNs are used, such as the arabinose-inducible promoter PBAD (Damron, F. H., et al. Applied and Environmental Microbiology 79, 718-721 (2013)). Additional promoters include Plac and PL, among others (Lale, R., et al. Broad-Host-Range Plasmid Vectors for Gene Expression in Bacteria in Strain Engineering, Vol. 765 (ed. Williams, J. A.) 327-343 (Humana Press, 2011)). The strength of these expression systems are tested by performing quantitative RT-PCR on the RGN RNA. GFP is placed under the control of these expression systems and output is quantified using flow cytometry.
Plasmid-exclusion assays are performed to determine the efficacy of programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) against plasmid-borne target genes. Vectors (e.g., phagemids) carrying programmable nuclease circuits are transformed into target cells containing the plasmid-borne target genes, transformants are selected with antibiotics and are tested with genotypic assays and phenotypic assays. DNA sequencing is used to determine to what extent transformed cells maintain an intact target gene. In addition, antibiotic-resistance assays are used to determine whether transformed cells maintain antibiotic resistance. Effective RGNs may reduce the number of cells that exhibit the antibiotic-resistant phenotype. Phenotype-specific assays for biothreat genes such as toxins are also performed.
Transformation assays are performed to determine the efficacy of programmable nuclease circuits against chromosomally integrated target genes. Vectors carrying the programmable nuclease circuits are transformed into target cells with chromosomally integrated target genes, and transformants are selected with antibiotics. Transformation efficiencies are calculated and compare to transformation reactions where vectors carrying non-cognate programmable nuclease circuits are transformed into target-containing cells as well as when vectors carrying programmable nuclease circuits are transformed into non-target-containing cells. Effective programmable nuclease circuits may show reduced transformation efficiencies compared to the controls because the programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) induce specific chromosomal DNA breaks that lead to cell death.
Phagemids that can be packaged into engineered phages are engineered. Phage packaging signals are incorporated into broad-host-range phagemids carrying programmable. For example, phagemids derived from P1 phage are used. P1 phage can target many species, with its adsorption to cells being tied to a terminal glucose residue in Gram-negative LPS (Yarmolinksy, M. B., & Sternberg, N. The Bacteriophages First Edition (ed. Calendar, R.), 291-438 (Oxford University Press, 1988)). P1 phagemids have been used previously to package and deliver genetic information to species such as E. coli, Shigella spp., K. pneumonia, P. aeruginosa, and Citrobacter freundii (Yarmolinksy, M. B., & Sternberg, N. The Bacteriophages First Edition (ed. Calendar, R.), 291-438 (Oxford University Press, 1988)). In some cases, target bacterial populations have some cells that receive the target plasmid from transducing particles while others are infected by wild-type virulent phage particles that can propagate and spread. To circumvent this problem, a packaging strain of lysogenic bacteria has been engineered with the packaging signal from the native P1 deleted. This signal has been shown to be required for specific packaging of virulent P1 DNA into phage heads (Sternberg, N., et al. Journal of molecular biology 194, 469-479 (1987). Thus, this system more efficiently packages phagemids into P1 particles.
Using programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) delivered via the P1 phagemid system, both defensive and offensive assays are performed against K. pneumoniae and P. aeruginosa, as described in the above Examples. The effectiveness of P1-delivered programmable nuclease circuits are characterized with genotypic and phenotypic assays.
Programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) are engineered to be deliverable via conjugation from bacterial probiotics to target recipients by including plasmid mobilization and conjugation signals. Such systems have been used previously to move plasmids between a wide range of gram-negatives and gram-positives, yeast and mammalian cells.
The plasmid pRK2013 was used to mobilize the conjugative transfer of the plasmid pBBR1MCS-1 into E. coli MG1655Pro. Adapting a mechanism such as the promiscuous pRK system permits conjugation into many different species, while using a mechanism such as the well-studied F-plasmid of E. coli results in a more narrow range of delivery. The pRK machinery can transfer into many gram-negatives tested and human pathogens, such as E. coli, P. aeruginosa, Bordetella pertussis, V. cholerae and K. pneumoniae.
