Delivery systems for functional nucleases

Abstract

To provide better delivery systems for delivering functional effector proteins into cells in order to target intracellular biomolecules.SOLUTION: A composition comprises a supercharged protein coupled to a functional effector protein, where the supercharged protein has an overall positive charge that is greater than its corresponding unmodified protein and that is in sufficient quantity, and is formulated for penetration into a cell. Such functional effector proteins include, without limitation, transcriptional modulators (e.g., repressors or activators), recombinases, nucleases (e.g., RNA-programmable nucleases, such as Cas9 proteins, TALE nucleases, and zinc finger nucleases), deaminases, and other gene modifying / editing enzymes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. 365(c) to U.S. application USSN 14 / 462,189, filed August 18, 2014, and U.S. application USSN 14 / 462,163, filed August 18, 2014, and also claims priority under 35 U.S.C. 365(c) to U.S. provisional patent application USSN 61 / 874,746, filed September 6, 2013, each of which is incorporated herein by reference. [Background technology]

[0002] Background of the Invention Macromolecule delivery into mammalian cells is an attractive approach for cellular engineering because it allows for the modulation of gene expression and genome modification, which in turn opens new avenues of research and the therapeutic targeting of molecules currently considered "undruggable" by small molecules. In particular, recombinant nucleases targeting genes or alleles associated with disease hold great potential as therapeutic agents. Current methods for macromolecule delivery include viral delivery of nucleic acid molecules, receptor-mediated delivery of nucleic acids or proteins, and the use of protein fusions with cell-penetrating peptides such as TAT, Arg9, or penetratin for protein delivery. Each of these delivery systems offers benefits for specific applications. However, in most cases, issues regarding efficacy, cytotoxicity, and ease of preparation remain. Developing easily prepared reagents that can effectively deliver macromolecules (e.g., functional effector proteins) to a variety of cell lines without significant cytotoxicity or other adverse side effects remains a considerable challenge.

[0003] Most proteins do not spontaneously enter mammalian cells, and therefore their use as research tools and their potential as therapeutic agents is naturally limited. Techniques for the delivery of proteins into mammalian cells have only recently been developed to address intracellular targets. These techniques include lipid-based reagents (Zelphati et al., J. Biol. Chem. 276, 35103-35110, 2001), nanoparticles (Hasadsri et al., J. Biol. Chem., 2009), vault ribonucleoprotein particles (Lai et al., ACS Nano 3, 691-699, 2009), genetic or chemical fusion to receptor ligands (Gabel et al., J. Cell Biol. 103, 1817-1827, 1986; Rizk et al., Proc. Natl. Acad. Sci. USA 106, 11011-11015, 2009), and fusion to cell-penetrating peptides (Wadia et al., Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al., J. Cell Biol. 103, 1817-1827, 1986). These include the use of HIV-1 proteins (e.g., HIV-1 fusion protein transduction domains (PTDs) containing the HIV-1 Tat (transcriptional transactivator) peptide and polyarginine peptides. These cationic PTDs facilitate the binding of foreign proteins to negatively charged cell surface structures and subsequent endocytosis.Both Tat and polyarginine have been used to deliver various macromolecules into cells both in vitro and in vivo (Wadia et al., Curr. Protein Pept. Sci. 4, 97-104, 2003; Zhou et al., Cell Stem Cell 4, 381-384, 2009; Myou et al., J. Immunol. 169, 2670-2676, 2002; Bae et al., Clin. Exp. Immunol. 157, 128-138, 2009; Schwarze et al., Science 285, 1569-1572, 1999). Despite these advances, intracellular targets remain difficult to affect using exogenous proteins. Even limited success can require toxic concentrations of the respective transduction agents due to low efficiency of functionally delivering proteins into cells (Zhou et al., Cell Stem Cell 4, 381-384, 2009; Wang et al., Nat. Biotechnol. 26, 901-908, 2008). Therefore, there remains a need for better delivery systems to get functional effector proteins into cells for targeting intracellular biomolecules. Summary of the Invention

[0004] The present disclosure provides novel systems, compositions, preparations, kits, and related methods for delivering functional effector proteins (e.g., site-specific proteins that bind to nucleic acids) into cells using supercharged proteins (e.g., positively charged supercharged proteins), cationic polymers, or cationic lipids. In some embodiments, the nuclease is a TALE nuclease, an RNA-programmable nuclease, or an engineered RNA-programmable genome editing enzyme (e.g., Cas9 and its variants or fusions), or a zinc finger nuclease. In some embodiments, the transcription factor is a TALE transcriptional activator or repressor. In some embodiments, the effector protein is a recombinase. As described in more detail herein, fusing or conjugating a functional effector protein (e.g., a nuclease, a transcriptional activator / repressor, a Cas9 protein (including variants and fusions thereof), etc.) to a positively charged supercharged protein allows for the delivery of the protein to the interior of a cell, for example, to affect gene expression or genome modification. It has also been found that fusing or conjugating a functional effector protein to a negatively charged supercharged protein allows the protein to be conjugated to a cationic lipid or cationic polymer, which provides potent delivery of the protein to the interior of a cell. Additionally, naturally negatively charged functional effector proteins (e.g., VP64 transcriptional activator, anionic 3xFLAG peptide tag, and fusions thereof) or functional effector proteins (e.g., Cas9 protein and variants and fusions thereof) associated with naturally negatively charged nucleic acids (e.g., guide RNA, "gRNA") can be associated with cationic lipids or cationic polymers for delivery to cells (e.g., in the absence of supercharged proteins).

[0005] While delivery of effector proteins has proven effective for extracellular targets, their use to address intracellular targets is relatively underdeveloped due to the inability of most proteins to spontaneously enter mammalian cells. Enabling foreign proteins to access intracellular targets is most commonly achieved by delivering their encoding DNA sequences via chemical transfection, electroporation, or viral delivery. However, introducing foreign DNA into cells results in permanent modifications to the genome, potential disruption of endogenous genes, and the potential for long-term exposure to the encoded drug. For some research or therapeutic applications (including genome editing applications that aim to achieve one-time, permanent modifications of genomic DNA), functional delivery of non-replicating protein drugs may offer improved safety and broader applicability. Furthermore, while protein delivery using cationic compounds such as lipids and polymers remains technically challenging and often induces cytotoxicity, it has surprisingly been found that using the compositions and methods provided herein, certain functional effector proteins (e.g., Cas9 protein and its variants and fusions, recombinases, transcriptional activators / repressors, etc.) can be delivered to cells with no or minimal toxicity, and in some cases, mediate genome modification with significantly improved efficiency and reduced off-target effects. For example, as described in Example 7, delivery of Cas9:gRNA complexes by cationic lipids is highly efficient (modifying up to 80% of cultured human cells from a single treatment) and also induces higher genome modification specificity compared to plasmid transfection, typically resulting in a >10-fold higher on-target:off-target DNA modification ratio in human cells.

[0006] Thus, in one aspect, supercharged proteins are used to deliver functional effector proteins (e.g., nucleases, transcriptional activators / repressors, Cas9 proteins (including fusions and variants thereof), etc.) into cells. In some embodiments, the supercharged proteins are engineered to exhibit an increased total surface charge compared to the corresponding unmodified protein. In other embodiments, the supercharged proteins are engineered to exhibit a decreased total surface charge compared to the corresponding unmodified protein. In other embodiments, the supercharged proteins used in the context of the present disclosure are naturally occurring supercharged proteins. The supercharged proteins can be associated with the protein to be delivered through covalent or non-covalent interactions. Without wishing to be bound by any particular theory, the Cas9 protein, variant, or fusion protein associated with the gRNA has a net negative charge, facilitating association with the positively charged supercharged protein. In certain embodiments, the functional effector protein associated with the supercharged protein is further associated with a cationic polymer or cationic lipid to form a composition suitable for delivery into cells. Examples of suitable engineered or naturally occurring supercharged proteins are described in International PCT Patent Application No. PCT / US07 / 70254 (filed June 1, 2007, published December 13, 2007 as WO2007 / 143574), International PCT Application No. PCT / US09 / 041984 (filed April 28, 2009, published November 5, 2009 as WO2009 / 134808), and International PCT Application No. PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), the entire contents of each of which are incorporated herein by reference. Further examples of supercharged proteins for use in delivering nucleases to cells are described herein. Additional examples of suitable functional effector proteins (e.g., nucleases and RNA-programmable effector proteins, such as Cas9 proteins) are described in U.S. Pat. No. 6,929,299, filed Aug. 22, 2013, entitled "Engineered Transcription Activator-Like Effector (TALE) Domains and Uses Thereof."US Provisional Patent Application USSN 61 / 868,846, entitled "Cas9 Variants and Uses Thereof," filed September 6, 2013; US Provisional Patent Application USSN 61 / 874,609, entitled "Switchable Cas9 Nucleases and Uses Thereof," filed September 6, 2013; US Non-Provisional Patent Application USSN 14 / 320,519, entitled "Engineered Transcription Activator-Like Effector (TALE) Domains and Uses Thereof," filed June 20, 2014; US Non-Provisional Patent Application USSN 14 / 320,498, entitled "Cas9-FokI Fusion Proteins And Uses Thereof," filed June 30, 2014; Thereof,” filed June 30, 2014, U.S. non-provisional application USSN 14 / 320,467, entitled “Switchable gRNAs Comprising Aptamers,” filed July 8, 2014, U.S. non-provisional application USSN 14 / 326,329, entitled “mRNA-Sensing Switchable gRNAs,” filed July 8, 2014, U.S. non-provisional application USSN 14 / 326,340, entitled “Extended DNA-Sensing gRNAs,” filed July 8, 2014, U.S. non-provisional application USSN 14 / 326,361, entitled “Fusions Of Cas9 Domains And Nucleic Acid-Editing Domains,” filed July 8, 2014, U.S. non-provisional application USSN 14 / 325,815, entitled “Methods For Nucleic Acid US non-provisional application USSN 14 / 326,109, filed July 8, 2014, entitled "Methods For Correcting PI3K Point Mutations," and US non-provisional application USSN 14 / 326,109, filed July 8, 2014, entitled "Methods For Correcting PI3K Point Mutations."US non-provisional application USSN 14 / 326,140, ​​entitled "Methods For Correcting Presenilin Point Mutations," filed July 9, 2014; US non-provisional application USSN 14 / 326,269, entitled "Methods For Correcting α-Antitrypsin Point Mutations," filed July 8, 2014; US non-provisional application USSN 14 / 326,290, entitled "Methods For Correcting Von Willebrand Factor Point Mutations," filed July 8, 2014; US non-provisional application USSN 14 / 326,318, entitled "Methods For Correcting Von Willebrand Factor Point Mutations," filed July 8, 2014; US non-provisional application USSN 14 / 326,303, entitled "Methods For Correcting Caspase-9 Point Mutations," filed July 8, 2014; and US non-provisional application USSN 14 / 326,318, entitled "Methods For Correcting Caspase-9 Point Mutations," filed July 8, 2014. and US Provisional Application No. USSN 62 / 030,943, entitled "Internes," the entire contents of each of which are incorporated herein by reference.

