WO2017048995A1 - Systèmes de crispr/cas9 et d'arni inductibles et procédés d'utilisation - Google Patents

Systèmes de crispr/cas9 et d'arni inductibles et procédés d'utilisation Download PDF

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WO2017048995A1
WO2017048995A1 PCT/US2016/051992 US2016051992W WO2017048995A1 WO 2017048995 A1 WO2017048995 A1 WO 2017048995A1 US 2016051992 W US2016051992 W US 2016051992W WO 2017048995 A1 WO2017048995 A1 WO 2017048995A1
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expression
cell
gene
cas9
shrna
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Prem PREMSRIUT
Chia-Lin Wang
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Mirimus, Inc.
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • This invention relates to methods and systems for gene targeting, genome editing and transient gene silencing in the field of molecular biology and genetic engineering. More specifically, the invention describes the use of CRISPR-associated nuclease to specifically and efficiently edit DNA sequences coupled with RNA interference to mimic drug therapy.
  • RNA interference post-transcriptional gene silencing
  • quelling these different names describe similar effects that result from the overexpression of transgenes encoding double- stranded RNA precursors, or from the deliberate introduction of double-stranded RNA into cells.
  • the present invention attempts to address issues with gene targeting and genome editing.
  • FIG. 2 is a diagram of a vector for the Inducible CRISPR/Cas9 and RNAi and treatment with CRE or CRE-ER + tamoxifen, according to one embodiment.
  • FIG. 3 is a diagram of a vector for the Inducible CRISPR/Cas9 and RNAi and treatment with tamoxifen, according to one embodiment.
  • FIG. 4 is a diagram of a vector for the Inducible CRISPR/Cas9 and RNAi and treatment with CRE or CRE-ER + tamoxifen, according to one embodiment.
  • FIG. 5 is a diagram of a vector for the Inducible CRISPR/Cas9 and RNAi and treatment with tamoxifen, including the TRE3G promoter and without the shCAS9 # , according to one embodiment.
  • FIG. 6 is a diagram of a vector for the Inducible CRISPR/Cas9 and RNAi and treatment with tamoxifen, including the TRE3G promoter and without the shCAS9 # , according to one embodiment.
  • FIGS. 7-18 are diagrams of a vector for the Inducible CRISPR/Cas9 and RNAi FLEx system that is Cre-loxP based.
  • FIG. 19 is a schematic representation of Collal homing cassette targeting FLEXi vectors.
  • FIG. 20 is a schematic diagram showing the incorporation of the FLEXi vectors into an Embryonic Stem Cells (ESC) system in which both CRISPR/Cas9 and RNAi are induced successively in the same animal, enabling stochastic introduction of somatic mutations in adult mice that can later be treated with shRNA therapy.
  • ESC Embryonic Stem Cells
  • references to "one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched poly
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • chimeric RNA refers to the polynucleotide sequence comprising the guide sequence, the tracr sequence and the tracr mate sequence.
  • guide sequence refers to the about 20 bp sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer”.
  • tracr mate sequence may also be used interchangeably with the term “direct repeat(s)”.
  • FIG. 1 An exemplary CRISPR-Cas system is illustrated in FIG. 1.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • variable should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • Complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part 1, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self 17 hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
  • expression refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as "gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • polypeptide refers to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • the terms "subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • the terms "therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject.
  • the beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.
  • treatment or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • the term "effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • Recombination refers to a process of exchange of genetic information between two polynucleotides.
  • homologous recombination refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, uses a "donor” molecule to template repair of a "target” molecule (i.e., the one that experienced the double-strand break), and is variously known as “non-crossover gene conversion” or “short tract gene conversion,” because it leads to the transfer of genetic information from the donor to the target.
  • such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis-dependent strand annealing," in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes.
  • Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.
  • Cleavage refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, fusion polypeptides are used for targeted double-stranded DNA cleavage.
  • a "cleavage domain” comprises one or more polypeptide sequences which possesses catalytic activity for DNA cleavage.
  • a cleavage domain can be contained in a single polypeptide chain or cleavage activity can result from the association of two (or more) polypeptides.
  • regulatory element is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • IRES may be substituted for P2A.
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • a tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell- type specific.
  • a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol I promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g.
  • pol III promoters include, but are not limited to, U6 and HI promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41 :521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • WPRE WPRE
  • CMV enhancers the R-U5' segment in LTR of HTLV-I
  • SV40 enhancer SV40 enhancer
  • the intron sequence between exons 2 and 3 of rabbit ⁇ -globin Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Promoter s/enhancers which may be used to control the expression of a shRNA construct in vivo include, but are not limited to, the PolIII human or murine U6 and HI systems, the cytomegalovirus (CMV) promoter/enhancer, the human ⁇ -actin promoter, the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR), the SV40 early or late region promoter, the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (RSV), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer, and the herpes simplex virus LAT promoter.
  • CMV cytomegalovirus
  • HMV herpes simplex virus
  • HSV herpes simplex virus
  • Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. Inducible systems, such as Tet promoters may be employed. In addition, recombinase systems, such as Cre/lox may be used to allow excision of shRNA constructs at desired times. The Cre may be responsive (transcriptionally or post-transcriptionally) to an external signal, such as tamoxifen.
  • viruses such as polyoma virus, fowlp
  • “Inhibition of gene expression” refers to the absence or observable decrease in the level of protein and/or mRNA product from a target gene. “Specificity” refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopaline synthase
  • OCS octopine synthase
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • RMCE Recombinase Mediated Cassette Exchange
  • SSRs site-specific recombination processes
  • Fn mutants of the naturally occurring 48 bp FRT-site
  • a gene cassette is flanked by a set of these sites (F and Fn, for example) it can change places, by double- reciprocal recombination, with a second cassette that is part of an exchange plasmid (Figure 1, part A).
  • Figure 1, part A A model experiment is shown in part C, in which an 'empty' cell is modified by either a standard transfection approach or by RMCE. Please note that in the first case multiple genomic sites are hit, each giving rise to a different expression level (cf. the broad distribution of green dots). If a pre-defined genomic address is used to introduce the same gene reporter, each clone derived from such an event shows comparable expression characteristics.
  • Recombinases are genetic recombination enzymes. DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression. These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30-40 nucleotides) target site sequences that are specific to each recombinase. These reactions enable four basic functional modules, excision/insertion, inversion, translocation and cassette exchange, which have been used individually or combined in a wide range of configurations to control gene expression.
  • the "tet inducible system” is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline).
  • the Ptet promoter expresses TetR, the repressor, and TetA, the protein that pumps tetracycline antibiotic out of the cell.
  • Tet-On and Tet- Off is not whether the transactivator turns a gene on or off, as the name might suggest; rather, both proteins activate expression.