Implementation of conjugative plasmids as delivery vehicles will demonstrates their therapeutic potential for in situ genome editing. To render them mobile, programmable nuclease circuits are cloned into the pIP501 and RK2 plasmids, which mediate high efficiency gene transfer in gram-positive and gram-negative bacteria, respectively (Kolatka, K., et al. Plasmid 64, 119-134 (2010); Grohmann, E., et al. Microbiol Mol Biol Rev 67, 277-301, table of contents (2003)). Due to the large size of these plasmids, the constructs are introduced into pIP501 and RK2 in E. coli using recombination-based genetic engineering referred to as λ-Red recombineering (Sharan, S. K., et al. Nat Protoc 4, 206-223 (2009)). To model the effects of disseminating a toxic RGN in a naïve microbial population, a strain possessing a mobile programmable nuclease circuit is introduced into populations of VRE or NDM-1 E. coli. The rate of conjugative transfer and the kinetics of resistance reversal is measured by plating the population on media selective for conjugants or antibiotic resistance.
As described above, programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) can be effective against E. coli containing undesirable genes with in vitro assays. Furthermore, in vivo assays have demonstrated delivery of synthetic gene circuits via phages to potentiate the activity of antibiotics in animal models of infection (Lu, T. K., et al. (2009)). These experiments are extended to test programmable nuclease circuits against K. pneumoniae, P. aeruginosa, and VRE to validate the generalizability of this strategy in vivo.
A mouse tissue cage model is used to study the effects of programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) on K. pneumoniae and P. aeruginosa infections (Fernandez, J. Curr Protoc Pharmacol Chapter 13, Unit13A, 15 (2006)). Tissue cage models allow the growth of bacteria in animals over many days without killing the animals, and thus can mimic abscess conditions. K. pneumoniae and P. aeruginosa infections are established in these conditions and then treated with programmable nuclease circuits and associated controls delivered through P1 phagemids. Bacterial cells are sampled periodically before and after treatment to determine the effectiveness of therapy using genotypic and phenotypic assays.
To demonstrate both the ability of programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) to be used as therapeutics in antibiotic resistant infections and to carve out niches in a complex microbial population, an engineered probiotic Lactobacillus rhamnosus GG possessing the pIP501-RGN plasmid is used to prevent and treat VRE in a murine colonization model. Recent studies suggest that administration of probiotic L. rhamnosus GG can reduce VRE colonization in human patients (Szachta, P., et al. Journal of clinical gastroenterology 45, 872-877 (2011); Manley, K. J., et al. The Medical journal of Australia 186, 454-457 (2007)). This protective effect can be potentiated through the use of programmable nuclease circuits. The VRE colonization mouse model has been widely used and accurately depicts the antibiotic-dependent colonization of humans observed in the clinic (Rice, L. B., et al. J Infect Dis 199, 342-349 (2009)). Mice fed wild-type or engineered probiotics, or left untreated, are first orally dosed for two days with vancomycin and then various inocula of VRE on the third day. For the next 14 days, wild-type and engineered probiotics are administered to all three groups and levels of VRE colonization are monitored by plating fecal content on selective media. Changes in phylogenetic and metagenomic content of the mouse gut are observed throughout the experiment by 16S sequencing and high-throughput Illumina® sequencing of mouse feces, respectively.
To demonstrate the ability of programmable nuclease circuits (e.g., programmable RNA circuits, programmable TALEN circuits, or programmable ZFN circuits) to be used as therapeutics in antibiotic resistant infections, programmable nuclease loaded particles are used to save a murine model from septicemia. Carbapenem-resistant E. coli and K. pneumoniae are estimated to cause 50% mortality rates in the U.S. (Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, 2013 (2013)). Bacteria are injected via the intraperitoneal route into mice at an inoculum sufficient to induce mortality and are subsequently administered the treatment with appropriately tuned nuclease circuits. Antibiotics to which the infecting bacteria are resistant or non- or off-targeting particles will show no saving effects while the described particles can be shown to improve the survival outcome of infected mice.