[0007] In some embodiments, engineered or naturally occurring supercharged proteins are positively charged. In other embodiments (e.g., those involving delivery of certain effector proteins using cationic lipids and / or cationic polymers), the supercharged protein is negatively charged. In certain embodiments, the superpositively or supernegatively charged protein is non-covalently associated with the effector protein. Alternatively, the superpositively or supernegatively charged protein can be covalently bound to the effector protein. In some embodiments, the effector protein is fused to the supercharged protein. In certain embodiments, the resulting fusion protein includes a linker (e.g., a cleavable linker) between the supercharged protein and the effector protein.

[0008] Some aspects of the present disclosure provide compositions comprising a supercharged protein associated with a functional effector protein (e.g., a nuclease, a transcriptional activator / repressor, a recombinase, a Cas9 protein (including variants and fusions thereof), etc.). In some embodiments, the composition further comprises a cationic lipid. In some embodiments, the composition further comprises a cationic polymer. In some embodiments, the composition further comprises a buffer or excipient. In some embodiments, the supercharged protein has a positive net charge that is greater than and sufficient for delivery to and penetration into cells, and is formulated for cellular delivery and penetration. In other embodiments (e.g., involving delivery of certain effector proteins using cationic lipids and / or cationic polymers), the supercharged protein has a negative net charge that is greater than the negative net charge for the corresponding unmodified protein. In some embodiments, the functional effector protein is a site-specific enzyme, such as a nuclease, a Cas9 protein, a recombinase, etc. In some embodiments, the Cas9 protein is a wild-type Cas9 protein, a Cas9 nickase, or comprises a nuclease-inactive (dCas9) protein. In some embodiments, the Cas9 protein is a fusion protein comprising dCas9. In some embodiments, the fusion protein comprises a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., KRAB, SID), a nuclease domain (e.g., FokI), a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g., cytidine deaminase or adenosine deaminase), or an epigenetic modifier domain (e.g., TET1). In some embodiments involving a nuclease, the nuclease is a TALE nuclease, Cas9 nuclease, Cas9 nickase, or zinc finger nuclease. In some embodiments, the nuclease specifically binds and cleaves a nucleic acid sequence. In some embodiments, the targeted nucleic acid sequence is the sequence of a gene that is a therapeutic target (e.g., a gene whose inactivation is desirable in the treatment of a disease).In some embodiments, the targeted nucleic acid sequence is a PRDM16, PPARγ, VEGF-A, Oct-4, PI3K, presenilin, α-antitrypsin, von Willebrand factor, or caspase-9 gene sequence.

[0009] In some embodiments, the functional effector protein is a transcription factor. In some embodiments, the functional effector protein is a TALE transcriptional activator or repressor. In some embodiments, the transcription factor, transcriptional activator, or transcriptional repressor specifically binds to and activates or represses a gene. In some embodiments, the gene is a therapeutic target. In some embodiments, the functional effector protein is a TALE effector. In some embodiments, the supercharged protein is covalently bound to the functional effector protein, thus forming a fusion protein. In some embodiments, the supercharged protein is associated with the functional effector protein via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a UV-cleavable linker or a linker cleaved by a lysosomal enzyme. In some embodiments, the supercharged protein is non-covalently associated with the functional effector protein, thus forming a complex. In some embodiments, the supercharged protein has a net positive overall charge. In other embodiments, the supercharged protein has a net negative overall charge, and the protein(s) are associated with a cationic lipid. In other embodiments, the supercharged protein has a net negative total charge, and the protein(s) are associated with a cationic polymer. In some embodiments, the net positive total charge is about +5 to about +40, or the net negative total charge is about -5 to about -50. In some embodiments, the supercharged protein is more positively or more negatively charged at physiological pH than its corresponding unmodified protein. In some embodiments, the corresponding unmodified protein is a naturally occurring protein. In some embodiments, the supercharged protein is at least +5 more positively or at least -5 more negatively charged at physiological pH than its corresponding unmodified protein. In some embodiments, the supercharged protein is a fluorescent protein. In some embodiments, the supercharged protein is green fluorescent protein (GFP). In some embodiments, the supercharged protein is superpositively charged GFP. In some embodiments, the supercharged protein is superpositively charged GFP (+36GFP) and has the sequence: GGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYK (SEQ ID NO: 1) In some embodiments, the supercharged protein comprises at least 20 consecutive amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the supercharged protein consists of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated for administration to a subject and comprises the supercharged protein and a functional effector protein in an amount effective for delivery to at least one cell of the subject. In some embodiments, the composition comprises the supercharged protein and a functional effector protein in an amount effective to induce a measurable therapeutic effect after administration to a subject.

[0010] Some aspects of the present disclosure provide compositions comprising a Cas9 protein associated with a gRNA and a cationic lipid. It has surprisingly been discovered that when the Cas9 protein is associated with the gRNA, the complex can be encapsulated by the cationic lipid and effectively delivered to cells. This can be accomplished with or without a supercharged protein. In some embodiments, the composition comprises a Cas9 protein associated with a supernegatively charged protein (e.g., supernegatively charged GFP) and a cationic lipid, again enabling successful delivery to cells. In some embodiments, the composition exhibits low toxicity when delivered to a population of cells. For example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells are viable after administration of the composition. In some embodiments, the Cas9 protein is a wild-type Cas9 protein, a Cas9 nickase, or comprises a nuclease-inactive (dCas9) protein. In some embodiments, the Cas9 protein is a fusion protein comprising dCas9. In some embodiments, the fusion protein comprises a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., KRAB, SID), a nuclease domain (e.g., FokI), a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g., cytidine deaminase or adenosine deaminase), or an epigenetic modifier domain (e.g., TET1).

[0011] Another aspect of the present disclosure provides compositions comprising a Cas9 protein associated with a gRNA and a cationic polymer. Similar to cationic lipids, when the Cas9 protein is associated with a gRNA, the complex can be effectively delivered to cells in association with a cationic polymer. This can be accomplished with or without a supercharged protein. In some embodiments, the composition comprises a Cas9 protein associated with a supernegatively charged protein (e.g., supernegatively charged GFP) and a cationic polymer, again enabling successful delivery to cells. In some embodiments, the composition exhibits low toxicity when delivered to a population of cells. For example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells are viable after administration of the composition. In some embodiments, the Cas9 protein is a wild-type Cas9 protein, a Cas9 nickase, or comprises a nuclease-inactive (dCas9) protein. In some embodiments, the Cas9 protein is a fusion protein comprising dCas9. In some embodiments, the fusion protein comprises a transcriptional activator (e.g., VP64), a transcriptional repressor (e.g., KRAB, SID), a nuclease domain (e.g., FokI), a recombinase domain (e.g., Hin, Gin, or Tn3), a deaminase (e.g., cytidine deaminase or adenosine deaminase), or an epigenetic modifier domain (e.g., LSD1, TET1).

[0012] Some aspects of the present disclosure provide methods for administering a composition provided herein to a subject. In some embodiments, the method comprises administering a composition described herein to a subject. In some embodiments, the subject is susceptible to, suffering from, or exhibiting one or more symptoms of a disease, disorder, or condition. In some embodiments, the composition is administered to a subject in an amount sufficient and under suitable conditions such that at least one sign or symptom is ameliorated as a result of administration. In some embodiments, the administering step is carried out under conditions sufficient for functional effector proteins to penetrate the subject's cells. In some embodiments, the disease, disorder, or condition is associated with abnormally elevated levels of mRNA, protein, or a combination thereof. In some embodiments, the composition comprises a nuclease that specifically binds to and cleaves a genomic sequence (e.g., a normal or pathogenic allele, a gene associated with disease susceptibility or onset or progression, a gene encoding a pathogenic RNA or protein, or a gene encoding an RNA or protein expressed at abnormally high levels in diseased cells or tissues). In some embodiments, the administering step comprises a route of administration selected from the group consisting of oral, intravenous, intramuscular, intraarterial, subcutaneous, intracerebroventricular, topical, inhalation, and mucosal delivery.