  • Tet-Off activates expression in the absence of Dox
  • Tet-On activates in the presence of Dox
  • the Tet-On Advanced transactivator also known as rtTA2S-M2
  • rtTA2S-M2 is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-Off.
  • its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing 3 minimal transcriptional activation domains.
  • Tet-On 3G (also known as rtTA-V16[Clontech Laboratories, Inc.]) is similar to Tet-On Advanced but was derived from rtTA2S-S2 rather than rtTA2S-M2. It is also human codon optimized and composed of 3 minimal VP 16 activation domains. However, the Tet-On 3G protein has 5 amino acid differences compared to Tet-On Advanced which appear to increase its sensitivity to Dox even further. Tet-On 3G is sensitive to 100-fold less Dox and is 7-fold more active than the original Tet-On. Other systems such as the T- REx system by Life Technologies work in a different fashion. The gene of interest is flanked by an upstream CMV promoter and two Tet02 sites.
  • TetR homodimers Expression of the gene of interest is repressed by the high affinity binding of TetR homodimers to each Tet02 sequences in the absence of tetracycline. Introduction of tetracycline results in binding of one tetracycline on each TetR homodimer followed by release of Tet02 by the TetR homodimers. Unbinding of TetR homodimers and Tet02 result in derepression of the gene of interest.
  • Transduction of foreign DNA material is the process by which genetic material, e.g. DNA or siRNA, is inserted into a cell by a virus.
  • genetic material e.g. DNA or siRNA
  • Common techniques in molecular biology are the use of viral vectors (including bacteriophages), electroporation, or chemical reagents that increase cell permeability. Transfection and transformation are also common ways to insert DNA into a cell.
  • Bostocyst injection generate of chimeric mice, i.e. mixtures of ES cell-derived and host blastocyst-derived tissues. The goal is a chimera with high contribution of ES cell-derived tissue, including the germline.
  • ES cells for injection can be prepared. Blastocysts (from strain C57BL/6 for 129-derived ES cells; from strain albino C57BL/6 for C57BL/6-derived ES cells) may be injected with gene-modified ES cells and implanted into recipient dams. Chimeric males may then be used for experimentation.
  • the Inducible CRISPR/Cas9 and RNAi method 100 described here is the novel combination of specific gene editing events (via CRISPR/Cas9, zinc fingers, TALENs, etc.) and RNA interference to be used sequentially and/or in combination in the same biological system (or organism or animal model).
  • the first method is the CRISPR/Cas9 genome editing tool, which initiates DNA cleavage at precise genomic locations to induce DNA repair by one of two mechanisms: NHEJ (non-homologous end joining) or HDR (homology directed repair).
  • these gene editing events are used to generate gene mutations by random insertion or deletions of nucleotides (INDELS) at desired genomic regions that may predispose the biological system or animal model to disease pathogenesis or expression of a desired phenotype.
  • INDELS nucleotides
  • HDR a donor template containing homologous regions along with the desired mutation is also delivered to induce a homologous recombination event and incorporation of the donor template into the genome.
  • the donor template may contain any number of transgene cassettes to alter the genomic DNA including but not limited to cDNAs, point mutation sequences, reporters, miRNAs, etc.
  • Cas9-mediated DNA cleavage can be induced at a precise time by expressing Cas9 from an inducible promoter, such as a TRE (tet-responsive element) promoter as illustrated in FIGS 1-6.
  • This configuration will drive Cas9 expression by the addition of doxycycline (a tetracycline analog) to the system or food or drinking water of an animal (Dow, L.E., Fisher, J., O'Rourke, K.P., Muley, A., Kastenhuber, E.R., Livshits, G., Tschaharganeh, D.F., Socci, N.D., and Lowe, S.W. (2015).
  • doxycycline a tetracycline analog
  • the tGFP-shRNA construct can be engineered in the opposite orientation (as shown in FIG. 1) and not in frame with the promoter, so its expression will not be induced initially following doxycycline treatment.
  • the second method to be applied is a recombination system, such as CRE/Lox or FLP/FRT or DRE/Rox, whereby inverted repeats flank the Cas9-CRE ERT2 construct (as shown in FIG.
  • loxP and lox2272 may be substituted for additional inverted repeats and recombination systems (ie. Flp/FRT, PhiC31/attP/B systems/Dre/Rox). Tamoxifen may be replaced by other estrogen or hormone molecules depending on the recombinase selected.
  • tGFP may be substituted for any reporter or DNA sequence to monitor inhibition of gene expression.
  • the tet-inducible and recombinase systems may be used in combination to create a conditional and inducible CRISPR/Cas9 and RNAi model.
  • an inverted Cas9 cassette may be flanked by opposing loxP sequences, and thus expression of Cas9 may be dependent on CRE recombinase expression. This configuration allows for each component of the system to be independently activated, thus allowing for independent control of expression for both CRISPR/Cas9 and RNAi.
  • both CRISPR/Cas9 and RNAi can be induced successively in the same animal, enabling stochastic introduction of somatic mutations in adult mice that can later be treated with shRNA therapy. It also allows for in situ delivery of CRE+gRNAs (or gRNA libraries) via adeno or lenti-viral delivery to introduce complex driver mutations in adult animals, which greatly advances the ability to model complex mutational patterns found in human disease. Together, this approach will dramatically expedite the rate of producing genetically complex in vivo models of disease with a mode for therapeutic evaluation ail within the same animal.
  • the inducible/ ' condition CRISPR/Cas9-RNAi approach described is rapid, inexpensive and creates a portable system for easy transfer, making it feasible for use in the drug discovery process and perform impactful preclinical validation studies necessary for successful novel and effective drug development.
  • the Inducible CRISPR/Cas9 and RNAi 100 method enables delivery of a single DNA construct into a biological system to facilitate efficient CRISPR/Cas9 mediated gene editing and RNAi interference-mediated gene silencing in combination.
  • Such a system would enable, for example, the induction of a specific disease or phenotype in a biological system or animal model, followed by RNAi-mediated gene silencing, which can effectively model therapeutic intervention.
  • the simplicity of the all-in-one design enables rapid generation of animal models of disease such that only 2 alleles are required for activation of the system: (1) the all-in-one FLEx system (FIG.
  • FLEXi vectors allow for the rapid generation of combination CRISPR/Cas9 and RNAi mice.
  • the Inducible CRISPR/Cas9 and RNAi method is unique in that it enables both inducible or conditional CRISPR/Cas9 and inducible RNAi to be used in the same system. It is conceivable that inducible CRISPR/Cas9 and inducible RNAi in combination could be achieved by combining two unique inducible expression systems, such as the SparQTM cumate switch (System Biosciences, Inc.) or the RheoSwitch inducible expression system (New England BioLabs), however, these systems have not been thoroughly tested in vivo animal models and are not as routinely utilized as the Tet-inducible system (Abe, T., and Branzei, D. (2014).