In light of the rising tide of antibiotic resistance, interest in engineered cellular and viral therapeutics as potential biological solutions to infectious disease has resurged. Synthetic biology has conferred new functionalities to living systems, such as tumor detection (Xie, Z. et al. Science 333, 1307-11 (2011)), generation of therapeutic small molecules (Paddon, C. J. et al. Nature 496, 528-32 (2013)), novel tools for drug screening (Weber, W. et al. Proc. Natl. Acad. Sci. U.S.A. 105, 9994-8 (2008)), next-generation probiotics (Duan, F. & March, J. C. Proc. Natl. Acad. Sci. U.S.A. 107, 11260-4 (2010)), and bacteriophage therapeutics that can eradicate biofilms (Lu, T. K. & Collins, J. J. Proc. Natl. Acad. Sci. U.S.A. 104, 11197-202 (2007)), inhibit conjugation (Lin, A. et al. PLoS One 6, e19991 (2011)), and potentiate antibiotic activity in resistant populations (Lu, T. K. & Collins, J. Proc. Natl. Acad. Sci. U.S.A. 106, 4629-34 (2009); Edgar, R. et al. Appl. Environ. Microbiol. 78, 744-51 (2012)). Herein, transmissible CRISPR-Cas systems were shown to act as a platform for programmable antimicrobials that harness site-specific cleavage to induce cytotoxicity, activate toxin-antitoxin systems, resensitize bacterial populations to antibiotics and sculpt bacterial consortia. The present disclosure complements the recent understanding that the Vibrio cholerae phage, ICP1, encodes its own CRISPR-Cas system to counteract a host-encoded phage defense locus (Seed, K. D. et al. Nature 494, 489-91 (2013)) and that CRISPR-Cas constructs transformed into electrocompetent cell populations are incompatible with cells that contain cognate target sequences (Gomaa, A. A. et al. MBio 5, (2013); Jiang, W. et al. Nat. Biotechnol. 31, 233-9 (2013); Vercoe, R. B. et al. PLoS Genet 9, e1003454 (2013)). In contrast to previous data, the data provided herein shows that programmable nuclease circuits efficiently delivered via bacteriophages or conjugation can elicit sequence-specific toxicity even in the absence of artificial selection, such that CRISPR-Cas technology can be applied in situ for the removal of undesired genes from microbial populations and in vivo to treat infection. Moreover, programmable nuclease circuits of the present disclosure can be used, in some instances, to artificially activate plasmid-borne toxin-antitoxin systems, which has recently become an attractive antimicrobial strategy (Williams, J. J. & Hergenrother, P. J. Trends Microbiol. 20, 291-8 (2012)). Using DNA damage-responsive reporter constructs, programmable nuclease circuits were used in a diagnostic capacity to detect single nucleotide sequence differences. Phage-based therapies are dependent on their ability to deliver nucleic acids into bacteria, which can be resisted through a variety of mechanisms (Labrie, S. J. et al. Nat. Rev. Microbiol. 8, 317-27 (2010)). These delivery vehicles can be limited to a subset of bacteria defined by the chosen phage, such that the design of programmable antimicrobials may require additional considerations as to the phage platform chosen. However, rational modification to phage host range through tail fiber alterations (Lin, T.-Y. et al. PLoS One 7, e30954 (2012)) or the use of bacteriophage cocktails (Abedon, S. T. et al. Bacteriophage 1, 66-85 (2011)) can mitigate the host-range limitations of phage-based therapies. In order to augment the demonstration of programmable nuclease circuit applications, an additional delivery strategy was devised by using a mobilizable broad-host-range system to introduce programmable nuclease circuits to recipient cells via conjugation. The use of conjugative delivery from probiotics into target bacteria enables a platform where engineered cells could integrate complex environmental cues and execute lethal payload delivery, akin to previously described sentinel cells (Saeidi, N. et al. Mol. Syst. Biol. 7, 521 (2011)).