[0013] Some aspects of the present disclosure provide methods for introducing a functional effector protein into a cell. In some embodiments, the method comprises contacting a cell with a composition comprising a supercharged protein described herein and a functional effector protein under conditions suitable for the functional effector protein to enter the cell, thereby introducing the functional effector protein into the cell. In some embodiments, the method comprises contacting a cell with a composition comprising a Cas9 protein and a cationic lipid and / or cationic polymer under conditions suitable for the Cas9 protein to enter the cell, thereby introducing the Cas9 protein into the cell. In some embodiments, the Cas9 protein enters the nucleus of the cell. For example, the Cas9 protein is directed to the nucleus by including a nuclear localization signal (NLS) in the protein. In some embodiments, the method further comprises confirming that the functional effector protein (e.g., including Cas9) has penetrated the cell. In some embodiments, the cell is in a subject and the contacting occurs in vivo. In some embodiments, the subject is diagnosed with or at risk of developing a disease associated with an abnormal expression level of the gene, and a functional effector protein (including, for example, Cas9) regulates the expression level of the gene. In some embodiments, the method further comprises detecting a change in the level of expression of the gene or detecting a therapeutic response in the subject. In some embodiments, the cell is a somatic cell. In some embodiments, the cell is contacted with the composition or pharmaceutical composition in an amount, for a time, and under conditions sufficient to induce programming of the cell toward a desired cell fate. In some embodiments, the method further comprises using the programmed cell in a cell replacement therapy procedure. In some embodiments, the cell is a cell harboring a genomic allele associated with the disease, and the functional effector protein specifically targets the allele. In some embodiments, the cell is contacted ex vivo and is readministered to the subject after successful targeting of the undesired allele with the functional effector protein.

[0014] Some aspects of the present disclosure provide kits comprising a composition described herein (e.g., a composition comprising a supercharged protein associated with a functional effector protein). In some embodiments, the kit comprises a Cas9 protein and a supercharged protein. In some embodiments, the kit comprises a Cas9 protein and a cationic lipid. In some embodiments, the kit comprises a Cas9 protein and a cationic polymer. In some embodiments, the kit further comprises instructions for using the components included in the kit.

[0015] These and other aspects and embodiments of the present invention, as well as various advantages and utilities, will become more apparent with reference to the drawings and detailed description of the invention.

[0016] definition As used in this specification and claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "agent" includes a single agent and multiple agents.

[0017] The term "associated with," as used herein in the context of two or more moieties (e.g., proteins or protein domains), refers to the fact that the moieties are physically associated or connected to one another, either directly or through one or more additional moieties that act as linkers, to form a sufficiently stable structure so that the moieties remain physically associated under the conditions in which the structure is used (e.g., physiological conditions). A supercharged protein can associate with a functional effector protein (e.g., a nuclease, a transcriptional activator / repressor, a recombinase, a Cas9 protein (including variants and fusions thereof), etc.) through noncovalent interactions (e.g., electrostatic interactions). In certain embodiments, a supercharged protein can associate with a functional effector protein through electrostatic interactions to form a complex. In some embodiments, a sufficient number of weaker interactions can provide sufficient stability for the moieties to remain physically associated under a variety of different conditions. In certain embodiments, a supercharged protein associates with a functional effector protein through a covalent bond (e.g., an amide bond). In some embodiments, the functional effector protein is associated with the supercharged protein directly by a peptide bond or indirectly through a linker.

[0018] The term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active or inactive DNA-cleavage domain of Cas9 or a partially inactive DNA-cleavage domain (e.g., a Cas9 "nickase") and / or a gRNA-binding domain of Cas9). In some embodiments, the term "Cas9" refers to a fusion protein comprising Cas9 or a fragment thereof.

[0019] In some embodiments, Cas9 is used to inhibit Corynebacterium ulcerans (NCBI Ref: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI Ref: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI Ref: NC_021284.1), Prevotella intermedia (NCBI Ref: NC_017861.1), Spiroplasma taiwanense (NCBI Ref: NC_021846.1), Streptococcus iniae (NCBI Ref: NC_021314.1), Belliella baltica (NCBI Ref: NC_018010.1), Psychroflexus torquisl (NCBI Ref: NC_018721.1), Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria meningitidis (NCBI Ref: YP_002342100.1).

[0020] The term "cationic lipid" refers to a lipid that has a cationic (or positive) charge at physiological pH. Cationic lipids can take various forms, including, but not limited to, liposomes or micelles. Cationic lipids useful in certain aspects of the present disclosure are known in the art and generally contain both polar and non-polar domains, bind to polyanions (e.g., nucleic acid molecules or negatively charged proteins), and typically facilitate the delivery of nucleic acids into cells. Examples of useful cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE® (e.g., LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, LIPOFECTAMINE® RNAiMAX, LIPOFECTAMINE® LTX), SAINT-RED (Synvolux Therapeutics, Groningen, Netherlands), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectin (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and dimethyldioctadecylammonium bromide (DDAB).Cationic lipids have been used in the art to deliver nucleic acid molecules into cells (see, e.g., U.S. Pat. Nos. 5,855,910, 5,851,548, 5,830,430, 5,780,053, 5,767,099, 8,569,256, 8,691,750, 8,748,667, 8,758,810, 8,759,104, 8,771,728; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15: 1). In addition, other lipid compositions are also known in the art, including those taught in, for example, US Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 4,737,323.

[0021] The term " cationic polymer " as used herein refers to a polymer that has a net positive charge.Cationic polymers are well known in the art, and include those described in Samal et al., Cationic polymers and their therapeutic potential.Chem Soc Rev. 2012 Nov 7;41(21):7147-94, published US patent applications US2014 / 0141487A1, US2014 / 0141094A1, US2014 / 0044793A1, US2014 / 0018404A1, US2014 / 0005269A1 and US2013 / 0344117A1, and USPat.No.8,709,466, 8,728,526, 8,759,103 and 8,790,664, the entire contents of each of which are incorporated herein by reference. Exemplary cationic polymers include, but are not limited to, polyallylamine (PAH), polyethyleneimine (PEI), poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyvinylamine homo- or copolymers, poly(vinylbenzyl-tri-C1-C4-alkylammonium salts), polymers of aliphatic or araliphatic dihalides and aliphatic N,N,N',N'-tetra-C1-C4-alkyl-alkylenediamines, poly(vinylpyridine) or poly(vinylpyridinium salts), poly(N,N-diallyl-N,N-di-C1-C4-alkyl-ammonium halide), homo- or copolymers of quaternized di-C1-C4-alkyl-aminoethyl acrylate or methacrylate, POLYQUAD™, polyaminoamides, and the like.

[0022] The term "deaminase" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively.

[0023] The term "effective amount," as used herein, refers to an amount of a bioactive agent sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a functional effector protein (e.g., a nuclease, a transcriptional activator / repressor, a recombinase, a Cas9 protein (including variants and fusions thereof), etc.) can refer to an amount of protein sufficient to induce a detectable effect (e.g., cleavage of a target site, modification of a target site, modulation of gene expression, etc.). Such an effect can be detected in a suitable assay (e.g., in a cell-free assay or in a target cell, tissue, or subject organism). As will be recognized by those skilled in the art, the effective amount of an agent (e.g., a functional effector protein) can vary depending on various factors, such as the desired biological response, the particular allele to be targeted, the genome, target site, cell, or tissue to be targeted, and the supercharged protein to be used.

[0024] The term "effector protein" refers to a protein that, when introduced into a cell, modulates a biological function of the cell (e.g., by modifying (e.g., cleaving, deaminating, recombining, etc.) a nucleic acid molecule within the cell, or modulating (e.g., increasing or decreasing) the expression or expression level of a gene within the cell).

[0025] The term "engineered," as used herein, refers to a protein molecule, complex, substance, or entity that is designed, generated, prepared, synthesized, and / or manufactured by humans. Thus, an engineered product is a product that does not exist in nature. In some embodiments, an engineered protein or composition (e.g., an engineered supercharged protein linked to a functional effector protein, such as a nuclease, a Cas9 protein (including variants and fusions thereof)) is a supercharged protein designed to meet specific requirements or have specific desired characteristics (e.g., to have a defined net charge, to specifically bind and / or cleave or modify a target sequence of interest, to have a specific minimum or maximum cleavage or enzymatic activity, and / or to have a specific stability).

[0026] The term "epigenetic modifier," as used herein, refers to a protein or catalytic domain thereof having enzymatic activity that causes epigenetic modifications of DNA (e.g., chromosomal DNA). Epigenetic modifications include, but are not limited to, DNA methylation and demethylation, histone modifications (e.g., methylation and demethylation (e.g., mono-, di-, and trimethylation), histone acetylation and deacetylation, and histone ubiquitination, phosphorylation, and sumoylation).

[0027] The term "functional protein" refers to a protein in a form in which it exhibits a property and / or activity by which it is characterized.

[0028] The term "fusion protein" refers to a protein that contains multiple heterologous proteins, protein domains, or peptides (e.g., supercharged proteins and functional effector proteins) linked together via peptide bonds, thereby forming a single amino acid sequence. In certain embodiments, fusion proteins are encoded by genes.

[0029] The term "gene" has its meaning as understood in the art. It will be recognized by those skilled in the art that the term "gene" can encompass gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will further be recognized that the definition of gene encompasses reference to nucleic acids that do not encode proteins but rather encode functional RNA molecules (e.g., RNAi agents, ribozymes, tRNAs, etc.). For purposes of clarity, it should be noted that the term "gene," as used herein, generally refers to a portion of a nucleic acid that encodes a protein. The term may optionally encompass regulatory sequences, as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to expression units that do not encode proteins, but rather to clarify that in most cases, the term, as used in this document, refers to a nucleic acid that encodes a protein.

[0030] The term "isolated" refers to a molecule, complex, substance, or entity that is (1) separated from at least some of the components with which it was originally associated when produced (whether in nature or in an experimental setting) and / or (2) produced, prepared, synthesized, and / or manufactured by humans. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0031] The term "linker," as used herein, refers to a chemical group or molecule that links two molecules or moieties (e.g., a supercharged protein and a nuclease). Typically, a linker is positioned between or flanked by two groups, molecules, or other moieties, and is connected to each other via a covalent bond, thus connecting the two. In some embodiments, the linker comprises one amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is a cleavable linker. For example, the linker comprises a bond that can be cleaved by exposure to a cleaving activity (e.g., UV light or a hydrolytic enzyme, such as a lysosomal protease). In some embodiments, the linker is any stretch of amino acids, having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or 51 or more amino acids. In some embodiments, the peptide linker comprises repeats of the tripeptide Gly-Gly-Ser, e.g., the sequence (GGS) nwhere n represents at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more repeats. In some embodiments, the linker comprises the sequence (GGS) (SEQ ID NO: 2). In some embodiments, the peptide linker is a 16-residue "XTEN" linker or a variant thereof (see, e.g., Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 3), SGSETPGTSESA (SEQ ID NO: 4), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 5). In some embodiments, the peptide linker is one or more selected from VPFLLEPDNINGKTC (SEQ ID NO: 6), GSAGSAAGSGEF (SEQ ID NO: 7), SIVAQLSRPDPA (SEQ ID NO: 8), MKIIEQLPSA (SEQ ID NO: 9), VRHKLKRVGS (SEQ ID NO: 10), GHGTGSTGSGSS (SEQ ID NO: 11), MSRPDPA (SEQ ID NO: 12), or GGSM (SEQ ID NO: 13).