  • SparQTM cumate switch System Biosciences, Inc.
  • RheoSwitch inducible expression system New England BioLabs
  • the Inducible CRISPR/Cas9 and RNAi method uses Cas9 and shRNA expression to be induced sequentially rather than simultaneously. The purpose of this is to allow mutagenesis to occur initially and reserving the induction of shRNA expression following disease pathogenesis or phenotype manifestation.
  • the Inducible CRISPR/Cas9 and RNAi system may also include a novel shRNA targeting Cas9 (shCas9) to prevent high levels of Cas9 expression from the TRE promoter (FIG. 1). It has been shown that high and/or continuous levels of Cas9 can be detrimental to cells, and therefore to limit its expression, the Inducible CRISPR/Cas9 and RNAi method may include an shRNA on the 3 ' UTR of the Cas9 expression cassette.
  • shCas9 novel shRNA targeting Cas9
  • the shCas9 also serves to control any leaky expression from the TRE promoter itself in the absence of doxycycline (for the Tet-on system) (McJunkin, K., Mazurek, A., Premsrirut, P.K., Zuber, J., Dow, L.E., Simon, J., Stillman, B., and Lowe, S.W. (201 1). Reversible suppression of an essential gene in adult mice using transgenic RNA interference. Proc. Natl. Acad. Sci. USA 108, 71 13-71 18).
  • TRE In a number of cases, the original TRE promoter has been demonstrated to be leaky, such that minimal expression does occur in the absence of doxycycline, and therefore multiple newer generations of promoters (TREtight and TRE3G) have been developed (Abe and Branzei, 2014; Loew et al., 2010). Unfortunately, while these promoters serve to control leakiness, there regulation can be too tight in some cases, such that expression becomes restricted in specific tissues in animal models (McJunkin et al., 2011). Nonetheless, TRE can be replaced with any promoter including the newer TREtight and TRE3G promoters.
  • the Inducible CRISPR/Cas9 and RNAi system is also unique in that it is highly adaptable. A number of versions can be utilized but the system is not limited to only what has been depicted.
  • the CRISPR/Cas9 and RNAi system may comprise more than one small or synthetic guide RNA (gRNA).
  • gRNA small or synthetic guide RNA
  • U6-gRNA cassettes may be expressed upstream of the TRE promoter.
  • U6-RNAs may be cloned in tandem, such as U6-gRNA-gRNA— U6-gRNA-gRNA— U6-gRNA-gRNA.
  • U6 may be substituted by other pol III promoters or regulatory elements, as described previously.
  • the gRNAs may be directed to multiple genes (Dow, L.E., Fisher, J., O'Rourke, K.P., Muley, A., Kastenhuber, E.R., Livshits, G., Tschaharganeh, D.F., Socci, N.D., and Lowe, S.W. (2015). Inducible in vivo genome editing with CRISPR-Cas9. Nat Biotechnol 33, 390-394). Synthetic guide RNAs may be delivered directly to adult animals in situ to generate somatic mutations.
  • the TRE promoter may be replaced by a TREtight (Clontech Laboratories, Mountain View, CA) or TRE3G (Clontech Laboratories, Mountain View, CA) promoter as shown in FIGS. 5-6, or ultimately another inducible promoter once tested and characterized.
  • the TRE promoter may be replaced with a tissue-specific or ubiquitous promoter or regulatory element.
  • the shCas9 may or may not be present depending on the promoter and whether abundant overexpression and/or leakiness is a concern.
  • CRE or CRE ERT2 may be delivered ectopically, for example, in the form of adenoviruses or lentiviruses containing CRE. In normal cells, CreER T2 is cytoplasmic and inactive, however addition of tamoxifen activates the recombinase activity of the fusion protein.
  • tetracycline (tet)-regulated system controls expression of RNAi constructs from tetracycline-responsive promoters (TRE) (Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I, Hannon, G. J., & Lowe, S. W. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nature Genetics. 37 (2005) 1289-95). Briefly, the tet- based system requires the additional expression of a tet-transactivator protein (tTA or rtTA) (Furth, P. A., St.
  • tTA or rtTA tet-transactivator protein
  • KD KD, R, B2, B3 or DRE recombinases
  • CRE/loxP CRE/loxP
  • the IRES sequence may also be interchangeable with P2A (Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A., and Choi, S.Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, el 8556) or any other ribosomal entry sequence.
  • the turboGFP (tGFP) depicted maybe substituted for any other reporter such as an antibiotic resistance cassette, fluorescence reporter, or even another cDNA. In fact, it may be replaced by random DNA sequence so long as it provides a spacer element between the promoter and the shRNA to induce increased RNAi efficiency (Premsrirut et al., 2011).
  • CRISPR cluster regularly interspaced short palindromic repeats
  • the CRISPR-associated nuclease is part of adaptive immunity in bacteria and archaea.
  • the Cas9 endonuclease a component of Streptococcus pyogenes type II CRISPR/Cas system, forms a complex with two short RNA molecules called CRISPR RNA (crRNA) and transactivating crRNA (transcrRNA), which guide the nuclease to cleave non-self DNA on both strands at a specific site.
  • crRNA CRISPR RNA
  • transcrRNA transactivating crRNA
  • the crRNA-transcrRNA heteroduplex could be replaced by one chimeric RNA (so-called guide RNA (gRNA)), which can then be programmed to targeted specific sites.
  • gRNA guide RNA
  • the minimal constrains to program gRNA-Cas9 is at least 15-base-pairing between engineered 5 ' - RNA and targeted DNA without mismatch, and an NGG motif (so-called protospacer adjacent motif or PAM) follows the base-pairing region in the targeted DNA sequence.
  • PAM protospacer adjacent motif
  • 15-22 nt in the 5 ' -end of the gRNA region is used to direct Cas9 nuclease to generate DSBs at the specific site.
  • the CRISPR/Cas system has been demonstrated for genome editing in human, mice, zebrafish, yeast and bacteria.
  • the said method may comprise gene editing and expressing DNA molecules encoding the one or more gene products an engineered, non-naturally occurring vector system comprising one or more vectors comprising: a) a first regulatory element operably linked to one or more Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) system guide RNAs that hybridize with target sequences in genomic loci of the DNA molecules encoding the one or more gene products, b) a second regulatory element operably linked to a Type-II Cas9 protein, wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNAs target the genomic loci of the DNA molecules encoding the one or more gene products and the Cas9 protein cleaves the genomic loci of the DNA molecules encoding the one or more gene products, whereby expression of the one or more gene products is altered; and, wherein the Cas9 protein and the guide RNAs do not naturally occur together.
  • CRISPR Cluster
  • the CRISPR/Cas-like sequence can be derived from a CRISPR/Cas type I, type II, or type III system.