In summary, programmable nuclease circuits of the present disclosure address multidrug-resistant bacterial infections by enabling transmissible site-specific nucleases that can be programmed to enact selective pressure against specific genetic contexts. Due to the modularity and simplicity of CRISPR-Cas engineering, libraries of multiplexed ΦRGNs (or other Φ programmable nuclease circuits) can be rapidly constructed to simultaneously target a plethora of antibiotic resistance and virulence determinants, as well as to sculpt complex microbial communities. Moreover, because CRISPR-Cas is widely conserved in prokaryotes, the use of a variety different delivery systems enables programmable nuclease circuits that can address other clinically important multidrug-resistant pathogens, including Gram-positive organisms such as Staphylococcus aureus that currently have similarly limited treatment options (Van Hal, S. J. & Fowler, V. G. Clin. Infect. Dis. 56, 1779-88 (2013)). The addition of facile, sequence-informed rational design to a field dominated by time- and cost-intensive screening for broad-spectrum small-molecule antibiotics could have the potential to reinvigorate the dry pipeline of new antimicrobials.
Unless otherwise noted, bacterial cultures were grown at 37° C. with LB medium (BD Difco). Where indicated, antibiotics were added to the growth medium to the following final concentrations: 100 μg/mL carbenicillin (Cb), 30 μg/mL kanamycin (Km), 25 μg/mL chloramphenicol (Cm), 100 μg/mL streptomycin (Sm), and 150 ng/mL ofloxacin (Ofx).
E. coli EMG2 SmR was generated by plating an overnight culture of E. coli EMG2 onto LB+Sm. Spontaneous resistant mutants were re-streaked onto LB+Sm and an isolated colony was picked and used as the recipient for conjugation of the multidrug resistance plasmids. Overnight cultures of EMG2 SmR, E. coli CDC1001728, and K. pneumoniae K6 were washed in sterile PBS and 100 μL of donor and recipient were spotted onto LB agar plates and incubated at 37° C. overnight. Transconjugants were harvested by scraping the cells in 1 mL of sterile PBS and plating onto LB+Sm+Cb. The chromosomal integrations of the β-lactamase genes and generation of EMG2 gyrAD87G were performed by λ-Red recombineering using the pSIM9 system (Datta, S. et al. Gene 379, 109-15 (2006)).
Templates for integration at the non-essential lacZYA locus were generated by amplifying the blaNDM-1 and blaSHV-18 genes from lysates of CDC1001728 and K6 using the primers rcD77/78 and rcD73/74, respectively. Templates for construction of EMG2 gyrAD87G were obtained by amplifying gyrA from RFS289 using primers mmD155/161.
To create the dual-guide RGN plasmids, an intermediate vector pZA-RGNØ which lacks a crRNA locus was created. The tracrRNA and PL(tetO-1) promoter were synthesized (Genewiz) and amplified using primers mmD98/99, Cas9 was amplified from pMJ806 (Jinek, M. et al. Science 337, 816-21 (2012)) using primers mmD74/75 and the vector backbone was amplified from pZA11G using primers mmD82/83. Each PCR product was purified and ligated by Gibson assembly (Gibson, D. G. et al. Nat. Methods 6, 343-5 (2009)). To create the final backbone vector for the RGN plasmids, the pBBR1 origin, chloramphenicol resistance marker, tL17 terminator, and crRNA locus cloning site were amplified from an intermediate vector pBBR1-MCS1-tL17 using mmD151/154, digested with NheI and SacI--HF, and ligated with pZA-RGNØ digested with SacI-HF and Avrll to create pZB-RGNØ. Digestion of this vector with PstI-HF and XbaI allowed for the insertion of assembled crRNA loci. The ΔtracrRNA pZB-RGNndm-1 plasmid was created by amplification of pZB-RGNndm-1 with mmD162/163, ClaI digestion, and self-ligation. The Cas9D10A mutant plasmids were constructed through site-directed mutagenesis of pZB-RGNndm-1 with primers mmD108/109 and the KAPA HiFi PCR kit (KAPA Biosystems).