[0032] The term "nuclease," as used herein, refers to an agent (e.g., a protein or small molecule) capable of cleaving the phosphodiester bonds connecting nucleotide residues in a nucleic acid molecule. In some embodiments, a nuclease is a protein (e.g., an enzyme capable of binding to a nucleic acid molecule and cleaving the phosphodiester bonds connecting nucleotide residues in a nucleic acid molecule). A nuclease can be an endonuclease, which cleaves phosphodiester bonds within a polynucleotide chain, or an exonuclease, which cleaves phosphodiester bonds at the end of a polynucleotide chain. In some embodiments, a nuclease is a site-specific nuclease, which binds and / or cleaves a specific phosphodiester bond in a specific nucleotide sequence, also referred to herein as a "recognition sequence," "nuclease target site," or "target site." In some embodiments, a nuclease recognizes a single-stranded target site, while in other embodiments, a nuclease recognizes a double-stranded target site (e.g., a double-stranded DNA target site). The target sites of many naturally occurring nucleases (e.g., many naturally occurring DNA restriction nucleases) are well known to those of skill in the art. In many cases, DNA nucleases (e.g., EcoRI, HindIII, or BamHI) recognize palindromic double-stranded DNA target sites 4-10 base pairs in length and cleave each of the two DNA strands at specific positions within the target site. Some endonucleases cleave double-stranded nucleic acid target sites symmetrically, i.e., cleave both strands at the same position, resulting in ends containing base-paired nucleotides (also referred to herein as blunt ends). Other endonucleases cleave double-stranded nucleic acid target sites asymmetrically, i.e., cleave each strand at a different position, resulting in ends containing unpaired nucleotides. Unpaired nucleotides at the ends of a double-stranded DNA molecule are also referred to as "overhangs" (e.g., "5' overhangs" or "3' overhangs," depending on whether the unpaired nucleotide(s) form the 5' or 3' end of each DNA strand). The ends of a double-stranded DNA molecule that terminate in unpaired nucleotide(s) are also referred to as sticky ends.Nuclease proteins are known for their ability to "stick" to the ends of other double-stranded DNA molecules that contain complementary unpaired nucleotide(s). Nuclease proteins typically contain a "binding domain" (which mediates the protein's interaction with the nucleic acid substrate) and a "cleavage domain" (which catalyzes the cleavage of phosphodiester bonds in the nucleic acid backbone). In some embodiments, nuclease proteins can bind to and cleave nucleic acid molecules in a monomeric form, while in other embodiments, nuclease proteins must dimerize or multimerize to cleave target nucleic acid molecules. Binding and cleavage domains of naturally occurring nucleases, as well as modular binding and cleavage domains that can be combined to create nucleases that bind to specific target sites, are well known to those of skill in the art. For example, a transcription activator-like element can be used as a binding domain that specifically binds to a desired target site and fused or conjugated to a cleavage domain (e.g., the cleavage domain of FokI) to create an engineered nuclease that cleaves a desired target site.

[0033] The terms "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a compound comprising a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single- and / or double-stranded DNA. Nucleic acids can occur naturally, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, such as a recombinant DNA or RNA, an artificial chromosome, an engineered genome or fragment thereof, or a synthetic DNA, RNA, or DNA / RNA hybrid, or may contain non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms encompass nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids may be purified from natural sources, produced and optionally purified using recombinant expression systems, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids may contain nucleoside analogs (e.g., analogs with chemically modified bases or sugars) and backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, nucleic acids are composed of natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C ...bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5-bromouridine, C5 -aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalating bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0034] The term "pharmaceutical composition," as used herein, refers to a composition that can be administered to a subject, for example, in the context of treating a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient (e.g., a supercharged protein linked to a functional effector protein, such as a nuclease, or a nucleic acid encoding the supercharged protein and the functional effector protein, for example, in the form of a fusion protein) and a pharmaceutically acceptable excipient.

[0035] The term "physiological pH," as used herein, refers to the pH value found in normal, non-pathological cells or subjects. In some embodiments, physiological pH is pH 5-8. In some embodiments, physiological pH is pH 7-7.5, e.g., pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, or pH 7.5. In some embodiments, physiological pH is pH 6.5-7.5. In some embodiments, physiological pH is pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, or pH 8.

[0036] The terms "prevention" or "preventing" refer to the prophylactic treatment of a subject at risk of developing a disease, disorder, or condition (e.g., at elevated risk compared to a control subject or control group of subjects, or at elevated risk compared to the average risk of age-matched and / or gender-matched subjects), resulting in a reduced probability (compared to the probability without prevention) that the subject will develop the disease, disorder, or condition, and / or the inhibition of further development of an already established disorder.

[0037] The term "proliferative disease," as used herein, refers to any disease in which cell or tissue homeostasis is disturbed, in that a cell or cell population exhibits an abnormally elevated rate of proliferation. Proliferative diseases include hyperproliferative diseases such as preneoplastic hyperplastic conditions and neoplastic diseases. Neoplastic diseases are characterized by abnormal proliferation of cells and include both benign and malignant neoplasms. Malignant neoplasms are also referred to as cancer.

[0038] The term "protein" is used interchangeably with "peptide" and "polypeptide" herein and refers to a polymer of amino acid residues linked by peptide (amide) bonds. The term refers to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids in length. A protein, peptide, or polypeptide can refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide can be modified, for example, by the addition of a chemical entity (e.g., a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker, etc., for conjugation, functionalization, or other modification). A protein, peptide, or polypeptide can be a single molecule or a multimolecular complex. A protein, peptide, or polypeptide can be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide can be naturally occurring, recombinant, synthetic, or any combination thereof. A protein may comprise different domains, for example, a TALE effector protein may comprise a nucleic acid binding domain and an effector domain (e.g., a nucleic acid cleavage domain or a transcriptional activator or repressor domain). In some embodiments, a protein comprises a proteinaceous portion (e.g., an amino acid sequence constituting the nucleic acid binding domain) and an organic compound (e.g., a compound that can act as a nucleic acid cleavage agent).

[0039] The terms "RNA-programmable nuclease" and "RNA-guided nuclease" are used interchangeably herein to refer to a nuclease that forms a complex (e.g., binds or associates with) one or more RNA molecules that are not the target of cleavage. In some embodiments, when an RNA-programmable nuclease is complexed with RNA, it can be referred to as a nuclease:RNA complex. RNA-programmable nucleases include Cas9. Typically, the bound RNA(s) is referred to as a guide RNA (gRNA). A gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule can be referred to as a single guide RNA (sgRNA), although "gRNA" is used interchangeably to refer to a guide RNA that exists either as a single molecule or as a complex of two or more molecules. Typically, the gRNA exists as a single RNA species and contains two domains: (1) a domain that shares homology to the target nucleic acid (e.g., and directs binding of the Cas9 complex to the target) and (2) a domain that binds to the Cas9 protein. The gRNA contains a nucleotide sequence complementary to the target site, which mediates binding of the nuclease / RNA complex to the target site and provides sequence specificity for the nuclease:RNA complex.

[0040] The term "recombinase," as used herein, refers to a site-specific enzyme that mediates the recombination of DNA between recombinase recognition sequences, resulting in the excision, integration, inversion, or exchange (e.g., translocation) of DNA fragments between the recombinase recognition sequences. Recombinases can be classified into two distinct families: serine recombinases (e.g., resolvases and invertases) and tyrosine recombinases (e.g., integrases). Examples of serine recombinases include, but are not limited to, Hin, Gin, Tn3, β-six, CinH, ParA, γδ, Bxb1, φC31, TP901, TG1, φBT1, R4, φRV1, φFC1, MR11, A118, U153, and gp29. Examples of tyrosine recombinases include, without limitation, Cre, FLP, R, lambda, HK101, HK022, and pSAM2. The names of serine and tyrosine recombinases come from the conserved nucleophilic amino acid residue that the recombinase uses to attack DNA and become covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the creation of gene knockouts / knockins and gene therapy applications. For example, Brown et al., “Serine ricombinases as tools for genome engineering.” Methods. 2011;53(4):372-9, Hirano et al., “Site-specific recombinases as tools for heterologous gene integration.” Appl. Microbiol. Biotechnol. 2011; 92(2):227-39, Chavez and Calos, “Therapeutic applications of the φC31 integrase system.” Curr. Gene Ther. 2011;11(5):375-81, Turan and Bode, “Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications.” FASEB J.2011; 25(12):4088-107, Venken and Bellen, “Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and φC31 integrase.” Methods Mol. Biol. 2012; 859:203-28, Murphy, “Phage recombinases and their applications.” Adv. Virus Res. 2012; 83:367-414, Zhang et al., “Conditional gene manipulation: Cre-ating a new biological era.” J. Zhejiang Univ. Sci. B. 2012; 13(7):511-24, Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination.” DNA Repair (Amst). 2012; 1;11(10):781-8, the entire contents of each of which are incorporated herein by reference in their entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that may be used in embodiments of the present invention. The methods and compositions of the present invention can be expanded by mining databases for new orthogonal recombinases or by designing synthetic recombinases with defined DNA specificity (see, e.g., Groth et al., "Phage integrases: biology and applications." J. Mol. Biol. 2004; 335, 667-678; Gordley et al., "Synthesis of programmable integrases." Proc. Natl. Acad. Sci. USA.2009; 106, 5053-5058, the entire contents of each of which are incorporated herein by reference in their entirety. Other examples of recombinases useful in the methods and compositions described herein will be known to those of skill in the art, and any new recombinases discovered or produced are expected to be capable of use in different embodiments of the invention. In some embodiments, the recombinase (or its catalytic domain) is fused to a Cas9 protein (e.g., dCas9).

