WO2014093479A1 - Contrôle de la régulation génétique guidé par arn crispr (répétitions palindromiques groupées, courtes régulièrement espacées ) - Google Patents

Contrôle de la régulation génétique guidé par arn crispr (répétitions palindromiques groupées, courtes régulièrement espacées ) Download PDF

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WO2014093479A1
WO2014093479A1 PCT/US2013/074372 US2013074372W WO2014093479A1 WO 2014093479 A1 WO2014093479 A1 WO 2014093479A1 US 2013074372 W US2013074372 W US 2013074372W WO 2014093479 A1 WO2014093479 A1 WO 2014093479A1
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crispr
target
sequences
cascade
cas
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Blake Wiedenheft
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Montana State University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • the invention relates to programmable gene modulating systems that offer a novel approach for systemically controlling the expression of a single gene or dozens of genes simultaneously.
  • CRISPRs Clustered regularly interspaced short palindromic repeats
  • Each CRISPR/cas locus is comprised of a series of direct repeats that are separated by nonrepetitive spacer sequences derived from foreign genetic elements.
  • An a denine - and thymiiie-rich leader sequence that contains a transcriptional promoter is located at one end of the locus.
  • CRISPR-associated (cas) genes which is typically located adjacent to a CRISPR/cas locus.
  • the Cas proteins encoded by these genes are required for new sequence acquisition, CRISPR RNA biogenesis and target sequence identification and destruction.
  • CRISPR/cas loci are transcribed and the long primary transcript is processed into a library of short CRJSPR-derived RNAs (crRNAs), also known as spacer sequences, that contain a unique sequence complementary to a foreign nucleic acid challenger.
  • CRISPR/cas loci are transcribed and processed into small RNAs that are incorporated into a multi-subumt surveillance complex called Cascade (CRISPR-associated complex for antiviral defense), which is required for protection against bacteriophages via high affinity binding to DNA targets that contain a sequence complementary to the crRNA-guide (spacer sequences).
  • Cascade CRISPR-associated complex for antiviral defense
  • Base pairing extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change. This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) that is required for target degradation.
  • CRISPR systems In other CRISPR systems, target DNA is degraded by Cas9, or other dedicated nucleases, CRISPR systems are in turn regulated by suppressors of CRISPR systems, such as anti-CRISPR proteins, which are encoded by some bacteriophages and which inactivate the CRISPR system, thereby evading the bacterial system for surveillance and targeting of foreign DNA.
  • suppressors of CRISPR systems such as anti-CRISPR proteins, which are encoded by some bacteriophages and which inactivate the CRISPR system, thereby evading the bacterial system for surveillance and targeting of foreign DNA.
  • RNAi RN A interference
  • dsR As long double- stranded RNAs
  • siRNAs 21-nt small interfering RNAs
  • AGO Argonaute protein
  • RNAi systems are limited by difficulties in designing effective RNAi sequences; problems with identifying safe and efficient methods of delivery of the RNAi sequence to the target cell; and non-specific or off-target effects of the RNAi system. Such issues have limited the use of RNAi systems, particularly with regard to therapeutic applications.
  • Other technologies for gene regulation such as TALENs (Trans activator-like endonucleases) and Zinc finger nucleases are associated with a high degree of off-target effects. Moreover, none of these technologies can be used to positively modulate, or turn on, gene expression.
  • RNAi The technology provided herein is also distinct from, the recently described systems for genetic modulation utilizing Cas9 CRISPR. systems. There is a need to provide simple, inexpensive, safe, versatile, and effective means to turn off or turn on gene expression and function in a programmable fashion.
  • the present invention provides a method of modulating the expression or function of one or more target DNA. or R.NA sequences in a cell.
  • the one or more target DNA sequences are not cleaved or degraded.
  • the method comprises expressing a synthetic CRISPR/cas locus in a cell comprising one or more target DNA sequences, wherein the CRISPR. comprises one or more spacer sequences complementary to the one or more target DNA sequences, and wherein the spacer sequences hybridize to said one or more target DNA sequences.
  • the synthetic CRISPR/cas system is a Cascade or a Cascade-like CRISPR/cas locus.
  • the CRISPR/cas system is capable of multiplexed modulation of target DNA sequences.
  • the method comprises expressing a synthetic CRISPR/cas system in a ceil comprising two or more target DNA sequences, wherein the CRISPR comprises two or more spacer sequences complementary to the two or more target DNA sequences, and wherein the spacer sequences hybridize to said two or more target DNA sequences and modulate expression or function of said target DNA sequences.
  • the method comprises modulating expression or function of multiple target sequences, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more target DNA sequences.
  • the multiple target sequences are modulated by expression of a single CRISPR/cas locus in the cell.
  • expression of one or more target DNA sequences is diminished. In another embodiment, the expression of one or more target DNA sequences is increased. In one embodiment, the method provided turns on a gene that was not previously expressed.
  • the CRISPR/cas system is a Cascade or Cascade-like system. In one embodiment, the Cascade or Cascade-like system does not comprise an active nuclease. In a further embodiment, the Cascade or Cascade-like system does not comprise Cas3.
  • the CRISPR/cas complex is any type of CRISPR/cas complex.
  • the CRISPR/cas complex is a Cascade-like CRISPR/cas complex.
  • Cascade-like CRISPR/cas complexes include, but are not limited to, Cascade, aCASCADE, the Csy-complex, and I-C Dvulg Cascade (see, for example, Bourns et al. Science (2008);321 (5891):960-4; lore et al Nat Struct Mo! Biol 2011; 18(5) :529-36; Wiedenheft et al Nature 2011 ;477(7365):486-9; Lintner et al J Biol Chem.
  • the CRISPR/cas complex is a Type I CRISPR/cas complex.
  • the Type 1 CRISPR/cas complex is a CR1SPR Cascade complex.
  • the CRISPR/cas system is a Type II CRISPR/cas system In another embodiment, the CRISPR/cas system is a Type III CRISPR/cas system.
  • the CRISPR/cas system is expressed in a cell and the Cas proteins and crRNA are expressed and self-assemble into a CR1SPR complex capable of binding to target DNA sequences and recruiting Cas nucleases for target DNA sequence destruction.
  • the components of the CRISPR/cas system are introduced into the ceil on separate expression vectors. In another embodiment, the components of the CRISPR/cas system are introduced into the cell on a single expression vector.