  • suitable CRISPR/Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, C
  • the CRISPR/Cas-like protein of the fusion protein is derived from a type II CRISPR/Cas system.
  • the CRISPR/Cas-like protein of the fusion protein is derived from a Cas9 protein.
  • the Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus s
  • CRISPR/Cas proteins comprise at least one RNA recognition and/or RNA binding domain.
  • RNA recognition and/or RNA binding domains interact with the guiding RNA.
  • CRISPR/Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • the CRISPR/Cas-like protein can be a wild type CRISPR/Cas protein, a modified CRISPR/Cas protein, or a fragment of a wild type or modified CRISPR/Cas protein, or Cpf 1.
  • the CRISPR/Cas protein can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease i.e., DNase, RNase
  • the CRISPR/Cas protein can be truncated to remove domains that are not essential for the function of the fusion protein.
  • the CRISPR/Cas protein can also be truncated or modified to optimize the activity of the effector domain of the fusion protein.
  • the CRISPR/Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof.
  • the CRISPR/Cas-like protein of the fusion protein can be derived from modified Cas9 protein.
  • the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein.
  • domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein.
  • a Cas9 protein comprises at least two nuclease (i.e., DNase) domains.
  • a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double- stranded break in DNA. (Jinek et al., Science, 337: 816-821).
  • the Cas9- derived protein can be modified to contain only one functional nuclease domain (either a RuvC- like or a HNH-like nuclease domain).
  • the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • the Cas9-derived protein is able to introduce a nick into a double-stranded nucleic acid (such protein is termed a "nickase"), but not cleave the double-stranded DNA.
  • nickase a double-stranded nucleic acid
  • an aspartate to alanine (D10A) conversion in a RuvC-like domain converts the Cas9-derived protein into a nickase.
  • a histidine to alanine (H840A) conversion in a HNH domain converts the Cas9-derived protein into a nickase.
  • both of the RuvC-like nuclease domain and the HNH-like nuclease domain can be modified or eliminated such that the Cas9-derived protein is unable to nick or cleave double stranded nucleic acid.
  • all nuclease domains of the Cas9- derived protein can be modified or eliminated such that the Cas9-derived protein lacks all nuclease activity.
  • any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and/or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • the CRISPR/Cas-like protein of the fusion protein is derived from a Cas9 protein in which all the nuclease domains have been inactivated or deleted.
  • compositions and methods for making and using CRISPR-Cas systems are described in U.S. Pat. No. 8,697,359, entitled “CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS,” which is incorporated herein in its entirety.
  • sequence-specific nucleases have been developed to increase the efficiency of gene targeting or genome editing in animal and plant systems.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • the programmable DNA binding domain can specifically bind to a corresponding sequence and guide the chimeric nuclease (e.g., the Fokl nuclease) to make a specific DNA strand cleavage.
  • a pair of ZFNs or TALENs can be introduced to generate double strand breaks (DSBs), which activate the DNA repair systems and significantly increase the frequency of both nonhomologous end joining (NHEJ) and homologous recombination (HR).
  • TALENs with 16-24 tandem repeats can specifically recognize 16-24 by genomic sequences and the chimeric nuclease can generate DSBs at specific genomic sites.
  • TALEN- mediated genome editing has already been demonstrated in many organisms including yeast, animals, and plants.
  • This example describes a system for creating genetically defined RNAi using Cre- mediated recombination to stably invert an integrated a single RNAi expression cassette into the desired orientation at a defined locus in the mouse genome.
  • This technique will minimize clonal variation due to random integration events seen in other studies and should allow for the efficient creation of "epi-allelic" series of RNAi constructs, as well as an inducible RNAi system.
  • Applicants have adapted a system developed for chromosomal engineering in mice to mediate the integration of a single short hairpin RNA (shRNA) expression cassette in mouse ES cells.
  • shRNA short hairpin RNA
  • ERT2-CRE-ERT2 may or may not be present; tamoxifen may be replaced for CRE when ERT2-CRE-ERT2 is not present - TRE may be substituted for any promoter or inducible promoter.
  • loxP and lox2272 may be substituted for additional inverted repeats and recombination systems (ie. Flp/FRT, PhiC31/attP/B, Dre/Rox systems); tamoxifen may be replaced when using a non-ERT2 system.
  • additional inverted repeats and recombination systems ie. Flp/FRT, PhiC31/attP/B, Dre/Rox systems
  • tamoxifen may be replaced when using a non-ERT2 system.
  • the invention provides systems which use RNA interference to stably, conditionally (e.g., with spatial, temporal, and/or reversible control) and specifically target and decrease the expression of one or more target genes in cells.
  • RNA interference effects of exogenously provided dsRNAs can be recapitulated in mammalian cells by the expression of single RNA molecules which fold into stable "hairpin" structures (Paddison, P. J., A. A. Caudy, and G. J. Hannon, Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA, 2002. 99(3): p. 1443-8).
  • shRNAs small "hairpin" RNAs
  • Applicants have now demonstrated that shRNAs can be stably introduced into mammalian cells, preferably in a site-specific manner, introduced into a living organism and propagated without significant loss of the RNA interference effect.
  • the stably integrated RNAi constructs may be conditionally expressed (e.g., expression may be turned on or off in a tissue-specific or reversible manner).
  • shRNA molecules of this type may be encoded in RNA or DNA vectors.
  • the term "encoded” is used to indicate that the vector, when acted upon by an appropriate enzyme, such as an RNA polymerase, will give rise to the desired shRNA molecules (although additional processing enzymes may also be involved in producing the encoded shRNA molecules).
  • vectors comprising one or more encoded shRNAs may be transfected into cells ex vivo, and the cells may be introduced into mammals. The expression of shRNAs may be constitutive or regulated in a desired manner.
  • RNA interference in vivo was unreliable; certain constructs were expressible in stem cells but not in differentiated cells, or vice versa.
  • Technology described herein makes it possible to achieve either constitutive or highly regulated expression of shRNAs in vivo across the spectrum of cell types, thereby permitting tightly controlled regulation of target genes in vivo.
  • a double-stranded structure of an shRNA is formed by a single self-complementary RNA strand.
  • RNA duplex formation may be initiated either inside or outside the cell.
  • Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
  • shRNA constructs containing a nucleotide sequence identical to a portion, of either coding or non-coding sequence, of the target gene are preferred for inhibition.
  • RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
  • the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing).
  • the length of the duplex-forming portion of an shRNA is at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage.
  • the shRNA construct is at least 25, 50, 100, 200, 300 or 400 bases in length.
  • the shRNA construct is 400-800 bases in length. shRNA constructs are highly tolerant of variation in loop sequence and loop size.
  • An endogenous RNA polymerase of the cell may mediate transcription of an shRNA encoded in a nucleic acid construct.