[0041] The terms "recombinate" or "recombination" are used in the context of nucleic acid modification (e.g., genome modification) to refer to a process in which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a recombinase protein. Recombination can result in, among other things, the insertion, inversion, excision, or translocation of nucleic acid sequences within or between one or more nucleic acid molecules.

[0042] The term "subject," as used herein, refers to an individual organism. In some embodiments, the subject is a human of either sex or at any stage of development. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is an experimental animal, such as a mouse, rat, gerbil, guinea pig, fish, frog, or fly. In some embodiments, the subject is a livestock animal, such as a sheep, goat, pig, or cow. In some embodiments, the subject is a companion animal, such as a cat or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject.

[0043] The term "supercharging" refers to any modification of a protein that results in an increase or decrease in the protein's overall net charge. Modifications include, but are not limited to, changes in the amino acid sequence or the addition of charged moieties (e.g., carboxylic acid, phosphate, sulfate, amino groups). Supercharging also refers to the association of a drug with a naturally occurring or modified charged protein to form a complex that has an increased or decreased charge relative to the drug alone.

[0044] The term "target site," as used herein in the context of functional effector proteins (e.g., nucleases and transcriptional activators or repressors) that bind to nucleic acid molecules, refers to a sequence in a nucleic acid molecule that is bound and acted upon by the effector protein (e.g., cleaved by a nuclease, or transcriptionally activated or repressed by a transcriptional activator or repressor, respectively). A target site can be single-stranded or double-stranded. In the context of an RNA-guided (e.g., RNA-programmable) nuclease (e.g., Cas9), the target site typically comprises a nucleotide sequence complementary to the gRNA of the RNA-programmable nuclease and a protospacer adjacent motif (PAM) at the 3' end adjacent to the gRNA-complementary sequence. For the RNA-guided nuclease Cas9 (or a variant or fusion comprising the gRNA-binding activity), the target site, in some embodiments, can be 20 base pairs plus a 3 base pair PAM (e.g., NNN, where N represents any nucleotide). Typically, the first nucleotide of the PAM can be any nucleotide, while the two downstream nucleotides are defined depending on the particular RNA-guided nuclease. Exemplary target sites for RNA-guided nucleases, such as Cas9, are known to those skilled in the art and include, without limitation, NNG, NGN, NAG, and NGG, where N represents any nucleotide. Additionally, Cas9 nucleases from different species (e.g., S. thermophilus instead of S. pyogenes) recognize PAMs containing the sequence NGGNG. Additional PAM sequences are known, including, but not limited to, NNAGAAW and NAAR (see, e.g., Esvelt and Wang, Molecular Systems Biology, 9:641 (2013), the entire contents of which are incorporated herein by reference). For example, a target site for an RNA-guided nuclease, such as Cas9, can include the structure [NZ]-[PAM], where each N independently represents any nucleotide and Z is an integer between 1 and 50.In some embodiments, Z is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50. In some embodiments, Z is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, Z is 20. In some embodiments, "target site" can also refer to a sequence in a nucleic acid molecule that is bound but not cleaved by a nuclease. For example, certain embodiments described herein provide a protein comprising an inactive (or inactivated) Cas9 DNA cleavage domain. Such a protein (e.g., when it also includes a Cas9 RNA-binding domain) is capable of binding to a target site defined by a gRNA. However, because the DNA cleavage site is inactivated, the target site is not cleaved by the protein. However, such proteins described herein are typically conjugated, fused, or attached to another protein (e.g., a nuclease, transcription activator, recombinase, deaminase, etc.) or molecule that mediates modification of the nucleic acid molecule. In some embodiments, the actual sequence cleaved will depend on the protein (e.g., nuclease) or molecule that mediates cleavage of the nucleic acid molecule. In some cases, this will be related, for example, to the proximity or distance to which the inactive Cas9 protein(s) bind.In the context of dimerizing nucleases (e.g., a protein dimer comprising an inactive Cas9 (or the RNA-binding domain of Cas9) and a DNA cleavage domain (e.g., a FokI cleavage domain or an active Cas9 cleavage domain)), a target site typically comprises a left half-site (bound by one protein), a right half-site (bound by a second protein), and a spacer sequence between the half-sites (where cleavage occurs). In some embodiments, either the left half-site or the right half-site (and not the spacer sequence) is cleaved. This structure ([left half-site]-[spacer sequence]-[right half-site]) is referred to herein as an LSR structure. In some embodiments, the left half-site and / or the right half-site correspond to an RNA-guided target site (e.g., a Cas9 target site). In some embodiments, either or both half-sites are shorter or longer, e.g., than a typical region targeted by Cas9, e.g., by more than 20 nucleotides. In some embodiments, the left and right half-sites comprise different nucleic acid sequences. In some embodiments, the target site is a sequence comprising three (3) RNA-guided nuclease target site sequences (e.g., three sequences corresponding to Cas9 target site sequences), where the first and second and second and third Cas9 target site sequences are spaced apart by spacer sequences. In some embodiments, the spacer sequences are at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, or at least 250 bp in length.

[0045] The terms "transcriptional activator" and "transcriptional repressor" refer to agents such as proteins (e.g., transcription factors or fragments thereof) that bind to a target nucleic acid sequence and cause an increase or decrease in the level of expression of a gene product associated with the target nucleic acid sequence. For example, if the target nucleic acid sequence is located within the regulatory region of a gene, a transcriptional activator causes an increase in the level of expression of the gene product encoded by the gene (conversely, a transcriptional repressor causes a decrease in the level of expression of the gene product encoded by the gene). A gene product can be RNA (e.g., mRNA) transcribed from the gene or a polypeptide translated from mRNA transcribed from the gene. Typically, an increase or decrease in the level of mRNA results in a decrease or increase in the level of the polypeptide translated from it. The level of expression can be determined using standard techniques for measuring mRNA or protein.

[0046] The term "transcription activator-like effector" (TALE), as used herein, refers to an effector protein that comprises a DNA-binding domain containing a highly conserved 33-34 amino acid sequence that includes a highly variable two-amino acid motif (repeated variable dinucleotide, RVD).The RVD motif determines the binding specificity for a nucleic acid sequence and can be engineered according to methods well known to those skilled in the art to specifically bind to a desired DNA sequence (e.g., Miller, Jeffrey; et.al. (February 2011). "A TALE nuclease architecture for efficient genome editing". Nature Biotechnology 29 (2): 143-8; Zhang, Feng; et.al. (February 2011). "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription". Nature Biotechnology 29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, SE; Boch, J. (2011), Shiu, Shin-Han. ed. "Transcriptional Activators of Human Genes with Programmable DNA-Specificity". PLoS ONE 6 (5): e19509; Boch, Jens (February 2011). "TALEs of genome targeting". Nature Biotechnology 29 (2): 135-6; Boch, Jens; et.al. (December 2009). "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors". Science 326 (5959): 1509-12; and Moscow, Matthew J.; Adam J. Bogdanove (December 2009). "A Simple Cipher Governs DNA Recognition by TAL Effectors". Science 326 (5959): 1501, the entire contents of each of which are incorporated herein by reference.The simple relationship between amino acid sequence and DNA recognition allows for the engineering of specific DNA-binding domains by selecting combinations of repeat segments containing appropriate RVDs. TALE effector proteins include, but are not limited to, TALE nucleases (TALENs) and TALE transcriptional activators and repressors.

[0047] The term "transcription activator-like element nuclease" (TALEN), as used herein, refers to an artificial nuclease that contains a transcription activator-like effector DNA binding domain in a DNA cleavage domain (e.g., a FokI domain). Several modular assembly schemes for generating engineered TALE constructs have been reported (Zhang, Feng; et.al. (February 2011). "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription". Nature Biotechnology 29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, SE; Boch, J. (2011), Shiu, Shin-Han. ed. "Transcriptional Activators of Human Genes with Programmable DNA-Specificity". PLoS ONE 6 (5): e19509; Cermak, T.; Doyle, EL; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C; Bailer, JA; Somia, NV et al. (2011). "Efficient design and "assembly of custom TALEN and other TAL effector-based constructs for DNA targeting". Nucleic Acids Research, Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). "Assembly of custom TALE-type DNA binding domains by modular cloning". Nucleic Acids Research, Li, T.; Huang, S.; Zhao, X.; Wright, DA; Carpenter, S.; Spalding, MH; Weeks, DP; Yang, B. (2011). "Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes". Nucleic Acids Research., Weber, E.; Gruetzner, R.; Werner, S.; Engler, C; Marillonnet, S. (2011). Bendahmane, Mohammed, ed. "Assembly of Designer TAL Effectors by Golden Gate Cloning". PLoS ONE 6 (5): el9722, each of which is incorporated herein by reference.

[0048] The term "transcriptional repressor" refers to a transcription factor (e.g., a protein) that binds to a nucleic acid sequence and causes a reduction in the level of expression of a gene product associated with the target nucleic acid sequence. For example, if the target nucleic acid sequence is located within the regulatory region of a gene, the transcriptional repressor causes a reduction in the level of expression of a gene product encoded by the gene. A gene product can be an RNA (e.g., mRNA) transcribed from the gene or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of mRNA results in a reduction in the level of the polypeptide translated from it. The level of expression can be determined using standard techniques for measuring mRNA or protein.