The crRNA loci were constructed through isothermal annealing and ligation of short, single-stranded oligonucleotides (Integrated DNA Technologies). Each spacer and repeat piece was built by a corresponding oligo duplex connected to adjacent pieces by 6 bp overhangs. In addition, the terminal repeats are designed to contain a 17 bp extension comprised of a BsaI restriction site that generates an overhang that allows insertion into the pUC57-Km-crRNAØ backbone vector synthesized by Genewiz. The oligos used to build each RGN are listed in Table 2 and 3.
To assemble the CRISPR loci, 500 pmol of sense and antisense oligos in a given duplex were annealed by boiling for 10 minutes at 99° C. and cooled to room temperature. 300 pmol of each annealed duplex were combined with 15U of T4 polynucleotide kinase (Affymetrix), 400U of T4 DNA ligase (NEB), T4 ligase buffer (NEB) and ddH2O to a volume of 20 μL. Following incubation at 25° C. for 1 hour, the reaction products were purified using a Qiagen QIAquick PCR Purification Kit. Purified products were digested for three hours with Bsal-HF and re-purified using QIAquick. To prepare the crRNA backbone vector, pUC57-Km-crRNAØ was amplified using primers mmD104/105, subsequently digested with BbsI to generate compatible overhangs, and ligated with the assembled crRNA loci. Positive clones of the crRNA loci were digested from the entry vector using PstI-HF and XbaI and ligated into pZB-RGNØ digested with the same enzymes to create the final RGN plasmids.
Escherichia coli EMG2
E. coli CJ236
E. coli RFS289
E. coli EMG2::NDM-1
E. coli EMG2::SHV-18
E. coli EMG2 gyrAD87G
E. coli EMG2 SmR
E. coli DH5αPRO
E. coli CDC 1001728
E. coli S17-1 λpir
E. coli EMG2 pNDM-1
Klebsiella pneumoniae K6
E. coli EMG2 pSHV-18
E. coli O157:H7 43888
E. coli O157:H7 43888 F′
Phagemid vector pZEf-gfp was created previously by adding the f1 origin amplified from the yeast shuttle pRS series (Sikorski, R. S. & Hieter, P. Genetics 122, 19-27 (1989)) into pZE22-gfp (Lutz, R. & Bujard, H. Nucleic Acids Res. 25, 1203-10 (1997)). The RGN constructs consisting of the genes encoding the tracrRNA, Cas9, and a sequence-targeting crRNA were amplified as a single product from the respective pZB-RGN vectors using KAPA HiFi polymerase (Kapa Biosystems) with primers rcD169/183 and digested with AvrII and XmaI (New England Biolabs). These inserts were ligated with a backbone derived from amplifying the kanamycin resistance cassette, ColE1 replication origin, and the fl origin required for packaging into M13 particles off of pZEf-gfp with primers rcD184/185 and digesting with the same enzymes. Ligated plasmids were transformed into E. coli DH5αPro for sequence verification and plasmid purification.
The pZE-blaNDM-1-gfp and pZA-blaNDM-1-gfp vectors were constructed by swapping the antibiotic resistance cassette of the Lutz-Bujard vectors pZE12-gfp and pZA12-gfp (Lutz, R. & Bujard, H. (1997)). The blaNDN-1 gene was amplified from a lysate of CDC1001728 using primers mmD8/9 and the PCR product was digested with SacI-HF and XhoI. The digested product was ligated into the Lutz-Bujard vectors digested with the same enzymes.