  • the present invention provides a method of modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR'cas locus comprising spacer sequences complementary to said one or more target DNA sequences to a cell, wherein the spacer sequences hybridize to said one or more target DNA sequences, and wherein expression of said one or more target DNA sequences is turned on or increased.
  • multiple target DNA sequences are turned on or increased.
  • one or more of the Cas proteins is tethered to a transcriptional initiation factor.
  • the CRISPR/cas system directs expression of the target sequence.
  • the protein that is tethered to a transcriptional initiation factor is CasA, and the transcriptional initiation factor is tethered to the C-terminus of CasA.
  • the protein that is tethered to a transcriptional initiation factor is CasE, and the transcriptional initiation factor is tethered to the N-terminus of CasE.
  • the transcriptional initiation factor is a sigma factor.
  • the transcriptional initiation factor is sigma 32 ( ⁇ 32 ).
  • the transcriptional initiation factor is a eukaiyotic transcriptional initiation factor
  • Eukaiyotic transcription factors are any factors that can activate or repress transcription of a single eukaryotic gene or a number of eukaryotic genes, and include DNA binding proteins, proteins that bind DNA binding proteins, protein kinases, protein phosphatases, protein methyltransferases, GTP-binding proteins, and the like.
  • Such transcription factors are well known in the art and include, but are not limited to, STAT family proteins (e.g., STATs 1 , 2, 3, 4, 5, and 6), fos/jun, NFkappaB, HIV-Tat, and the E2F family.
  • the present invention provides a method of modulating the expression of a target DNA sequence comprising expressing a synthetic CRISPR/cas locus in a eukaiyotic cell comprising a target DNA sequence.
  • the CRISPR/cas locus is codon optimized for eukaiyotic expression.
  • the CRISPR/cas locus further comprises a nuclear localization signal.
  • a method for modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR/cas locus in a cell comprising said one or more target DNA sequences, wherein the method provided induces fewer off-target effects as compared to other methods known in the art, such as TALENs and Zinc finger nucleases.
  • the method induces at least 80% fewer off-target effects as compared to other methods of gene targeting known in the art.
  • the method provided induces at least 100% fewer off-target effects as compared to a. other methods of gene targeting known in the art.
  • the method provided does not induce off-target effects.
  • the present invention provides a method of modulating the expression or function of a target DNA sequence in a cell wherein the target DNA sequence is not cleaved or degraded, comprising expressing a synthetic CRISPR/cas system in a cell comprising a target DNA sequence, wherein said CRISPR comprises one or more spacer sequences complementary to the target DNA sequence, wherein said spacer sequences hybridize to said target DNA sequence, and wherein the spacer sequence is at least 85% complementary to the target DNA sequence.
  • the spacer sequence is at least 95% complementary to the target sequence.
  • the spacer sequence is 100% complementary to the target DNA. sequence.
  • transcriptional repression or activation of the target gene is tunable based on the extent that the spacer sequence is complementary to the target sequence.
  • a spacer sequence that is less than 100% complementary to the target sequence may repress or activate expression of a DNA. sequence to a lesser extent compared to a spacer sequence that is 100% complementary to the target sequence.
  • the synthetic CRISPR/cas locus is specific for more than one site on a target gene.
  • the number of sites within a gene that are targeted may affect the extent of repression or activation of a target gene.
  • a CRISPR may comprise spacer sequences complementary to 1 , 2, 3, 4, 5, 6, 7, 8 or more sites on either the coding or template stand of a gene or in a promoter region of the gene.
  • a CRISPR comprising spacer sequences complementary to multiple genes affects repression or activation of a target gene to a larger extent than a CRISPR comprising spacer sequences complementary to one site on a gene.
  • the synthetic CRISPR/cas system can be used to regulate gene expression by targeting a promoter, enhancer of gene expression, repressor of gene expression, or any other sequence involved in the control of gene expression.
  • the synthetic CRISPR/cas locus is specific for a promoter sequence. In another embodiment, the synthetic CRISPR/cas locus is specific for an enhancer or repressor of gene expression.
  • the methods provided further comprise the use of suppressors of CRISPRs to reverse the synthetic CRISPR-mediated gene regulation.
  • Suppressors of CRISPR may be anti-CRISPR proteins, including virally-encoded proteins, non-coding RNA, or other materials that interfere with the CRISPR system.
  • the synthetic CRISPR/cas system may be used to turn on or turn off gene expression, and the effect of the synthetic CRISPR/cas system may be immediately reversed through the expressions of one or more suppressors of CRISPR in the cell.
  • anti-CRISPR proteins bind directly to the Cascade or Cascade-like complex, thereby interfering with gene regulation.
  • compositions and methods provided herein provide a programmable system, for modulation of gene expression that includes the ability to turn on as well as turn off genes; allows modulation that may be multiplexed; is associated with few or no off-target effects; is tunable based on the number of targeted sites on a gene or the location of the targeted sites (e.g., promoter targets); and is reversible via suppressors of CRISPR.
  • the method comprises expressing two or more synthetic products
  • the two or more synthetic CRISPR/cas loci comprise spacer sequences that hybridize to different DNA target sequences.
  • the DNA target sequences are in the same biological pathway.
  • the DNA target sequences are in different biological, pathways.
  • the present in vention pro vides a method of modulating the expression or function of one or more target DNA sequences in a cell wherein the cell is a prokaryotic cell, bacterial or archaeal.
  • a target DNA may be dsDNA or ssDNA.
  • the present invention provides a method of modulating the expression or function of one or more ssRNA target.
  • a method for modulating one or more target RNA sequence in a cell comprising expressing a Cascade or Cascade-like complex in a cell comprising the one or more target RNA sequences, wherein the CRISPR is specific for the target RNA sequence, wherein the Cascade or the Cascade-like complex comprises an RNase, and the RN A target is degraded.
  • the target RNA sequence is a microRNA.
  • the cell is a eukaryotic cell. In another embodiment, the cell is present in a subject. In another embodiment, the subject has a disease or condition caused in whole or in part by the one or more target DNA sequences. In another embodiment, the cell is a. eukaryotic cell, and the cell is present in a. subject. In a further embodiment, the subject is a human.