[0049] The term "zinc finger nuclease" as used herein refers to a nuclease comprising a nucleic acid cleavage domain conjugated to a binding domain comprising a zinc finger array. In some embodiments, the cleavage domain is the cleavage domain of the type II restriction endonuclease FokI. Zinc finger nucleases can be designed to target almost any desired sequence in a given nucleic acid molecule for cleavage, and the possibility of designing zinc finger binding domains to bind to unique sites in complex genomic contexts allows for targeted cleavage of single genomic sites in living cells, achieving, for example, targeted genomic changes of therapeutic value. Targeting double-strand breaks to desired genomic loci can be used to introduce frameshift mutations in the coding sequence of genes due to the error-prone nature of non-homologous DNA repair pathways. Zinc finger nucleases can be generated to target sites of interest by methods well known to those skilled in the art. For example, zinc finger binding domains with desired specificity can be designed by combining individual zinc finger motifs of known specificity. The structure of the zinc finger protein Zif268 bound to DNA has informed much of the research in this field. The concept of obtaining a zinc finger for each of the 64 possible base pair triplets and then mixing and matching these modular zinc fingers to design proteins with any desired sequence specificity has been described (Pavletich NP, Pabo CO (May 1991). "Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A". Science 252 (5007): 809-17, the entire contents of which are incorporated herein). In some embodiments, separate zinc fingers, each recognizing a three-base pair DNA sequence, are combined to generate three-, four-, five-, or six-finger arrays that recognize target sites ranging from 9 to 18 base pairs in length. Longer arrays are contemplated in some embodiments.In other embodiments, two-finger modules recognizing 6-8 nucleotides are combined to generate 4-, 6-, or 8-zinc finger arrays. In some embodiments, bacterial or phage display is used to develop zinc finger domains that recognize desired nucleic acid sequences (e.g., desired nuclease target sites 3-30 bp in length). Zinc finger nucleases, in some embodiments, comprise a zinc finger binding domain and a cleavage domain fused or otherwise conjugated to each other via a linker (e.g., a polypeptide linker). The length of the linker determines the distance of cleavage from the nucleic acid sequence bound by the zinc finger domain. If a shorter linker is used, the cleavage domain will cleave nucleic acids closer to the bound nucleic acid sequence. Meanwhile, a longer linker will result in a greater distance between the cleaved and bound nucleic acid sequences. In some embodiments, the cleavage domain of a zinc finger nuclease must dimerize to cleave the bound nucleic acid. In some such embodiments, the dimer is a heterodimer of two monomers, each of which contains a different zinc finger binding domain. For example, in some embodiments, a dimer can include one monomer comprising zinc finger domain A conjugated to a FokI cleavage domain and one monomer comprising zinc finger domain B conjugated to a FokI cleavage domain. In this non-limiting example, zinc finger domain A binds to a nucleic acid sequence on one side of a target site, zinc finger domain B binds to a nucleic acid sequence on the other side of the target site, and the dimerized FokI domain cleaves the nucleic acid between the zinc finger domain binding sites.

[0050] The term "zinc finger," as used herein, refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold. Zinc fingers encompass a variety of different protein structures (see, e.g., Klug A, Rhodes D (1987). "Zinc fingers: a novel protein fold for nucleic acid recognition." Cold Spring Harb. Symp. Quant. Biol. 52: 473-82, the entire contents of which are incorporated herein by reference). Zinc fingers can be designed to bind to specific sequences of nucleotides, and zinc finger arrays comprising fusions of a series of zinc fingers can be designed to bind to almost any desired target sequence. Such zinc finger arrays can form the binding domain of a protein (e.g., of a nuclease when conjugated to a nucleic acid-cleavage domain). Different types of zinc finger motifs are known to those skilled in the art, including, but not limited to, Cys2His2, Gag knuckle, Treble-clef, zinc ribbon, Zn2 / Cys6, and TAZ2 domain-like motifs (see, e.g., Krishna SS, Majumdar I, Grishin NV (January 2003). "Structural classification of zinc fingers: survey and summary." Nucleic Acids Res. 31 (2): 532-50). Typically, a single zinc finger motif binds to 3 or 4 nucleotides of a nucleic acid molecule. Thus, a zinc finger domain containing two zinc finger motifs can bind to 6-8 nucleotides, a zinc finger domain containing three zinc finger motifs can bind to 9-12 nucleotides, a zinc finger domain containing four zinc finger motifs can bind to 12-16 nucleotides, and so on.Any suitable protein engineering technique can be used to alter the DNA-binding specificity of a zinc finger and / or design novel zinc finger fusions to bind to almost any desired target sequence of 3 to 30 nucleotides in length (see, e.g., Pabo CO, Peisach E, Grant RA (2001). "Design and selection of novel cys2His2 zinc finger proteins". Annual Review of Biochemistry 70: 313-340; Jamieson AC, Miller JC, Pabo CO (2003). "Drug discovery with engineered zinc-finger proteins". Nature Reviews Drug Discovery 2 (5): 361-368; and Liu Q, Segal DJ, Ghiara JB, Barbas CF (May 1997). "Design of polydactyl zinc-finger proteins for unique addressing within complex genomes". Proc. Natl. Acad. Sci. USA 94 (11), the entire contents of each of which are incorporated herein by reference). Fusions between engineered zinc finger arrays and nucleic acid-cleaving protein domains can be used to generate "zinc finger nucleases." Zinc finger nucleases typically contain a zinc finger domain that binds to a specific target site in a nucleic acid molecule and a nucleic acid cleavage domain that cleaves the nucleic acid molecule within or proximal to the target site bound by the binding domain. Typical engineered zinc finger nucleases contain a binding domain with three to six individual zinc finger motifs that bind to target sites ranging from 9 to 18 base pairs in length. Longer target sites are particularly attractive in situations where it is desired to bind to and cleave a target site that is unique in a given genome.

[0051] The terms "treatment," "treat," and "treating" refer to clinical measures aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. As used herein, the terms "treatment," "treat," and "treating" refer to clinical measures aimed at reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms have developed and / or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay the onset of symptoms or inhibit the onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to prevent or delay their recurrence. [Brief explanation of the drawings]

[0052] [Figure 1] Schematic diagram of macromolecule delivery into mammalian cells. [Figure 2] Programming adipocyte cell fate: Switching from white adipose tissue (WAT) to brown adipose tissue (BAT). [Figure 3] A supercharged delivery platform is used to deliver TALE activators programmed to target PPARγ or PRDM16. [Figure 4] Schematic diagram of the fusion protein containing the +36 GFP fusion, the 18.5-mer TALE domain, and the VP64 activation domain. [Figure 5] Expression and purification of +36GFP-TALE activator-fusion protein.

[0053] [Figure 6]+36 Testing the activation of adipocyte regulator genes by delivery of GFP-PPARγ and PRDM16-TALE activator fusion proteins. [Figure 7] Delivery efficacy of +36GFP-TALE activator fusion protein at different concentrations. [Figure 8] Comparison of the delivery efficacy of two different +36GFP-PRDM16-TALE fusion proteins in NIH3T3 cells. [Figure 9] PPARγ gene expression after delivery of PPARγ-TALE activator fusions and comparison to various controls.

[0054] [Figure 10] PRDM16 gene expression after delivery of RDM16-TALE activator fusions and comparison to various controls. [Figure 11] Moderate TALE activity is observed in the presence of serum. [Figure 12] Validation of viral delivery of PPARγ followed by 7 days of treatment with an adipogenic cocktail. [Figure 13] Schematic of the assay for programming fibroblasts into WAT and BAT. [Figure 14] +36 Adipogenesis observed by treatment with GFP-TALE activator fusion protein. [Figure 15] LipidTOX red staining after 7 days of various treatments shows the formation of adipocytes after viral delivery and also after delivery of the supercharged PPARγ-TALE activator fusion protein.

[0055] [Figure 16] LipidTOX red staining after 7 days of various treatments shows the formation of adipocytes after viral delivery and also after delivery of the supercharged PPARγ-TALE activator fusion protein. [Figure 17] Expression of WAT biomarker genes after viral delivery and also after delivery of supercharged PPARγ-TALE activator fusion proteins. [Figure 18] Delivery of supercharged PRDM16-TALE activator fusion proteins to induce brown fat adipogenesis in vivo. Robust adipogenesis was observed after viral delivery of PPARγ and PRDM16, and also after delivery of supercharged TALE activator protein fusions. [Figure 19] Comparison of TALE / TALE, virus / TALE, and virus / virus-induced expression of brown fat markers with PPARγ and PRDM16 expression.

[0056] [Figure 20] RT-qPCR assessment is consistent with the adipocyte differentiation observed by LipidTOX staining. [Figure 21] Delivery of functional TALE activator fusion proteins as complexes with +36GFP improves TALE activator activity after delivery. [Figure 22] PRDM16 gene expression after delivery of TALE activator fusions either as fusions with +36GFP (+36GFP-PRDM16-TALE-3) or as complexes (+36GFP+PRDM16-TALE-3). Delivery of complexes tends to increase TALE activator activity.

[0057] [Figure 23] Effect of Aurein peptide fusion to +36GFP on PRDM16 gene expression after delivery of TALE activator fusions (either as fusions or complexes with +36GFP). [Figure 24] PRDM16 gene expression after delivery of TALE activator fusions as either fusions (+36GFP-PRDM16-TALE-3) or complexes (+36GFP+PRDM16-TALE-3) by Lipofectamine LTX. [Figure 25] Delivery of a supercharged fusion protein or complex with Cas9 into mammalian cells. (GGS)9-T-ALAL-PKKKRKV corresponds to SEQ ID NO: 251. [Figure 26]Purification of wild-type Cas9 protein and Cas9 fusion proteins with +36GFP and Aurein-GGS9.