The PemI antitoxin complementation plasmid pZA31-pemI was created by first amplifying pSHV-18 with mmD253/254. The PCR product was digested with BamHI and KpnI and ligated with the large fragment of a pZA31G digest with the same enzymes. The SOS-responsive pZA3LG reporter plasmid was derived from pZE1LG (Dwyer, D. J. et al. Mol Syst Biol 3, (2007)) by swapping the origin of replication and antibiotic resistance marker with pZA31G using AatII and AvrII as restriction enzymes.
Mobilizable RGNs were created by first amplifying the R1162 replication origin and oriT using mmD266/267. The chloramphenicol selection marker and RGN locus were amplified from pRGNndm-1 and pRGNshv-18 with mmD247/248. PCR products were digested with SpeI and XmaI, ligated and transformed into E. coli S17-1 λpir to create the donor cells used in matings.
MICs were determined by broth microdilution using LB broth according to the CLSI guidelines (Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Seventh Edition. 64 (2006)).
Overnight cultures were diluted 1:100 in fresh LB and grown to an optical density (OD600) of approximately 0.3-0.5. Following 15 minutes of incubation on ice, cultured cells were centrifuged at 3200×g, and pellets were resuspended in one tenth volume of TSS buffer (LB, 10% polyethylene glycol, 5% dimethyl sulfoxide, 50 mM Mg2+ at pH 6.5) (Chung, C. T. et al. Proc. Natl. Acad. Sci. U.S.A. 86, 2172-5 (1989)). A 100 μL aliquot of cells was incubated with 10 ng of RGN plasmid DNA. Plasmids were purified from the DH5αPro cloning host using a QIAGEN® QIAprep Spin Miniprep Kit and the concentration was determined using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). Following 30 minutes of incubation on ice, cells were heat shocked at 42° C. for 30 seconds, returned to ice for 2 minutes and recovered for 1.5 hours at 37° C. in 1 ml of SOC broth (HIMEDIA). For the chromosomal target assay, serial dilutions of cells were plated on LB+Cm to select for transformants. Plates were incubated overnight at 37 ° C., and the number of colony forming units (CFU) were enumerated the following day. Transformation efficiency was used to assess whether the given RGN plasmid was toxic to cells and was calculated as the CFU/mL per μg of DNA transformed.
For the episomal target assay, following recovery, cultures washed in fresh LB, diluted 1:100 in LB supplemented with chloramphenicol to select for transformants and incubated for 16 hours at 37° C. Samples washed in sterile PBS, serially diluted and plated on LB+Cm and LB+Cm+Cb or analyzed by flow cytometry. Colony counts were enumerated the following day and plasmid loss was inferred by calculating the ratio of CbR+CmR CFUs to CmR CFUs.
Overnight cultures of RGN transformants were also diluted 1:100 in sterile PBS, aliquoted in duplicates in a 96-well plate and immediately assayed using a BD LSRFortessa cell analyzer. Cells were consistently gated by side scatter and forward scatter across independent biological replicates. Fluorescence measurements were performed using a 488-nm argon excitation laser. The GFP gate and laser voltages were initially determined using untreated pZE-blaz-gfp and EMG2 cells as positive and negative controls, respectively, and implemented across biological replicates. BD FACSDIVA software was used for data acquisition and analysis.
Escape mutants from transformation assays were re-isolated by passaging surviving colonies onto LB+Cm+Cb. DNA isolation for escape sequencing analysis was performed by either extracting plasmid DNA from isolated escape mutants using the Qiagen QIAprep Spin Miniprep Kit or by amplifying the integrated target locus using primers mmD9/234 or mmD3/4, for blaNDM-1 and blaSHV-18, respectively. Sequencing was performed by Genewiz using the primers mmD112-115/153 and rcD11 for analysis of the RGN plasmids and mmD3 or mmD234 for examination of the integrated resistance genes.