  • the present invention provides a method of treating, affecting, or ameliorating a disease or condition caused in whole or in part by one or more target DNA sequences comprising expressing a synthetic CRISPR/cas complex in the cell of an animal or plant having the disease or condition, wherein said synthetic CRISPR/cas complex comprises a one or more CRISPR comprising one or more spacer sequences complementary to said one or more target DNA sequences, and wherein the spacer sequences hybridize to the one or more target DNA sequences.
  • the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex.
  • the activity, expression, or function of one or more target DNA sequences are modulated as a result of the hybridization to the spacer sequences.
  • the activity, expression, or function of multiple target DNA sequences associated with the disease or condition are modulated by the synthetic CRISPR/cas complex.
  • the one or more target DNA sequences are not, cleaved or degraded.
  • the animal is a human.
  • the animal is a livestock animal, such as, for example, a pig or a cow.
  • the plant is a plant grown for agricultural purposes.
  • the present invention provides a method of treating, preventing, or ameliorating a disease or condition caused in whole or in part by one or more target DNA and/or RNA sequences comprising expressing a synthetic CRISPR/cas complex in the cell of an animal or plant, wherein said synthetic CRISPR/cas complex comprises one or more spacer sequences complementary to one or more target DNA and/or RNA sequences, and wherein the spacer sequences hybridize to said one or more target DNA and/or RNA sequences.
  • the synthetic CRISPR/cas complex is a Cascade or Cascade-like complex.
  • the activity, expression or function of one or more target DNA and/or RNA sequences are modulated as a result of the hybridization to the spacer sequence.
  • the activity, expression or function of multiple target DNA and/or RNA sequences are modulated by the synthetic CRISPR/cas complex.
  • the animal is a human.
  • the animal is a livestock animal, such as, for example, a pig or a cow.
  • the plant is a plant grown for agricultural purposes.
  • the present invention provides a method of treating, preventing, or ameliorating an infection comprising expressing a synthetic CRISPR'cas locus in the cell of a subject infected by an infectious agent or at risk of infection by an infectious agent, wherein said synthetic CRISPR/cas locus comprises one or more spacer sequences complementary to one or more target DNA and/or R A seque ces from said infectious agent, and wherein the spacer sequences hybridize to said one or more target DNA and/or RNA sequences.
  • the synthetic CRISPR/cas locus is a. Cascade or Cascadelike complex.
  • the one or more target DNA and/or RNA sequences are modulated as a result of the hybridization to the spacer sequence.
  • multiple target DNA and/or RNA sequences from the infectious agent are modulated by the synthetic CRISPR/cas locus.
  • the target DNA sequences are not cleaved or degraded.
  • the infectious agent is a bacterium, virus, fungus, LINEs (Long interspersed elements); SINEs (Short, interspersed elements) or other genetic parasite.
  • the subject is an animal, for example, a human. In another embodiment, the subject is a plant or plant cell .
  • the present invention provides a method of inhibiting horizontal gene transfer.
  • Horizontal gene transfer includes, but is not limited to, phage transduction, DNA transformation, and plasmid conjugation.
  • a method is provided in which CRISPR/cas complex are expressed in a cell, tissue, or subject in order to inhibit horizontal gene transfer into the DNA of said cell, tissue, or subject.
  • the subject is an arc ea or bacteria.
  • Figure 1 shows Cascade protein subunits (CasA, CasB, CasC, CasD, and CasE) and crRNA following expression and purification of the self-assembling complex from E. coli cells.
  • Figure 2A shows purification of the holo Cascade, or subcomplexes that are missing either CasA (-A) or CasA and CasB (-B).
  • Figure 2B show's the purified Cascade complex from T, thermophilics.
  • Figure 3A depicts the regions of the GFP sequence for which complementary spacer sequences were generated (i.e., region 1 , 2, 3, and 4) to design synthetic CRISPR/cas loci that contain one or more spacer sequences (i.e., CRISPR/cas loci that contain 1 , 2, 3, or 4 complementary spacer sequences).
  • Figure 3B shows cell pellets from cells expressing the GFP expression plasmid (pGLO) alone (top row): cell pellets from cells expressing the pGLO plasmid and an anti-phage CRISPR (middle row); and cell pellets from cells expressing the pGLO plasmid and the anti-GFP CRISPR with the Cascade gene cassette (i.e., casA-E) (bottom row), in the presence of IPTG (induces Cascade and CRISPR) alone (left column); L-arabinose (induces GFP expression) alone (middle column); or both IPTG and L-arabinose (right column),
  • Figsire 3C depicts a quantitative analysis of GFP expression in cells expressing BL21 control plasmid; pGLO control plasmid; control plasmid comprising an anti-phage CRISPR; and plasmids comprising pGLO and the indicated anti-GFP CRISPR with the Cascade gene cassette. Black arrow
  • Figure 4 shows white light images (left column of images) as well as UV-light images (right column of images) of representative colonies of the E, coli cells from GFP- CRJSPR studies.
  • GFP+ or GFP- green or not green, respectively.
  • Figure 5 shows binding of an anti-CRISPR protein to the Cascade-like complex
  • Viruses that infect bacteria are the most abundant and diverse biological agents on the planet. Bacteriophages and plasmids are major purveyors of traits that confer antibiotic resistance, enhance bacterial adhesion, colonization, invasion, dissemination through human tissues, resistance to immune defenses, transmissibility among humans and exotoxin production.
  • the selective pressures imposed by bacteriophages and plasmids have a profound impact on the composition and behavior of microbial communities in every ecosystem.
  • microbes In response to these selective pressures microbes have evolved a sophisticated nucleic acid-based adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeat) (Al- Attar S. et al.
  • Bacteria and archaea acquire resistance to viral and piasmid challengers by integrating short fragments of foreign nucleic acids into the host chromosome at one end of the CRISPR (Barrangou et al Science 2007 3 i 5(5819): 1709-12; Cady et al J Bacterial 2012; Datsenko et al Nat Commun 2012 3:945; Pourcel et al Microbiology 2005 151(Pt 3):653; Swarts et al PloS One 2012 7(4):e35888; each of which is incorporated herein by reference in its entirety).
  • Each CRISPR/cas locus comprises of a series of repeat sequences of approximately 24-48 nucleotides that are separated by unique 'spacer' sequences derived from foreign genetic elements, like viruses and plasmids.
  • Each CRISPR/cas complex or system also includes CRJSPR-associated genes (cas genes) involved in surveillance for and acquisition and destruction of foreign DNA.