[0058] [Figure 27] Strategies for delivering proteins into mammalian cells by fusion or noncovalent complexation with polyanionic polymers and encapsulation by cationic lipids are presented. Figure 27(A) shows that recombinases, transcription activator-like effector (TALE) proteins, and Cas9 endonucleases bind nucleic acids, are naturally cationic (net theoretical charges are shown in black), and are not efficiently encapsulated by cationic lipids. However, these proteins can be made highly anionic by fusion to ultranegatively charged proteins such as (-30)GFP or by complexation with polyanionic nucleic acids. Figure 27(B) shows a schematic illustrating how cationic lipids commonly used to transfect DNA and RNA encapsulate the resulting highly anionic proteins or protein:nucleic acid complexes, mediating their delivery into mammalian cells.

[0059] [Figure 28]Delivery of Cre recombinase into cultured human cells. Figure 28(A) shows the fusion of either the highly cationic (+36)GFP or the highly anionic (-30)GFP to Cre recombinase. A HeLa reporter cell line that expresses DsRed upon Cre-mediated recombination was used to evaluate Cre delivery efficiency. (GGS)9 corresponds to SEQ ID NO: 252, and His6 corresponds to SEQ ID NO: 253. Figure 28(B) shows HeLa-dsRed cells treated with 10 nM (-30)GFP-Cre and the cationic lipid RNAiMAX. Cells were visualized after 48 hours of incubation in medium containing 10% fetal bovine serum (FBS). Figure 28(C) shows the delivery of (+36)GFP-Cre in 10% FBS medium or serum-free medium, and (-30)GFP-Cre with or without the cationic lipid RNAiMAX in full serum medium. Figure 28(D) presents the effect of cationic lipid dose on functional (-30)GFP-Cre delivery efficacy after 48 hours in 275 μL of medium containing 10% FBS. Figure 28(E) compares the functional delivery efficacy of (-30)dGFP-Cre with several commercially available cationic lipids and polymers. Figure 28(F) shows RNAiMAX-mediated delivery of multiple anionic peptide or protein sequences fused to Cre. Error bars reflect standard deviation from three biological replicates performed on different days.

[0060] [Figure 29]Delivery of TALE transcription activators into cultured human cells. Figure 29(A) shows the design of an 18.5-repeat TALE activator C-terminally fused to the VP64 activation domain and N-terminally fused to (-30)GFP and an NLS. (GGS)9 corresponds to SEQ ID NO: 251, and His6 corresponds to SEQ ID NO: 252. The net theoretical total charge of the fusion is -43. Figure 29(B) demonstrates activation of NTF3 transcription by conventional transfection of a plasmid encoding a TALE-VP64 activator targeting a site within the NTF3 gene or by RNAiMAX cationic lipid-mediated delivery of the corresponding NTF3-targeting (-30)GFP-TALE-VP64 protein. Gene expression levels were measured by qRT-PCR and normalized to GAPDH expression levels. Error bars reflect the standard deviation from three biological replicates performed on different days.

[0061] [Figure 30]Delivery of Cas9:sgRNA, Cas9D10A nickase, and dCas9-VP64 transcriptional activator into cultured human cells. Figure 30(A) demonstrates cationic lipid-mediated delivery of Cas9 protein variants complexed with an sgRNA targeting EGFP or an sgRNA targeting VEGF into U2OS EGFP reporter cells. Results are compared to those of standard transfection of Cas9 and sgRNA expression plasmids. Figure 30(B) shows the results of a T7 endonuclease I (T7EI) assay to measure EGFP modification. Figure 30(C) shows the results of a T7EI assay of genome modification in EGFP and three endogenous genes by single delivery of four sgRNAs and Cas9 complexed with RNAiMAX. The indel efficiency calculated by densitometry is shown below the gel image. Figure 30(D) shows the delivery of sgRNA pairs and Cas9D10A nickase by either plasmid transfection or RNAiMAX-mediated protein:RNA complex delivery. The sgRNAs GFPg1 + GFPg5 or GFPg3 + GFPg7 that disrupt EGFP are predicted to result in gene disruption, whereas GFPg5 + GFPg7 targets the same strand and is therefore predicted to be non-functional. Figure 30(E) shows the delivery of a catalytically incompetent (dCas9)-VP64 transcription activator targeting NTF3 by either plasmid transfection or RNAiMAX-mediated protein delivery. Delivery of both VEGFg3 and VEGFg5 sgRNAs served as negative controls for NTF3 gene activation.Error bars reflect the standard deviation from six biological replicates performed on different days.

[0062] [Figure 31] The DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells via plasmid transfection or cationic lipid-mediated protein:sgRNA delivery is shown. In Figure 31(A), T7EI assays were performed for on-target modification of the endogenous CLTA, EMX, and VEGF genes. In Figure 31(B-D), the on-target:off-target DNA modification ratios resulting from Cas9:sgRNA are shown for plasmid transfection or cationic lipid-mediated protein:sgRNA delivery. The conditions for each treatment were adjusted to result in approximately 10% on-target cleavage, allowing for comparison of DNA cleavage specificity between the two delivery methods under conditions where on-target gene modification efficiency was comparable. P values ​​are listed in Table 2. Each on- and off-target sample was sequenced once, with >10,000 sequences analyzed per on-target sample and an average of >111,000 sequences analyzed per off-target sample (Table 2).

[0063] [Figure 32]In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear is shown. In Figure 32(A), the scala media (cochlea) of P0 floxP-tdTomato mice (n=4) was injected with 0.3 μL of 23 μM (-30)GFP-Cre in 50% RNAiMAX or RNAiMAX alone (control). Five days later, tdTomato expression, indicative of Cre-mediated recombination, was visualized using immunohistology. Red = tdTomato, green = Myo7a, white = Sox2, blue = DAPI. The yellow bracket indicates the outer hair cell (OHC) layer. Figure 32(B) shows that 10 days after (-30)GFP-Cre delivery, intact espin (Esp)-expressing stereocilia of tdTomato-positive outer hair cells were present (arrows), similar to stereocilia in control cochleae. Red = tdTomato, green = Esp, white = Sox2, blue = DAPI. Figure 32(C) is identical to Figure 32(A) except that Lipofectamine 2000 was used instead of RNAiMAX (n=4). The top and bottom panels are images of mouse cochleae at low and high magnification, respectively, detailing the efficiency of delivery and its effect on cochlear structure and hair cell loss. Figure 32(D) shows the results when the scala media (cochlea) of P2 Atoh1-GFP mice (n=3) was injected with 0.3 μL of 33 μM sgRNA and 33 μM Cas9 in 50% RNAiMAX or Lipofectamine 2000. Cas9-mediated gene disruption results in loss of GFP expression when visualized 10 days later. The upper panel shows only the GFP signal, while the lower panel includes additional immunohistochemistry markers. The yellow box in the lower panel highlights hair cells that have lost GFP expression. Red = tdTomato, green = Myo7a, white / light blue = Sox2, blue = DAPI. All scale bars are shown in white and are 10 μm.

[0064] [Figure 33]Optimization of cationic lipid-mediated delivery of Cre recombinase. Figure 33(A) shows the optimization of (-30)GFP-Cre delivery in BSR-TdTomato cells (a second reporter cell line used to measure Cre recombination efficiency). Figure 33B demonstrates the effect of RNAiMAX dose on (-30)GFP-Cre recombination efficiency and toxicity in HeLa dsRed reporter cells, as measured by FACS. HeLa cells were sorted by forward and side scatter gating to identify viable cells that retained normal morphology. Figure 33(C) illustrates the relationship between the net charge of the protein fused to Cre recombinase and the functional cationic lipid-mediated Cre delivery efficiency. 25 nM of Cre recombinase fused to the listed domains was combined with 1.5 μL of RNAiMAX and incubated with HeLa dsRed reporter cells. After two days, recombination efficiency was measured by FACS. Error bars reflect the standard deviation from three biological replicates performed on different days.

[0065] [Figure 34]Protein uptake by cationic lipid-mediated delivery versus hyperpositive cationic protein delivery. Figure 34(A) quantifies GFP fluorescence from cells treated with (-30)GFP-Cre and either RNAiMAX or (+36)GFP-Cre after washing the cells with PBS + heparin (20 U / mL) to remove unbound protein. Figure 34(B) shows the functional Cre recombinase delivery efficiency of (-30)GFP-Cre + 1.5 μL RNAiMAX relative to the Cre recombinase delivery efficiency resulting from fusion with (+36)GFP. Figure 34(C) provides a comparison of mCherry uptake by (-30)GFP fusion + 1.5 μM RNAiMAX treatment versus (+36)GFP fusion by measuring the average mCherry fluorescence of the total cell population 48 hours after treatment, with cells washed with PBS + heparin. Figure 34(D) shows total cellular GFP fluorescence of (-30)GFP-Cre or (+36)GFP-Cre in the presence or absence of RNAiMAX.

[0066] [Figure 35] Delivery optimization of a TALE activator designed to target the NTF3 gene. HEK293T cells were treated with either NTF3-TALE plasmid via transfection or liposomal delivery of NTF3-TALE protein. Cells were collected for both treatments after experimentally determined optimal incubation times and analyzed for NTF3 mRNA levels by qRT-PCR. The optimal protein (25-50 nM) and lipid dose (1.5 µL of RNAiMAX) were selected for the comparison of the two delivery techniques in Figure 29B. Error bars reflect the standard deviation from six biological replicates performed on different days.

[0067] [Figure 36]Determination of gene disruption frequency of the EGFP reporter gene by delivery of Cas9:sgRNA and analysis by flow cytometry. Figure 36(A) provides a schematic of EGFP disruption in U2OS cells by NHEJ induced by a Cas9 double-strand break. Figure 36(B) shows delivery of an EGFP-targeting sgRNA or an off-target sgRNA complexed with (-30)dGFP-Cas9 using RNAiMAX, along with a positive plasmid transfection control (orange). Figure 36(C) provides confirmation that the disruption of EGFP fluorescence is not the result of cytotoxicity by treating samples with TO-PRO-3 live / dead stain (Life Technologies, Carlsbad, CA) and analyzing the resulting cells by flow cytometry. Figure 36(D) shows testing of TO-PRO-3 staining with the addition of a cell-permeabilizing, but not completely membrane-lytic, detergent (0.5% Tween).