  • the shRNA construct may also be synthesized by a bacteriophage RNA polymerase (e.g., T3, T7, SP6) that is expressed in the cell.
  • expression of an shRNA is regulated by an RNA polymerase III promoters; such promoters are known to produce efficient silencing. While essentially any PolII promoters may be used, desirable examples include the human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse HI RNA promoter and the human tRNA-val promoter.
  • a U6 snRNA leader sequence may be appended to the primary transcript; such leader sequences tend to increase the efficiency of sub-optimal shRNAs while generally having little or no effect on efficient shRNAs.
  • a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
  • Inhibition may be controlled by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
  • RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • the use and production of an expression construct are known in the art (see also WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references cited therein).
  • a shRNA construct is designed with 29 bp helices following a U6 snRNA leader sequence with the transcript being produced by the human U6 snRNA promoter.
  • This transcription unit may be delivered via a Murine Stem Cell Virus (MSCV)-based retrovirus, with the expression cassette inserted downstream of the packaging signal.
  • MSCV Murine Stem Cell Virus
  • An shRNA will generally be designed to have partial or complete complementarity with one or more target genes (i.e., complementarity with one or more transcripts of one or more target genes).
  • the target gene may be a gene derived from the cell, an endogenous gene, a transgene, or a gene of a pathogen which is present in the cell after infection thereof.
  • the procedure may provide partial or complete loss of function for the target gene. Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
  • “Inhibition of gene expression” refers to the absence or observable decrease in the level of protein and/or mRNA product from a target gene. “Specificity” refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Luc nopaline synthase
  • OCS octopine synthase
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
  • RNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.
  • shGOI shRNA targeting a gene of interest, may also be an shRNA within a miRNA backbone, such as miR30.
  • the present invention is not limited to any type of target gene or nucleotide sequence.
  • the following classes of possible target genes are listed for illustrative purposes: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Writ family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JTJN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, T
  • developmental genes e
  • Promoter s/enhancers which may be used to control the expression of a shRNA construct in vivo include, but are not limited to, the PolIII human or murine U6 and HI systems, the cytomegalovirus (CMV) promoter/enhancer, the human ⁇ -actin promoter, the glucocorticoid- inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR), the SV40 early or late region promoter, the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (RSV), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer, and the herpes simplex virus LAT promoter.
  • CMV cytomegalovirus
  • HMV herpes simplex virus
  • HSV herpes simplex virus
  • Transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. Inducible systems, such as Tet promoters may be employed. In addition, recombinase systems, such as Cre/lox may be used to allow excision of shRNA constructs at desired times. The Cre may be responsive (transcriptionally or post-transcriptionally) to an external signal, such as tamoxifen.
  • viruses such as polyoma virus, fowlp
  • a vector system for introducing shRNA constructs into cells are retroviral vector systems, such as lentiviral vector systems.
  • Lentiviral systems permit the delivery and expression of shRNA constructs to both dividing and non-dividing cell populations in vitro and in vivo.
  • Lentiviral vectors are those based on HIV, FIV and EIAV. See, e.g., Lois, C, et al., Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science, 2002. 295(5556): p. 868-72.
  • a highly transfectable 293 cell line may be used for packaging vectors, and viruses may be pseudotyped with a VSV-G envelope glycoprotein for enhanced stability and to provide broad host range for infection.
  • the invention provides novel vectors adapted for use with shRNA expression cassettes.
  • a Gateway recipient sequence may be inserted downstream of the packaging signal to facilitate movement of the shRNA construct to and from different vector backbones by simple recombination.
  • recombination signals may be inserted to facilitate in vivo transfer of shRNAs from, e.g., a genome-wide shRNA library.
  • vector and promoters to be employed should be selected, in part, depending on the organism and cell type to be affected. In the case of ex vivo stem cell therapy for human patients, a vector and promoter that are capable of transfection and expression in human cells should be selected.
  • retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • a retroviral plasmid vector may be employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14.times., VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art.
  • a producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express a polypeptide of the present invention.
  • cells are engineered using an adeno-associated virus (AAV).
  • AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos.
  • an AAV vector may include all the sequences necessary for DNA replication, encapsidation, and host-cell integration.
  • the recombinant AAV vector may be transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc.
  • Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses.
  • any method for introducing a nucleic acid construct into cells may be employed.
  • Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct.
  • a viral construct packaged into a viral particle may be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA.
  • Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like.
  • shRNA-encoding nucleic acid construct may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
  • the invention provides methods of creating specific genetic lesions and dampening gene expression in cells that may attenuate disease by way of CRISPR/Cas9-mediated gene engineering and shRNA expression, respectively.
  • the gRNAs, Cas9 and shRNAs may be reliably expressed in vivo in a variety of cell types.
  • the cells are administered in order to treat a condition. There are a variety of mechanisms by which genetic manipulation combined with shRNA expression in cells may be useful for treating a condition.
  • a condition may be caused in part by a population of cells expressing a combination of undesirable genes created by way of CRISPR/Cas9-mediated editing, some of which must be genetically altered and some which may only be quelled to achieve therapeutic benefits. These cells may be ablated and replaced with administered cells comprising the correct genes and shRNAs to "fix" specific genes and/or decrease expression of other undesirable genes, respectively; alternatively, the diseased cells may be competed away by the administered cells, without need for ablation.
  • a condition may be caused by a deficiency in a secreted factor. Amelioration of such a disorder may be achieved by administering cells expressing a shRNA that indirectly stimulates production of the secreted factor, e.g., by inhibiting expression of an inhibitor.
  • CRISPR/Cas9 may be used to alter the genetic makeup on nearly any gene, just as an shRNA may be targeted to essentially any gene, and in some instances, this combination will be required to achieve the gene expression profile which may be helpful in promoting disease pathogenesis as well as treating a condition.
  • the target genes may participate in a disease process in the subject.
  • the target genes may encode a host protein that is co-opted by a virus during viral infection, such as a cell surface receptor to which a virus binds while infecting a cell. HIV binds to several cell surface receptors, including CD4 and CXCR5.
  • the introduction of HSCs or other T cell precursors carrying specific genetic manipulations and an shRNA directed to an HIV receptor or coreceptor is expected to create a pool of resistant T cells, thereby ameliorating the severity of the HIV infection. Similar principles apply to other viral infections.
  • Immune rejection is mediated by recognition of foreign Major Histocompatibility Complexes.
  • the cells may be genetically altered and transfected with shRNAs that target any MHC components that are likely to be recognized by the host immune system.
  • the shRNA transfected cells will achieve beneficial results by partially or wholly replacing a population of diseased cells in the subject.
  • the transfected cells may autologous cells derived from cells of the subject, but carrying a shRNA that confers beneficial effects.