  • Foreign DNA sequences selected for integration are called protospacers (i.e.
  • protospacers are selected from regions of DNA that are flanked by a short sequence motif referred to as the protospacer adjacent motif (PAM) (Deveau et al J Bacterial 2008 190(4): 1390; Horvath et al J Bacterial 2008 190(4): 1401; Mojica et al Microbiology 2009 155(Pt 3):733 and Yosef et al Nucleic Acids Res 2012 40(12):5569; each of which is incorporated herein by reference in its entirety).
  • PAM protospacer adjacent motif
  • Type I and Type II CRISPR systems PAM sequences are essential for high affinity binding.
  • CRISPR-mediated adaptive immune systems proceed according three distinct stages: acquisition of foreign DNA, CRISPR RNA biogenesis, and target interference. Although these three basic stages appear to be common to all CRISPR systems, CRISPR/cas loci and the proteins that mediate each stage of adaptive immunity are remarkably diverse.
  • the CRISPR system is considered a Type 1 CRISPR system.
  • Cascade CRISPR-associated complex for antiviral defense
  • Cascade is a 405- kDa- ribonucleoprotein complex composed of 11 subunits of five functionally essential Cas proteins (one CasA, two CasB, six CasC, one CasD and one CasE, corresponding to SEQ ID NOs: 1 , 2, 3, 4, and 5:
  • Cas A-E are also known as Csel , Cse2, Cse4, Cas5e, and Cse3, respectively) and a 61- nucleotide crRNA.
  • the cas genes for the Cascade-like complexes are related to but distinct from tliese sequences.
  • Cascade finds and binds specific dsDNA sequences with high efficiency and affinity, in part through recognition of the Protospacer Adjacent Motif (PAM), which is a 2 to 5 sequence motif adjacent to the protospacer.
  • PAM Protospacer Adjacent Motif
  • the PAM is essential for high affinity dsDNA binding in the E. coli Cascade system, as well as in Type IA-F and Type IIA-B CRISPR systems.
  • Cascade but not Cas9 can bind to ssDNA and ssRNA targets that do not have an adjacent PAM.
  • dsDNA binding results in base pairing that extends along the crRNA, resulting in a series of short helical segments that trigger a concerted conformational change.
  • This conformational rearrangement may serve as a signal that recruits a trans-acting nuclease (Cas3) that, is required for target degradation.
  • Cas3 trans-acting nuclease
  • Type II CRISPR systems use a trans-encoded small RNA (tracrRNA) that pairs with the repeat fragment of the pre-erRNA, followed by degradation of the invading DNA by the nuclease Cas9.
  • Cas6 is responsible for the processing step (i.e., the processing of foreign DNA for integration of the foreign DNA), but the crRNAs are believed to be transferred to a distinct Cas complex (called Csm in subtype 1 ⁇ - ⁇ systems and Cmr in subtype III-B systems) for degradation of target sequences.
  • the two subtypes of CRISPR -Cas type III systems target either DNA (subtype III- A systems) or RNA (subtype III-B systems).
  • the synthetic CRISPRs provided herein utilize CRISPR-associated complexes known as Cascade, or Cascade-like complexes, which comprise a multi-unit architecture.
  • Cascade or Cascade-like CRISPR/cas loci can be loaded with multiple spacer sequences, or crRNA guides.
  • spacer sequences are encoded in a single Cascade or Cascade-like CRISPR/cas locus; the Cascade or Cascade-like complex comprises a subunit (e.g., the endoribunuciease Cas6) that processes the CRISPR RNA and then loads each Cascade molecule with the spacer sequences.
  • the single Cascade or Cascadelike CRISPR/cas complex is able to process and load multiple mature CRISPR RN As (crRNAs), thereby making possible a multiplexed CRISPR system for gene targeting.
  • crRNAs CRISPR RN As
  • compositions and methods for multiplexed gene targeting are meant targeting more than one different target on one or more different genes. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 15, 20, 30 or more different targets on genes on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 30 genes that are targeted by spacers on a single CRISPR-'cas locus.
  • CRISPR is hypothesized to accelerate target identification through complex binding behaviors that involve fast association and dissociation rates with non-target sequences. It is further hypothesized that crRNA targeted to a particular sequence may be utilized to silence or induce expression of the target sequence. Silencing effects on the expression of a target sequence are due to crRNA hybridization inhibiting the binding of RNA polymerase or other transcription factors, resulting diminished or silenced gene expression without destruction of the target sequence; induced or increased expression of a target sequence may occur when a transcription initiation factor is tethered to a CRISPR protein expressed with the crRNA.
  • the present invention provides methods, systems, and compositions for use in modulating the expression and/or function of one or more target DNA sequences using synthetic CRISPR/cas loci.
  • the present invention provides methods, systems, and compositions for manipulating the genomes of organisms including bacterial, plant, and animal organisms.
  • the one or more target DNA sequences are in a eukaryotic ceil.
  • the one or more target DNA sequences are in a prokaryotic cell.
  • the cell is in a subject.
  • the cell is in vitro, or outside a subject.
  • the provided methods, systems, and compositions relate to interfering with horizontal gene transfer.
  • the compositions and methods of the present invention relate to the use of CRISPR to interfere with an organism's ability to receive horizontal gene transfer of genetic material or provide resistance against horizontal gene transfer from bacteria, viruses, and plasmids.
  • the methods, systems, and compositions provided herein prevent the exchange of bacterial DNA by transduction.
  • the methods, systems, and compositions provided herein protect a subject, tissue, or cell from the transfer of foreign genes.
  • the subject, tissue, or ceil may be protected from horizontal transfer of foreign genes from plasmids, bacteria, viruses, and the like via the methods, systems, and compositions provided herein.
  • the present invention provides methods, systems, and compositions useful in biotechnology, medicine, veterinary medicine, research, commercial, industrial, and other fields.
  • the methods and compositions of the present invention provide programmable systems for targeted genome modification.
  • the programmable systems allow multiplexed gene targeting, i.e., targeting multiple genes with a. single synthetic CRISPR/cas locus.
  • the methods and compositions of the present invention provide programmable systems for targeting genome modification in organisms that lack facile mechanisms for generating gene knockouts.
  • Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have proven to be powerful tools for targeted genome modifications in mammalian cells. However, these technologies require elaborate and sometimes error prone methods for engineering proteins to recognize specific sequences.