Phagemids encoding the RGNs were purified using the Qiagen QIAprep Spin Miniprep Kit (Qiagen) and transformed into E. coli DH5αPro along with the m13cp helper plasmid for generation of phagemid-loaded M13 particles (Chasteen, L. et al. Nucleic Acids Res. 34, e145 (2006)). Strains were inoculated and grown overnight in 250 mL LB+Cm+Km to maintain m13cp and the phagemid, respectively. Cells were pelleted and the supernatant fluid containing the phagemid particles was passed through a 0.2 μm filter. M13 phagemid particles were precipitated by the addition of 5% polyethylene glycol (PEG-6000) and 0.5 M NaCl and incubation overnight at 4° C. (Westwater, C. et al. Antimicrob. Agents Chemother. 47, 1301-7 (2003)) and pelleted at 12,000×g for 1 h. Purified phagemid pellets were resuspended gently in SM buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM MgSO4) and stored at 4° C. Titers were measured by incubating sample dilutions with E. coli EMG2 for 30 minutes and enumerating transductants by plating on LB and LB+Km. Titers were defined in TFU100/mL, which is the concentration of phagemid at which ˜100% of a recipient population would be transduced at a multiplicity of infection (MOI) of 1.
Cultures were inoculated and grown overnight in LB with appropriate antibiotics at 37° C. with shaking. The following day, overnights were subcultured 1:100 into 3 mL LB (no antibiotics) and grown at 37° C. with shaking until the OD600 reached ˜0.8. Cultures were diluted into LB to ˜108 CFU/mL for pNDM-1 and pSHV-18 assays or ˜106 CFU/mL for gyrAD87G and EHEC assays and 245 μL of the suspension was added to 5 μL of purified phagemid stock in a 96-well plate and incubated static at 37° C. The number of viable cells in samples at each interval during the time-course or at 2 h for endpoint assays was determined by serial dilution and spot plating onto LB, LB+Cb, and LB+Km to analyze cytotoxicity, plasmid loss, and phagemid delivery, respectively. Initial suspensions were also diluted and plated onto LB to quantify the initial bacterial inocula. Colonies were enumerated after 8-9 h incubation at 37° C. to calculate cell viability (CFU/mL) and averaged over three independent experiments. Non-linear curve fitting of the time-course to an exponential decay curve was performed using GraphPad Prism.
Larvae of the model organism Galleria mellonella (Desbois, A. P. & Coote, P. J. Adv. Appl. Microbiol. 78, 25-53 (2012)) were purchased from Vanderhorst Wholesale, Inc. (St. Marys, Ohio, USA) and received in the final larval instar for survival assays. Larvae were removed from food source, allowed to acclimate for at least 24 h at room temperature in the dark, and used within 4 days of receipt. For all injections, a KDS100 (KD Scientific) or Pump 11 Elite (Harvard Apparatus) automated syringe pump was set to dispense a 10 μL volume at a flow rate of ˜1 μL/s through a 1 mL syringe (BD) and 26 G needle (BD). To prepare bacteria for injection, an overnight culture of E. coli O157:H7 43888 F′ was subcultured in Dulbecco's Modified Eagle Medium (Gibco) for 4 hours at 37° C. with shaking until OD600˜0.6. Cultures were washed twice in PBS and diluted to a concentration of approximately 4×105 CFU/mL. In accordance with other studies (Ramarao, N. et al. J. Vis. Exp. e4392 (2012)), twenty larvae per treatment group were randomly selected based on size (150-250 mg) and excluded based on poor health as evidenced by limited activity, dark coloration, or reduced turgor prior to experiments. Larvae were delivered injections without blinding of either PBS or bacteria behind the final left proleg. Approximately an hour after the first injection, SM buffer or DRGN treatment was administered behind the final right proleg. Larvae were incubated at 37° C. and survival was monitored at 12 hour intervals for 72 hours, with death indicated by lack of movement and unresponsiveness to touch (Desbois, A. P. & Coote, P. J. Adv. Appl. Microbiol. 78, 25-53 (2012)). Kaplan-Meier survival curves were generated on the data pooled from four independent experiments and analyzed with the log-rank test using GraphPad Prism.