  • One utility of the present invention is to generate animal models that have both the potential to initiate a disease process and also carry an shRNA or shRNAs that may be used to treat the disease itself.
  • ESC-derived animals can be generated by way of blastocyst injection.
  • CRISPR/Cas9-mediated mutagenesis may by induced by treating the animals with doxycycline. Mutagenesis may even be triggered in embryos by treating pregnant mothers with doxycycline as well. These induced mutations by be disease sensitizing and trigger a cascade of events that lead to disease pathogenesis.
  • inversion of the inserted cassette can be induced by treatment with CRE/tamoxifen. Subsequently, following the inversion event, treatment with doxycycline can induce shRNA expression and thus silencing of specific genes that may have therapeutic potential to treat the disease or attenuate the disease process.
  • the system provides a unique ability to induce multiple genetic manipulations at a specific time point without having to cross the mice to other disease-allele carrying strains. It is distinctive in that shRNAs that suppress gene function may also be used following the onset of disease progression to determine whether the target gene(s) have therapeutic potential or perhaps accelerate disease.
  • One utility of the present invention is as a method inducing a specific phenotype via CRISPR/Cas9 and identifying gene function in the specific phenotype context of an organism, especially higher eukaryotes, by comprising the use of double-stranded RNA to inhibit the activity of a target gene of previously unknown function.
  • functional genomics would envision determining the function of uncharacterized genes by employing the invention to reduce the amount and/or alter the timing of target gene activity.
  • the invention could be used in determining potential targets for pharmaceuticals, understanding normal and pathological events associated with development, determining signaling pathways responsible for postnatal development/aging, and the like.
  • the increasing speed of acquiring nucleotide sequence information from genomic and expressed gene sources, including total sequences for mammalian genomes, can be coupled with the invention to determine gene function in a cell or in a whole organism.
  • the preference of different organisms to use particular codons, searching sequence databases for related gene products, correlating the linkage map of genetic traits with the physical map from which the nucleotide sequences are derived, and artificial intelligence methods may be used to define putative open reading frames from the nucleotide sequences acquired in such sequencing projects.
  • a simple assay would be to inhibit gene expression according to the partial sequence available from an expressed sequence tag (EST). Functional alterations in growth, development, metabolism, disease resistance, or other biological processes would be indicative of the normal role of the EST's gene product.
  • EST expressed sequence tag
  • duplex RNA can be produced by an amplification reaction using primers flanking the inserts of any gene library derived from the target cell or organism. Inserts may be derived from genomic DNA or mRNA (e.g., cDNA and cRNA). Individual clones from the library can be replicated and then isolated in separate reactions, but preferably the library is maintained in individual reaction vessels (e.g., a 96 well microtiter plate) to minimize the number of steps required to practice the invention and to allow automation of the process.
  • mRNA e.g., cDNA and cRNA
  • the subject invention provides an arrayed library of RNAi constructs.
  • the array may be in the form of solutions, such as multi-well plates, or may be "printed" on solid substrates upon which cells can be grown.
  • solutions containing duplex RNAs that are capable of inhibiting the different expressed genes can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells/organisms in each well can be assayed for any changes or modifications in behavior or development due to inhibition of target gene activity.
  • the invention provides methods for evaluating gene function in vivo.
  • a cell containing an shRNA expression construct designed to decrease expression of a target gene may be introduced into an animal and a phenotype may be assessed to determine the effect of the decreased gene expression.
  • An entire animal may be generated from cells (e.g., ES cells) containing an shRNA expression construct designed to decrease expression of a target gene.
  • a phenotype of the transgenic animal may be assessed.
  • the animal may be essentially any experimentally tractable animal, such as a non-human primate, a rodent (e.g., a mouse), a lagomorph (e.g., a rabbit), a canid (e.g. a domestic dog), a feline (e.g., a domestic cat). In general, animals with complete or near complete genome projects are preferred.
  • a phenotype to be assessed may be essentially anything of interest. Quantitating the tendency of a stem cell to contribute to a particular tissue or tumor is a powerful method for identifying target genes that participate in stem cell differentiation and in tumorigenic and tumor maintenance processes. Phenotypes that have relevance to a disease state may be observed, such as susceptibility to a viral, bacterial or other infection, insulin production or glucose homeostasis, muscle function, neural regeneration, production of one or more metabolites, behavior patterns, inflammation, production of autoantibodies, obesity, etc.
  • a panel of shRNAs that affect target gene expression by varying degrees may be used, and phenotypes may be assessed. In particular, it may be useful to measure any correlation between the degree of gene expression decrease and a particular phenotype.
  • a heterogeneous pool of shRNA constructs may be introduced into cells, and these cells may be introduced into an animal.
  • the cells will be subjected to a selective pressure and then it will be possible to identify which shRNAs confer resistance or sensitivity to the selective pressure.
  • the selective pressure may be quite subtle or unintentional, for example, mere engraftment of transfected HSCs may be a selective pressure, with some shRNAs interfering with engraftment and others promoting engraftment. Development and differentiation may be viewed as a "selective pressure", with some shRNAs modulating the tendency of certain stem cells to differentiate into different subsets of progeny.
  • Treatment with a chemotherapeutic agent may be used as selective pressure, as described below.
  • the heterogeneous pool of shRNAs may be obtained from a library, and in certain preferred embodiments, the library is a barcoded library, permitting rapid identification of shRNA species.
  • the invention provides methods for identifying genes that affect the sensitivity of tumor cells to a chemotherapeutic agent.
  • the molecular mechanisms that underlie chemoresistance in human cancers remain largely unknown. While various anticancer agents clearly have different mechanisms of action, most ultimately either interfere with DNA synthesis or produce DNA damage. This, in turn, triggers cellular checkpoints that either arrest cell proliferation to allow repair or provoke permanent exit from the cell cycle by apoptosis or senescence.
  • a method comprises introducing into a subject a transfected stem cell comprising a nucleic acid construct encoding an shRNA, wherein the shRNA is complementary to at least a portion of a target gene, wherein the transfected stem cell exhibits decreased expression of the target gene, and wherein the transfected stem cell gives rise to a transfected tumor cell in vivo.
  • the stem cell may be derived from an animal that has a genetic predisposition to tumorigenesis, such as an oncogene over-expressing animal (e.g. ⁇ - myc mice) or a tumor suppressor knockout (e.g., p53 -/- animal).
  • an animal comprising the stem cells may be exposed to carcinogenic conditions such that tumors comprising cells derived from the stem cells are generated.
  • An animal having tumors may be treated with a chemotherapeutic or other anti-tumor regimen, and the effect of this regimen on cells expressing the shRNA may be evaluated.
  • An shRNA that is overrepresented following anti-tumor therapy is likely to be targeted against a gene that confers sensitivity.
  • An shRNA that is underrepresented following anti-tumor therapy is likely to be targeted against a gene that confers resistance.