  • compositions and methods of the present invention are simpler and/or more programmable than the CRISPR systems previously reported in the art.
  • the methods and compositions described herein can be easily manipulated to perform multiplexed gene regulation.
  • the programmable CRJSPR system described herein does not comprise a nuclease, or comprises an inactive nuclease (e.g., a nuclease that is catalyticaliy dead).
  • the programmable CRISPR system described herein comprises an inactive (e.g., catalyticaliy dead) nuclease.
  • the methods and compositions disclosed herein do not result in degradation of target sequences.
  • the methods and compositions described herein provide an easily manipulated tool for multiplexed gene targeting, including both turning on and turning off of specifically targeted genes.
  • the compositions and methods of the present invention use CRISPR RNA-guide complexes to block gene transcription.
  • the compositions and methods of the present invention may be used to modulate gene expression by turning a gene on.
  • the compositions and methods of the present invention in one embodiment, modulate gene expression with minimal toxicity and minimal off-target effects.
  • the compositions and methods of the present invention may be applied to a broad spectrum of CRISPR pathways.
  • the compositions and methods described herein harness Cascade or a Cascade-like complex, and therefore allow many distinct crRNAs to be loaded in a single CRISPR/cas locus.
  • the synthetic CRISPR functions to diminish or silence gene expression in the absence of nuclease activity. In one embodiment, the lack of nuclease activity results in a lower frequency of off-target effects. In one embodiment, a synthetic CRISPR/cas locus induces at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least, 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% fewer off-target effects when compared to an RNA interference system targeted to the same gene. In another embodiment, a synthetic CRISPR/cas locus does not induce any off-target effects.
  • the synthetic CRISPR/cas locus can be targeted to specific genes in order to block or augment expression of said genes.
  • the compositions and methods herein are unlike RNAi at least in that they act at the DNA level.
  • Cascade but not Cas9, can also act on ssDNA and ssRNA.
  • the synthetic CRISPR/cas is designed to target RNA.
  • Cascade is tethered to an RNase and an RNA is thereby specifically targeted and destroyed using the synthetic CRISPR/cas system.
  • the target RNA is a microRNA (e.g., a small non-coding RNA that functions as a post- transcriptionai regulator of gene expression).
  • CRISPR/cas locus complexes inhibit the recruitment and'Or elongation of the RNA polymerase due to fast on-rates and slow off-rates of crRN A binding, and thereby silence expression of genes to which the CRISPR is directed via spacer sequences complementary to the target gene.
  • the CRISPR localizes to the target gene to which it is directed via complementary spacer sequences, and initiates or augments expression of the target gene.
  • the knockdown of specific genes is reversible insofar as the Cas proteins and RNA guides are eventually diluted due to cellular growth.
  • the methods and compositions provided herein provide the ability to reverse the effects on target genes in a more controlled fashion, using suppressors of CRISPRs.
  • Suppressors of CRISPRs are agents that, alter or interfere with the activity of CRISPRs.
  • Suppressors of CRISPRs may be virally-encoded proteins, non-coding RNA, or other virus-derived materials that interfere with the CRISPR system.
  • Suppressors of CRISPRs may also be agents that mimic the activity of virally-encoded suppressors of CRISPR.
  • Suppressors of CRISPR include those described in the art, for example, in Bondy-Denomy, Nature 2013 493(7432):429-32, which is herein incorporated by reference in its entirety.
  • the synthetic CRISPR/cas locus may be used to turn on or turn off gene expression, and the effect of the synthetic CRISPR cas locus may be immediately reversed through the use of one or more suppressors of CRISPR.
  • a suppressor of the CRISPR system is an anti-CRISPR protein.
  • the anti- CRISPR protein binds directly to the Cascade or Cascade-like complex, thereby interfering with target binding, regulation, or nuclease recruitment.
  • any suppressor of CRISPR may be used in the methods described herein to turn off the synthetic Cascade-like CRISPR system-mediated gene regulation.
  • an anti-CRISPR protein that binds to a Cascade or Cascade-like complex or component of the complex may be used in the methods described herein to turn off the synthetic Cascade of Cascade-like CRISPR- mediated gene regulation system.
  • the CRISPR/cas system or complex is any type of
  • the CRISPR/cas complex is a Cascade or Cascade-like complex.
  • the CRISPR/cas complex is a Type I CRISPR/cas complex.
  • the Type I CRiSPR'cas complex is a CRISPR Cascade complex.
  • the CRISPR/cas system is a Type II CRiSPR'cas system.
  • the CRISPR/cas locus is a Type III CRiSPR'cas locus.
  • the synthetic CRISPR/cas complex is Cascade or a Cascadelike complex such as, for example, Cascade, aCASCADE, the Csy-complex, or I-C/Dvulg Cascade.
  • the CRISPR/cas complex comprises the Cascade complex.
  • the Cascade complex is the holo Cascade complex from a bacterial organism.
  • the Cascade complex is missing one or more Cas proteins that are normally found in the Cascade complex.
  • the Cascade complex is missing CasA (-A), CasB (-B), Ca.sC (-C), CasD (-D), CasE (-E), or any combination of Cas proteins (e.g., CasA. and CasB (-A-B)).
  • the Cascade complex is the Cascade complex or a Cascade- like complex from, any bacterial organism.
  • the Cascade complex is the Cascade complex from. Escherichia coli.
  • the Cascade complex is the Cascade complex from Streptococcus thermophilus or Thermus therniophilus.
  • the term, "gene,” as used herein, refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • the nucleic acid may also optionally include non-coding sequences such as promoter or enhancer sequences.
  • the term "intron” refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons.
  • the term “genome” refers to the DNA of an organism or the RNA of a virus.
  • the terms “genome” and “genomic DNA” encompass genetic materia! that may have undergone amplification, purification, or fragmentation. The genome may encompass the entirety of the genetic materia! from of an organism, or it may encompass one chromosome from an organism with a plurality of chromosomes.
  • nucleic acid or nucleic acid molecule refers to any DNA or
  • the DNA or RNA molecule is single stranded (i.e., ssDNA or ssRNA). in other embodiments, the DNA or RNA molecule is double stranded (i.e., dsDNA or dsR A).
  • isolated nucleic acid refers to DNA that is free of flanking genes present in the naturally-occurring genome of the organism from which the DNA is derived.