Overnight cultures of EMG2 WT, EMG2 pNDM-1 and EMG2 gyrAD87G containing the SOS-responsive reporter plasmid pZA3LG were diluted 1:50 in LB and incubated with either SM buffer, ΦRGNndm-1 and ΦRGNgyrAD87G at MOI ˜5 for two hours at 37° C. Cultures were diluted 1:5 in 250 μL of sterile PBS and analyzed using an BD LSR Fortessa cell analyzer, as above. BD FACSDIVA software was used for data acquisition and analysis was performed using FlowJo software.
Donor and recipient strains grown overnight in LB with appropriate antibiotics were diluted 1:100 in fresh media and grown to an OD600˜1.0. Cells were pelleted, resuspended in sterile PBS and mating pairs were mixed at a donor to recipient ratio of 340±66:1. Mating mixtures were pelleted, resuspended in 20 μL of PBS and spotted onto nitrocellulose filters placed on LB agar plates. Initial bacterial suspensions were serially diluted and plated on LB agar plates to quantify the initial inocula. Matings proceeded at 37° C. for 3 h with a single mixing step. At 90 minutes, mating mixtures were collected by vigorously vortexing the filters in 1 mL sterile PBS. Cells were pelleted, resuspended in 20 μL PBS and re-seeded onto filters and incubated as above for the remaining 90 minutes. At the end of the h mating, cells were again recovered by vigorously vortexing the filters in 1 mL sterile PBS. Mating mixtures were serially diluted in PBS and plated onto LB+Cb to select for total number of Cb-resistant recipient cells and LB+Cb+Cm to select for transconjugants. Colonies were enumerated following overnight incubation at 37 ° C. to determine viable cell counts and were averaged over nine independent biological replicates.
E. coli CJ236, EMG2 pNDM-1, and RFS289 strains grown overnight in LB with appropriate antibiotics were diluted 1:100 into fresh LB (no antibiotics) and grown to OD600˜0.8. Cultures were seeded into fresh LB such that the initial mixture contained ˜1×106 CFU/mL of each strain and 245 μL of the suspension was added to 5 μL of SM buffer or purified ΦRGNndm-1 or ΦRGNgyrAD87G in triplicate in a 96-well plate and spotted onto LB+Cm, +Sm, and +Ofx to quantify the initial concentration of CJ236, EMG2 pNDM-1, and RFS289, respectively. Samples were then incubated, plated, and enumerated as in phagemid kill assays. The composition of the synthetic ecosystem under each treatment condition was determined by counting viable colonies on plates selective for each strain as above and data were calculated as viable cell concentration (CFU/mL) averaged over three biological replicates.
Data points in figures indicate the mean of at least three biological replicates (n≥3), where applicable. Error bars shown depict the standard error of the mean. All data were recorded and analyzed using GraphPad Prism version 6.0 (GraphPad Software, San Diego, Calif., USA).
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements).
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
All references (e.g., published journal articles, books, etc.), patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which, in some cases, may encompass the entirety of the document.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.
This application is a divisional of U.S. application Ser. No. 14/475,785, filed Sep. 3, 2014, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 61/873,894, filed Sep. 5, 2013, and U.S. provisional application No. 62/010,976, filed Jun. 11, 2014, each of which is incorporated by reference herein in its entirety.
This invention was made with Government support under Grant No. OD008435 awarded by the National Institutes of Health and under Grant No. HDTRA1-14-1-0007 awarded by the Defense Threat Reduction Agency. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62010976 | Jun 2014 | US | |
| 61873894 | Sep 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14475785 | Sep 2014 | US |
| Child | 16936881 | US |