  • An shRNA that is underrepresented may be developed for use as a co-therapeutic to be coadministered with the chemotherapeutic agent in question and suppress resistance.
  • Overrepresentation and underrepresentation are generally comparative terms, and determination of these parameters will generally involve comparison to a control or benchmark.
  • a comparison may simply be to the same animal prior to chemotherapy administration.
  • a comparison may also be to a control subject that has not received the chemotherapeutic agent.
  • a comparison may be to an average of multiple other shRNA trials. Any control need not be contemporaneous with the experiment, although the protocol should be substantially the same.
  • a method may comprise introducing into a subject a plurality of transfected stem cells, wherein each transfected stem cell comprises a nucleic acid construct comprising a representative shRNA of an shRNA library, and wherein a representative shRNA of an shRNA library is complementary to at least a portion of a representative target gene, wherein a plurality of the transfected stem cells exhibits decreased expression of a representative target gene, and wherein a plurality of the transfected stem cells gives rise to transfected tumor cells in vivo.
  • each representative shRNA is associated with a distinguishable tag that permits rapid identification of each shRNA.
  • shRNAs may be obtained from a shRNA library that is barcoded.
  • cancer cells e.g., lymphoma cells
  • lymphoma cells e.g., lymphoma cells
  • This allows in vitro manipulation of tumor cells to create potentially chemoresistant variants that can be analyzed in vivo.
  • the invention exploits advantages of the ⁇ -myc system to undertake an unbiased search for genetic alterations that can confer resistance to chemotherapeutics, such as the widely used alkylating agent, CTX.
  • the invention provides a composition formulated for administration to a patient, such as a human or veterinary patient.
  • a composition so formulated may comprise a stem cell comprising the Cas9 protein and gRNAs to induce specific genetic alterations and a nucleic acid construct encoding an shRNA designed to decrease the expression of a target gene.
  • a composition may also comprise a pharmaceutically acceptable excipient. Essentially any suitable cell may be used, included cells selected from among those disclosed herein. Transfected cells may also be used in the manufacture of a medicament for the treatment of subjects.
  • Examples of pharmaceutically acceptable excipients include matrices, scaffolds or other substrates to which cells may attach (optionally formed as solid or hollow beads, tubes, or membranes), as well as reagents that are useful in facilitating administration (e.g. buffers and salts), preserving the cells (e.g. chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics), or promoting engraftment.
  • reagents e.g. buffers and salts
  • preserving the cells e.g. chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics
  • chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics
  • Cells may be encapsulated in a membrane or in a microcapsule. Cells may be placed in microcapsules composed of alginate or polyacrylates. Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat. No. 5, 106,627; U.S. Pat. No. 4,391,909; U.S. Pat. No. 4,353,888.
  • the site of implantation of insulin-producing cell compositions may be selected by one of skill in the art depending on the type of cell and the therapeutic objective.
  • Exemplary implantation sites include intravenous or intraarterial administration, administration to the liver (via portal vein injection), the peritoneal cavity, the kidney capsule or the bone marrow.
  • a gRNA targeting the tumor suppressor gene Trp53 has been inserted at the 5' end of the construct under the control of a U6 promoter and an shRNA targeting Kras, a GTPase commonly mutated in variety of cancer types, has been incorporated in the construct in the opposite orientation to the promoter.
  • the constructs will be transfected into NIH3T3 cells by iipofectamin. These transient transfected cells will first be treated with doxycycHne, harvested and then subjected to western blot to confirm appropriate expression Cas9. The next step will be to determine gDNA and Cas9-mediated cleavage of the Trp53 gene. For this, T7 endonuclease analysis and site specific PCR as well as DNA sequencing will be used to evaluate the efficiency of indels of the targeted gene locus (in this case, Trp53). Western Blotting will be performed to ensure the loss of expression of deleted gene of interest.
  • the transfected cells will be treated sequentially with tamoxifen first followed by doxycycHne and analyzed for tGFP expression under the fluorescent microscopes as well as Western Blotting with anti-GFP antibodies.
  • the reduction of RNAi targeted gene product will be verified (in this case, Kras) by western blot.
  • the construct described and Flp-recombinase expressing plasmid will be electroporated into the LSL- Kras G12D harboring ESCs to enable RMCE to occur at the CollAl locus for cassette integration.
  • the cassette integration will be further verified by PCR-based genotyping.
  • the validated ESCs will then be treated with doxycycline to confirm the proper expression Cas9.
  • T7 endonuciease analysis, site specific PGR, DNA sequencing and western blot procedures will be employed to examine the ESCs for p53 indel efficiency as previously described in our in vitro test. In the next stage, these ESCs will be treated with tamoxifen and confirm GFP expression via fluorescent microscopes and fluorescent assisted cell sorting. Western blot will also be performed for both GFP expression and knockdown of Kras.
  • the verified ESC clones will be used for blastocyst injection and embryo transfer for founder mice generation.
  • the pups will be verified by genotyping PGR and southern blotting.
  • the founder mice will be crossed with their parental strain and further genotyping will be conducted for germ line transmission.
  • the mice Once the mouse line is maintained, the mice will be treated with Adeno-Cre viral vapor via lung inhalation to turn on Kras G12D expression.
  • the mice will then be treated with doxycycline to generate indel s within the Trp53 gene and the mice will be monitored for lung carcinoma formation and development of tumor metastases.
  • the animals will be first treated with tamoxifen for FLEx switching, followed bv doxycycline for turning on GFP-shRNA expression.
  • GFP expression will be verified by fluorescence and collect GFP-positive tissue for further validation of Kras knockdown by RT-PCR for transcripts level and western blot for protein level. In doing so, evaluation can determine whether the tumor progression and metastasis are ameliorated by inducible Kras suppression.
  • Kras G12D ESCs 2) Generation of a novel Kras ⁇ /p53 mut lung adenocarcinoma model that can be readily adapted to involve different driver mutations (through virally delivered sgRNAs) and enables the direct evaluation of candidate targets through transgenic Tet-regulatable shRNAs; 3) Evaluation of the tumor-specific effects of RNAi-mediated Kras suppression in KrasGl 2D/p53mut j un g tumors; 4) Evaluation of systemic phenotypes associated with ubiquitous Kras suppression to investigate possible toxicities of Kras- targeted therapies; 5) Generation of a novel LSL-KrasG12D stra in in which additional driver mutations can be induced by in situ delivery of Cre+sgRNAs and evaluation of Kras inhibition can be evaluated.
  • RNAi mice can be used to mimic therapeutic administration by global suppression of a specific gene through RNAi-mediated gene silencing.
  • RNAi-mediated gene silencing Several limitations within this platform and additional ones with the new CRISPR/Cas9 platform may be expected.