  • isolated nucleic acid refers to an mR A molecule that is encoded by an isolated DNA molecule, or that is chemically synthesized, or that is separated or substantially free from at least some cellular components such as other types of RNA molecules or proteins.
  • isolated nucleic acid includes, for example, a recombinant DNA which is incorporated into a vector, such as a plasmid or virus; or incorporated into the genomic DNA of a prokaryoie or eukaryote; or which exists as a separate molecule.
  • a synthetic CRISPR/cas locus is expressed in a cell by methods well known in the art.
  • the term “express,” “expressed,” or “expressing” a gene or protein refers to the introduction of a nucleic acid or protein, or the presence of a nucleic acid or protein, in a ceil. Expression may be at the nucleic acid or protein level. In some embodiments, the cell is transfected with the nucleic acid.
  • modulate refers to a. change or an alteration in expression or activity of a gene. Modulation may be an increase or a decrease in expression. In some embodiments, modulation means that expression or activity of a gene is diminished, reduced, inhibited, or silenced. In other embodiments, modulation means that the expression of a gene is augmented, for example, turned on or increased. In some embodiments, the expression of the gene is reduced or increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more.
  • Expression of one or more genes is measured by methods well known in the art that include, but are not limited to, Northern blot, PGR, in situ hybridization, RNA-seq, and dot-blotting. In some embodiments, expression is reduced or increased by at least 1 % as measured by Northern blot. In some embodiments, expression is reduced or increased by at least 10% as measured by Northern blot. In other embodiments, expression is reduced or increased by at least 50% as measured by Northern blot.
  • off-target effects and “non-specific effects” are used interchangeably herein and refer to undesirable effects of a composition on genes and proteins that the composition is not designed to target.
  • the compositions described herein demonstrate reduced off target effects as compared to gene silencing techniques commonly known in the art. Off-target effects can be measured via methods known by those skilled in the art, including microarray analysis or RNA-seq (i.e., whole-transcriptome shotgun sequencing; see, for example, Qi et ah, 2013 Cell 152; 1 173-83). To measure the number of differentially expressed genes. The number of differentially expressed genes may be expressed as the frequency of off-target effects.
  • a differentially expressed gene may be defined as a gene having at least a 2-fold change in expression after treatment.
  • the change in expression may be an increase or a decrease in expression.
  • the term "eukaryote” as used herein refers to an organism with a nucleus and complex cellular structures enclosed within membranes.
  • the eukaryote is a yeast, metazoan, or animal.
  • the animal is a mammal.
  • the mammal may be human, non-human primate, canine, feline, rodent, bovine, equine, porcine, or any other mammal.
  • prokaryote refers to organisms such as bacteria and archaea that have cells lacking a nucleus.
  • a "vector” is a composition of matter, which can be used to deliver a nucleic acid of interest to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasrnids, and viruses.
  • the term “vector” includes an autonomously replicating or non-replicating plasmid or a virus.
  • viral vectors include, but are not, limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • the terms "expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.
  • a "promoter” refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat).
  • CMV human cytomegalovirus
  • the promoter is a tissue specific promoter. The promoter together with other transcriptional and translational regulatory nucleic acid sequences is necessary to express a given gene.
  • transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • promoter also refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a vector comprises a promoter that is operably linked to a polynucleotide.
  • operably linked means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • the polynucleotide is under transcriptional control of a promoter.
  • the promoter operably linked to a polynucleotide may be an inducible promoter.
  • Inducible promoters include, but, are not limited to, tetracycline promoter, metallothionein IIA promoter, heat, shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.
  • the present invention provides a method of modulating the expression of one or more target DNA sequences comprising expressing a synthetic CRISPR cas system comprising a CR ISPR locus comprising spacer sequences complementary to the one or more target DNA sequences, wherein said spacer sequences hybridize to said one or more target DNA sequences, and wherein expression of the one or more target DNA sequences is turned on or increased.
  • the CRISPR/cas system comprises one or more protein that is tethered to a transcriptional initiation factor.
  • Tethered refers attachment or connection of two compositions (e.g., a CRISPR-related protein and a transcriptional initiation factor) and may be used interchangeably herein with “fused” or “linked.”
  • Transcriptional initiation factors for prokaryotic transcription are well known in the art and include sigma factors
  • Transcriptional initiation factors for eiikaryotic transcription are well known in the art and include, for example, STAT family proteins (e.g., STATs 1 , 2, 3, 4, 5, and 6), fos/jun, NFkappaB, HIV-Tat, and the E2F family.
  • STAT family proteins e.g., STATs 1 , 2, 3, 4, 5, and 6
  • the transcriptional initiation factor is tethered to the C-terminus or the N-terminus of the CRISPR protein.
  • the transcriptional initiation factor is tethered to CasA, CasB, CasC, CasD, or CasE.
  • the transcriptional initiation factor is tethered to CasA or CasE. In further embodiments, the transcriptional initiation factor is tethered to the C-terminus of CasA or the N-terminus of CasE. In certain embodiments, the transcriptional activation factor and is tethered in such a way that will permit DNA binding and RNA polymerase recruitment without steric clashing.
  • the CRISPR/cas locus directs expression of the target sequence
  • the synthetic CRISPR comprises a nuclear localization sequence.
  • the nuclear localization sequence directs the CRISPR to the nucleus of a eukaryotic cell.
  • nuclear localization signal is used interchangeably with the term “nuclear localization sequence” and refers to an amino acid sequence, which directs a target protein to the cell nucleus by nuclear transport.
  • the signal comprises one or more short, amino acid sequences of positively charged lysines or arginines.
  • Exemplary nuclear localization signals include, but are not limited to, those from the SV40 Large T antigen or SV40 medium T-antigen, influenza virus, and viral Tat proteins such as HIV Tat.
  • the synthetic CRISPR targets a single site in a genome or a single gene within a cell.
  • the synthetic CRISPR or CRISPRs are capable of targeting multiple sites in a genome or multiple genes within a cell.
  • the CRISPR or CRISPRs are capable of targeting multiple sites within a single gene.
  • a CR ISPR is designed to target one or more promoters. Without wishing to be bound by theory, CRISPRs designated to target promoters will block promoter recognition by transcriptional initiators, and these sites will be the most potent inhibitors.