  • heterogeneity in the expression of the shRNA cassette in specific tissues may be seen, particular when using the weaker ubiquitous Rosa26- M2rtTA strain.
  • Rosa26- M2rtTA strain To address this, the CAG-rtTA3 strain is generated and strong ubiquitous expression across all tissues examined were noted, hence this allele in the newly derived ESCs in order to model systemic inhibition.
  • Mosaicism is expected in p53 expression or any other gene that is being targeted by sgRNAs, first representing the incomplete inversion of the Cas9 cassette by Cre, and secondly, reflecting the many in-frame deletions that do not cause loss of protein expression or cells that do not modify the locus. (Dow, L. E. et al. Inducible in vivo genome editing with CRISPR-Cas9.
  • FIG. 20 shows ESCs harboring the Collal homing cassette and CAG-rtTA3 allele will be targeted via CRJSPR/Cas9 FIDR for insertion of conditional mutant alleles. Following validation, these ESCs will be subsequently targeted using RMCE to insert an array of targeting vectors (FIG. 19). Mice will be generated using tetraploid embryo complementation and treated with Adeno- Cre+sgRNAs at 4 weeks of age. Mice will be monitored for disease and treated as indicated. FIG.
  • 20 may enable the development of LSL-Kras Mnl ESCs; development of KrasG12D/p53+/- lung tumors and treat with an shRNA targeting Kras; development of rasG12D/p53+/- lung tumors and treat with an shRNAs targeting Mekl/2 compared with Trametinib; and the development of KrasG12D/p53+/-/Smarca2 (KPS) and rasG12D/p53+/-/Aridl a (KPA) lung tumors and treat with an shRNA targeting Kras.
  • KPS KrasG12D/p53+/-/Smarca2
  • KPA rasG12D/p53+/-/Aridl a
  • adenoviral vectors will be generated encoding Cre, an sgRNA targeting p53, and an sgRNA targeting either Aridla (KPA model) or Smarca4 (KPS model), two components of the SWI/SNF complex that are frequently mutated in human NSCLC (Cancer Genome Atlas Research, N.
  • Adenoviral vectors encoding Cre, a p53- sgRNA, and an sgRNA targeting Aridla or Smarca4 will be delivered into the lung of FLEXi mice derived from ESC harboring shRNAs targeting Aridlb or Smarca2.
  • the power of the FLEXi models will be demonstrated by enabling the evaluation of additional cooperating lesions through Cas9-mediated mutagenesis by simple delivery of additional sgRNA s.
  • Kras inhibition will be assessed in these complex KPS and KPA models, providing new insights into the value of potential Kras inhibitors.
  • the FLEXi models enables: 1) Creation of viral vectors containing combination sgRNAs to target p53/Smarca4 and p53/Aridl a; 2) Creation KPS and KPA lung adenocarcinoma models.

Abstract

La présente invention concerne des systèmes et des procédés pour CRISPR/Cas9 et ARNi inductibles et conditionnels.
PCT/US2016/051992 2015-09-15 2016-09-15 Systèmes de crispr/cas9 et d'arni inductibles et procédés d'utilisation WO2017048995A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019108644A1 (fr) * 2017-11-28 2019-06-06 Mirimus, Inc. Procédés de génie génétique médié de modèles d'arni
WO2019178420A1 (fr) * 2018-03-15 2019-09-19 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie
WO2019178421A1 (fr) * 2018-03-15 2019-09-19 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie
US11091756B2 (en) 2018-10-16 2021-08-17 Blueallele Corporation Methods for targeted insertion of dna in genes
EP3889259A1 (fr) * 2020-03-30 2021-10-06 IMBA-Institut für Molekulare Biotechnologie GmbH Norme interne pour arn guide de crispr
WO2021242989A1 (fr) * 2020-05-29 2021-12-02 Health Research, Inc. Compositions et procédés pour induire une expression de protéine cas9 par l'utilisation d'une activité de promoteur pd-1 autonome
WO2023226856A1 (fr) * 2022-05-27 2023-11-30 上海科技大学 Construction d'acide nucléique fondée sur les techniques cre-loxp et crispr et son utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5885836A (en) * 1991-03-08 1999-03-23 The Salk Institute For Biological Studies FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5885836A (en) * 1991-03-08 1999-03-23 The Salk Institute For Biological Studies FLP-mediated gene modification in mammalian cells, and compositions and cells useful therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAVIS ED PH.D.: "Knockout by TALEN or CRISPR vs. Knockdown by shRNA or siRNA", GENECOPOEIA, 2014, pages 1 - 3, XP055371804, Retrieved from the Internet <URL:www.genecopoeia.com> *
MALINA A. ET AL.: "Repurposing CRISPR/Cas9 for in situ functional assays", GENES & DEVELOPMENT, vol. 27, no. 23, December 2013 (2013-12-01), pages 2602 - 2614, XP055177303 *
SHALEM O. ET AL.: "Genome-scale CRISPR-Cas9 knockout screening in human cells", SCIENCE, vol. 343, no. 6166, 3 January 2014 (2014-01-03), pages 11 - 14, XP055115506 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019108644A1 (fr) * 2017-11-28 2019-06-06 Mirimus, Inc. Procédés de génie génétique médié de modèles d'arni
CN111699256A (zh) * 2017-11-28 2020-09-22 米瑞莫斯公司 RNAi模型遗传介导工程的方法
US11957114B2 (en) 2017-11-28 2024-04-16 Mirimus, Inc. Methods of genetic mediated engineering of RNAi models
WO2019178420A1 (fr) * 2018-03-15 2019-09-19 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie
WO2019178421A1 (fr) * 2018-03-15 2019-09-19 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie
US11091756B2 (en) 2018-10-16 2021-08-17 Blueallele Corporation Methods for targeted insertion of dna in genes
US11254930B2 (en) 2018-10-16 2022-02-22 Blueallele Corporation Methods for targeted insertion of DNA in genes
US11365407B2 (en) 2018-10-16 2022-06-21 Blueallele Corporation Methods for targeted insertion of DNA in genes
EP3889259A1 (fr) * 2020-03-30 2021-10-06 IMBA-Institut für Molekulare Biotechnologie GmbH Norme interne pour arn guide de crispr
WO2021198233A1 (fr) * 2020-03-30 2021-10-07 Imba - Institut Für Molekulare Biotechnologie Gmbh Norme interne pour arn guide de crispr
WO2021242989A1 (fr) * 2020-05-29 2021-12-02 Health Research, Inc. Compositions et procédés pour induire une expression de protéine cas9 par l'utilisation d'une activité de promoteur pd-1 autonome
WO2023226856A1 (fr) * 2022-05-27 2023-11-30 上海科技大学 Construction d'acide nucléique fondée sur les techniques cre-loxp et crispr et son utilisation

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