  • a sequence is 100% complementary to a reference sequence if 100% of the nucleotides of the first sequence are able to form a base-pair with nucleotides of the reference sequence; and a sequence is 90% complementary to a reference sequence if 9 out of 10 nucleotides are able to form a base-pair with the nucleotides of the reference sequence.
  • the nucleotide sequence "5'-TATAC-3"' is 100% complementary to a reference sequence "5'-GTATA-3"'.
  • the nucleotide sequence "5'-TATAC-3"' is 80% complementary to a reference sequence "5'-GTATG-3" ⁇
  • the synthetic CRISPR/cas loci comprise sequences that are 100% complementary to their DNA targets. In other embodiments, the synthetic CRISPR/cas loci comprise sequences that are not 100% complementary to their DNA targets.
  • the synthetic CRISPR/cas loci comprise sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%), 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to their DNA targets.
  • transcriptional repression by CRISPR/cas loci is tunable based on the use of CRISPRs that are not 100% complementary to their DNA targets. For example, transcriptional repression or activation in some embodiments is decreased when CRISPR/cas loci contain sequences that are less than 100% complementary to the target DNA sequence.
  • transcriptional repression or activation by CRISPR/cas loci is tunable based on the number of target sites in a single gene. For example, repression or activation by CRISPR of a particular gene is increased when more than one site on the gene is targeted.
  • a CRISPR. may comprise spacer sequences complementary to 1 , 2, 3, 4, 5, 6, 7, 8 or more sites on a gene.
  • the CRISPR/cas systems useful in the invention are Cascade or Cascade-like CRISPR/cas complexes.
  • Cascade-like CRISPR/cas systems include any CRISPR-associated ribonucleoprotein including, but not limited to, Cascade, aCASCADE, the Csy-complex, and 1-C/Dvulg Cascade.
  • CRISPR CRISPR-associated ribonucleoprotein
  • Cascade Cascade-associated ribonucleoprotein
  • aCASCADE the Csy-complex
  • 1-C/Dvulg Cascade 1-C/Dvulg Cascade.
  • CRISPR CRISPR-associated ribonucleoprotein
  • CRISPR CRISPR-associated ribonucleoprotein
  • CRISPR/cas locus CRISPR/cas locus
  • CR ISPR/cas loci are used interchangeably herein and refer to a clustered regularly interspaced short palindromic repeat.
  • spacer sequence and “crRNAs” (“CRISPR-derived RNAs”) are used interchangeably herein and refer to the library of short RNAs that contain unique sequences complementary to a foreign nucleic acid.
  • Synthetic CRISPR or “synthetic CRISPR/cas loci” or “synthetic CRISPR/cas system” refer to CRISPRs that have been engineered to target a particular sequence. Synthetic CRISPRs may be engineered such that they do not include all Cas proteins associated with the CRISPR Type. Synthetic CRISPRs may further comprise transcription factors or transcription initiation factors, nuclear localization factors, or other factors involved in the transcription of sequences. Synthetic CRISPRs may be engineered such that different components of the CRISPR/cas locus are present on different expression vectors. For example, the cas genes, or cas gene cassette, may be present on a separate expression vector comprising the DNA targeting sequences. I one embodiment, the CR1SPR complex self-assembles upon expression of components of the CRISPR/cas locus and the cas genes in a host cell.
  • beneficial or desired results may include inhibiting or suppressing the growth of an infectious agent or killing an infectious agent; inhibiting one or more processes through which an infectious agent infects a cell or subject; inhibiting or ameliorating the disease or condition caused by an infectious agent; inhibiting or ameliorating the symptoms of a disease caused by an infectious agent; or a combination thereof.
  • beneficial or desired results may also include inhibiting or suppressing the development or progression of a disease or disorder that is caused in whole or in part by a target DNA sequence.
  • the target DNA sequence is associated with an infectious agent.
  • the target DNA sequence is not associated with an infectious agent.
  • the subject, plant, or animal has a disease or condition caused in whole or in part by said target DNA sequence.
  • has a disease or condition caused in whole or in part by the target DNA sequence is meant that inappropriate, excessive, or deficient expression of the gene is frequently, typically, or consistently occurs in the presence of the disease or condition.
  • the terms “treat,” “treatment,” or “treating” also refer to prophylaxis or prevention of an infection, disease, or disorder, as well as ameliorating of the symptoms of the infection, disease, or disorder.
  • subject may refer to animals, yeasts, metazoa, bacteria, archaea, cell lines such as human cell lines (e.g., HeLa, 293T ceils, RAW), embryonic stem cells, or hamster cells (e.g., CHO).
  • human cell lines e.g., HeLa, 293T ceils, RAW
  • embryonic stem cells e.g., hamster cells
  • animal refers to humans, non-human primates, sheep, goats, cattle, pigs, deer, elk, clogs, buffaloes, camels, horses, mules, donkeys, cats, bison, both wild-life and domestic, bison/cattle hybrids (beefalo and/or cattalo), antelope, bears, rodents (including mice and rats), monkeys, rabbits, reindeer, caribou, fish, birds, chickens, roosters, ducks, geese, turkeys, oxen, Llamas, alpacas, emus, ostriches, honey bees and other insects.
  • compositions and kits of the invention can be used to achieve methods of the invention.
  • the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
  • the use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or.”
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form, of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form, of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • the five cas genes were cloned into three different expression vectors and synthetic CRISPR/cas locus repeats containing seven identical spacer sequences were cloned downstream of a T7 promoter on a fourth vector that does not contain a ribosome binding site
  • the CasB subunit was engineered to include an N-terminal Strep II tag followed by a
  • One limitation to crystallization can be intrinsic flexibility.
  • limited proteolysis and mass spectrometry analysis may be used to identify regions susceptible to protease trimming.
  • Limited proteolysis is routinely used to establish stable core complexes that are often more amenable to crystallization, and combining this technique with denaturing gel electrophoresis and electrospray mass spectrometry can reveal the identity of flexible regions on the surface of the complex.
  • Our data suggest that Cascade from E. coli is amenable to structure determination using this technique.
  • Example 3 e ulation of expression of specific genes sssisig programmable Cascade [0067]
  • synthetic CRISPR/cas complexes were generated and tested to determine if Cascade can be used as a programmable delivery system for repression of gene expression.
  • a series of synthetic CRISPR/cas loci that contain one or more spacer sequences complementary to specific regions of a reporter gene i.e., green fluorescent protein; "GFP"
  • Spacer sequences were designed to target either the coding or the template stand of the target gene by annealing overlapping oligonucleotides followed by ligation and cloning.
  • Each spacer sequence of the CRISPR was designed to target regions of the GFP sequence with a TT motif 3' of the protospacer ( Figure 3A).
  • coli BL21 DE3 cells were co-transfected with a streptomycin resistant plasmid containing the Cascade gene cassette (i.e., casA-E; other Cas proteins, including Cas3, were not included in the synthetic CRISPR/cas loci), one of several different GFP-targeting synthetic CRISPR/cas loci on a Kanamycin resistant plasmid and an Ampicillin resistant GFP expression plasmid (i.e., pGLO) under the control of an arabinose inducible pBAD promoter.
  • Cascade genes and CRISPR RNA expression were induced with IPTG, and GFP expression was induced with L-arabinose ( Figure 3B).
  • Cells were cultured in a 96-well plate agitated at 220 rpms in a chamber maintained at 37°C. Shaking stopped every 15 mins for a total of 30 seconds while the optical density of each well was measured at 600 nm and then GPF expression was assessed by excitation at 395 nm and emission was recorded at 508 nm.
  • Cells transformed with three plasmids were cultured in media with thee antibiotics (i.e.. Step, Kan, and Amp) and these cells exhibited a protected lag -phase compared to cells that only contained the pGLO (pGLO control) plasmid (Amp only).
  • Figure 4 shows colonies of E. coli cells transfected with control (non-GFP) plasmid, pGLO plasmid, or pGLG plasniid with GFP-targeting CRISPR and cascade cassette.
  • GFP+ colonies of E. coli cells were evident in pGLOgroups (middle row), but were not evident in control (no pGLO) groups or groups transfected with the pGLO plasmid and GFP-targeting CRISPR. and Cascade cassette (bottom, row). The results of the study indicated that synthetic CRISPR/cas loci can be utilized to target specific genes for repression.
  • CRISPR Cascade system is further demonstrated.
  • the use of Cascade to silence a single gene or multiple genes simultaneously, and the use of Cascade to tune expression of the target gene(s) by adjusting the target site location (i.e. blocking recognition of the transcriptional promoter to serve as a potent inhibitor) and/or by designing spacers with that are imperfectly complementary (i.e., less than 100% complementary) to the target sequence is demonstrated.
  • Cas3 is not, included in the experiments, and thus the results of the study will show that gene expression can be repressed without cleaving of target genes via Cas nucleases. The results of the study will further show that repression of gene expression is tunable based on number of target sites, target site location, and extent that the spacer sequences are complementary to the target sequences.
  • RNA is isolated from cells containing synthetic CRISPRs that target a single site or multiple sites on a gene of interest.
  • cells contain synthetic CRISPRs that target a single site on GFP (e.g., position 1 , 2, 3, or 4 as shown in Fignre 3A), or multiple sites on GFP
  • cells contain synthetic CRISPRs that comprise sequences that are not 100% complementary to positions 1 , 2,
  • RNA from each ceil line are separated on 2% agarose gels containing formaldehyde, transferred to nitrocellulose membranes, and subjected to Northern blot analysis using 3z P-labeled probes generated by random hexamer amplification of the gene of interest, for example, GFP (Ready-to-go-DNA Labeling beads, GE Healthcare).
  • Hybridization, detection, and quantification of transcripts are performed using standard methods that are well known by those having ordinary skill in the art and are described in Sambrook, J. et a!., (1989) Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • the CRISPRs targeting one or more heat shock gene are used to demonstrate that Cascade can be engineered to deliver transcriptional initiation factors to the promoters of specific genes.
  • Sigma factor 32 (o J ⁇ ) is genetically tethered to the C terminus of
  • a j is engineered to dangle from the end of Cascade in a way that will permit DNA binding and RNA polymerase recruitment without steric clashing. These regions are selected by docking high-resolution crystal structures of CasA and CasE into the cryo-E map of Cascade, These two proteins are located at opposing ends of the Cascade complex and biochemical studies have shown that fusions at these locations do not perturb Cascade assembly. Similarly, the high-resolution structure of the transcriptional initiation complex from T.
  • thermophilus which includes the RNA polymerase, ⁇ , and a fragment of the promoter DNA, provides a molecular blueprint for predicting how o' 2 might interact with promoter DNA and the RNA polymerase in the context of a Cascade fusion. Using these structures to guide the design, synthetic CRISPRs that target a sequence adjacent to promoters recognized by ⁇ '" are designed.
  • Cells expressing a CRlSPR/cas locus targeting one or more genes are co- expressed in E. coli BL21 DE cells, with or without, the Cascade ⁇ 32 fusion constructs.
  • Virally-encoded suppressors of CRISPRs are described herein as a method for controlling synthetic CRISPR-mediated modulation of gene expression.
  • the Cascade-like complex sometimes referred to as the Csy-complex, was incubated with an anti-CRISPR, and the bound (Csy-complex + anti-CRISPR) or unbound (anti-CRISPR alone) complexes were purified.
  • the anti-CRISPR bound to the Cascadelike protein indicating that an anti-CRISPR protein may be used to regulate Cascade-like synthetic CRISPR-mediated gene modulation.
  • CRISPRs Clustered regularly interspaced short palindromic repeats
  • CRISPR clustered regularly interspaced short palindromic repeat
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Cas3 is a singlestranded DMA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 2011 ;30(7 ⁇ : 1335-42.

Abstract

La présente invention concerne des procédés, des systèmes et des compositions destinés à une modulation génétique programmable basée sur les de répétitions palindromiques groupées, courtes régulièrement espacées (CRISPR). Ces procédés comprennent l'expression d'un locus CRISPR/cas synthétique dans une cellule comprenant une ou plusieurs séquences d'ADN cibles, afin de moduler l'expression ou la fonction d'une ou de plusieurs séquences d'ADN cibles dans une cellule, lesdites une ou plusieurs séquences d'ADN cibles n'étant pas coupées ni dégradées.
PCT/US2013/074372 2012-12-11 2013-12-11 Contrôle de la régulation génétique guidé par arn crispr (répétitions palindromiques groupées, courtes régulièrement espacées ) WO2014093479A1 (fr)

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