[0068] [Figure 37] Optimization of functional delivery of Cas9:sgRNA. In Figure 37(A), cationic lipid-mediated delivery efficiency of the two tested constructs indicates that the more anionic (-30)dGFP-NLS-Cas9 facilitates more efficient delivery at lower protein and sgRNA concentrations compared to native Cas9. Figure 37(B) shows delivery optimization of (-30)dGFP-NLS-Cas9 as a function of protein and sgRNA concentration. Figure 37(C) shows delivery of either fusion or untagged Cas9 protein as a function of protein and sgRNA concentration. Figure 37(D) shows the optimal sgRNA-to-protein ratio for RNAiMAX-mediated delivery of (-30)dGFP-NLS-Cas9 and native Cas9. Error bars reflect standard deviation from three biological replicates performed on different days.

[0069] [Figure 38]Effect of NLS and / or (-30)dGFP on functional Cas9 delivery as a function of both sgRNA and Cas9 concentration. EGFP gene disruption was measured at three fixed sgRNA concentrations: 10 nM (Figure 38(A)), 25 nM (Figure 38(B)), and 50 nM (Figure 38(C)), along with various protein concentrations as indicated on the graph. Delivery was performed using 0.8 μL of RNAiMAX and assayed by FACS after 48 hours for loss of EGFP fluorescent signal.

[0070] [Figure 39] The effects of RNAiMAX and Lipofectamine 2000 on Cas9:sgRNA delivery efficiency and cytotoxicity. Figure 39(A) shows EGFP gene disruption at different Cas9 protein concentrations and a constant dose of 100 nM EGFP sgRNA in U2OS EGFP reporter cells treated with either RNAiMAX or Lipofectamine 2000 for 16 hours. After 16 hours, the medium was removed, and fresh medium was added to the cells until the assay endpoint of 48–72 hours after protein delivery treatment. Viable cell populations were determined by FACS using TO-PRO-3 Live / Dead staining. Figure 39(B) shows the toxicity profile of Cas9:sgRNA delivery into U2OS cells as a function of Lipofectamine 2000 dose. Figure 39(C) provides the toxicity profile for cells as a function of RNAiMAX dose. Error bars reflect the standard deviation from three biological replicates performed on different days.

[0071] [Figure 40]Optimization of dCas9-VP64 delivery targeting the NTF3 gene at various protein and sgRNA concentrations. HEK293T cells were treated with dCas9-VP64 activator and either NTF3-targeting gRNAg2 or a mixture of all six NTF3-targeting sgRNAs for 16 hours in a 48-well plate format (final volume of 275 μL) with 0.8 μL of RNAiMAX. NTF3 mRNA levels were determined by qRT-PCR and normalized to those of GAPDH. Error bars reflect the standard deviation from six biological replicates performed on different days.

[0072] [Figure 41] The indel frequencies (measured by high-throughput sequencing) of several human genes treated with either mock treatment, transfection of a linear DNA PCR product of the Cas9 plasmid and sgRNA, or cationic lipid-mediated protein:sgRNA delivery are shown. The mock treatment involved cationic lipid-mediated protein:sgRNA delivery of an sgRNA targeting EGFP in place of the sgRNAs targeting three human genes. Figure 41(A) shows the on-target and off-target indel frequencies for the CLTA gene. Figure 41(B) provides the on-target and off-target indel frequencies for the EMX gene. Figure 41(C) demonstrates the on-target and off-target indel frequencies for the VEGF gene. Each on- and off-target sample was sequenced once, with >10,000 sequences analyzed per on-target sample and an average of >111,000 sequences analyzed per off-target sample (Table 2).

[0073] [Figure 42]Delivery of Cas9 endonuclease into mouse embryonic stem cells. Figure 42(A) shows that floating spheres treated with Cas9 protein and RNAiMAX but not sgRNA (control) retained strong GFP fluorescence (right), while those treated with Cas9:sgRNA and RNAiMAX displayed reduced GFP fluorescence (left). Scale bar: 100 μm. Figure 42(B) shows control progenitor cells after cell attachment; virtually all control progenitor cells were GFP positive (right panel). Cas9:sgRNA treatment led to a significant reduction in GFP expression (left panel), and many progenitor cells showed complete GFP knockdown after cell attachment (arrows). Scale bar: 20 μm. Figure 42(C) shows that T7EI assay of stem cells collected after imaging confirms cleavage of the GFP reporter. Similar gene targeting efficiencies were observed from cationic lipid-mediated Cas9:sgRNA delivery and transfection of Cas9 and EGFP sgRNA plasmids.

[0074] [Figure 43]Genome modification induced by cationic lipid-mediated protein delivery of Cas9 nuclease and sgRNA at endogenous loci in vivo. Figure 43(A) shows a representative example of the genomic DNA sequence of an EGFP on-target locus modified in mouse hair cells after cationic lipid-mediated delivery of Cas9 and EGFP sgRNA. For each example shown, the unmodified genomic site is the first sequence, followed by the eight most abundant sequences containing deletions and three sequences containing insertions. The number before each sequence indicates the sequencing count. The sgRNA target site is bold, underlined in green, and inserted. Insertions and deletions are shown in red. PAM sites are shown in blue. Figure 43(B) shows the same analysis as Figure 42(A) for EMX on-target sites in mouse hair cells. The sequences shown in Figure 43(A) correspond, from top to bottom, to SEQ ID NOs: 223 to 236, and the sequences shown in Figure 43(B) correspond, from top to bottom, to SEQ ID NOs: 237 to 250. DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention provides complexes, compositions, preparations, kits, systems, and related methods for delivery of functional effector proteins (e.g., nucleases, recombinases, and Cas9 proteins (including variants and fusions thereof, e.g., Cas9 fusions to Cas9 nickases and deaminases, gene editing enzymes, transcriptional repressors and activators, epigenetic modifiers, etc.) to cells by associating the functional effector proteins with one or more of supercharged proteins, cationic polymers, and / or cationic lipids. Typically, the functional effector proteins are delivered inside a cell to elicit, for example, a biological effect within the cell (e.g., cleavage of a genomic target sequence or modulation of expression of a target gene). In some embodiments, the biological effect exerts a therapeutic benefit on the subject in which the cells are found. The complexes, compositions, preparations, systems, kits, and related methods for delivery of functional effector proteins are useful for introducing effector proteins into cells, for example, in the context of manipulating cells for research or therapeutic purposes. The compositions, preparations, systems, kits, and related methods for delivering functional effector proteins provided herein exhibit improved efficacy and reduced cytotoxicity, as well as ease of preparation, compared to current technologies. The delivery of site-specific proteins, such as TALEN or Cas9 proteins (or variants or fusions thereof), using the compositions, preparations, systems, kits, and related methods provided herein allows for targeted manipulation / modification of the genome of host cells in vitro or in vivo, while avoiding the use of more invasive delivery methods (e.g., viral delivery of vectors encoding site-specific proteins).

[0076] In some embodiments, the technology of the present invention uses supercharged proteins to deliver functional effector proteins into cells. In certain embodiments, the supercharged proteins are engineered proteins. In some embodiments, the supercharged proteins are naturally occurring supercharged proteins. Some aspects of the present invention are based on the recognition that supercharged proteins are endocytosed by cells, that functional effector proteins that can associate with the supercharged proteins are effectively taken up by cells along with the supercharged proteins, and that such functional effector proteins retain their biological function after intracellular uptake (e.g., in that they are capable of cleaving or modifying genomic target sites or modulating the transcription of target genes).

[0077] In some embodiments, compositions provided herein comprising a supercharged protein associated with a functional effector protein (e.g., a nuclease, a transcription activator / repressor, a recombinase, a Cas9 protein (including variants and fusions thereof), etc.) are useful as therapeutic agents, diagnostic agents, or research tools. In some embodiments, the functional effector protein (e.g., a nuclease or a transcription factor) can be therapeutically active, for example, in targeting a gene associated with a disease or disorder. In some embodiments, compositions provided herein comprising a supercharged protein and a functional effector protein (e.g., a nuclease or a transcription factor) are used to regulate the expression of a gene in a cell or regulate a biological pathway (e.g., a signaling pathway, a metabolic pathway) in the cell. In some embodiments, a cell is contacted with a composition of the invention described herein to introduce a functional effector protein into the cell. In some embodiments, a composition of the invention is administered to a subject in need thereof to introduce a functional effector protein into a cell within the subject (e.g., into a cell associated with a disease or disorder). Suitable cells and cell types for delivery of functional effector proteins according to some aspects of the present disclosure include, but are not limited to, human cells, mammalian cells, T cells, neurons, stem cells, progenitor cells, blood cells, fibroblasts, epithelial cells, neoplastic cells, and tumor cells.

[0078] Supercharged Proteins Supercharged proteins for use in the present invention can be generated by changing non-conserved amino acids on the surface of a protein to more polar or charged amino acid residues. In certain embodiments, non-conserved amino acids on the surface of a protein are mutated to become positively charged amino acids at physiological pH (about pH 7.4). The amino acid residues to be modified can be hydrophobic, hydrophilic, charged, or a combination thereof. Supercharged proteins can also be generated by attaching charged moieties to proteins to make them supercharged. Supercharged proteins are frequently resistant to aggregation, have an increased ability to refold, are resistant to improper folding, have improved solubility, and are generally more stable under a wide range of conditions (including denaturing conditions such as heat or the presence of detergents).

[0079] Supercharged proteins suitable for use in accordance with aspects of the present disclosure are known in the art and include, without limitation, the supercharged proteins disclosed in International PCT Patent Application No. PCT / US07 / 70254 (filed June 1, 2007, published December 13, 2007 as WO2007 / 143574), International PCT Application No. PCT / US09 / 041984 (filed April 28, 2009, published November 5, 2009 as WO2009 / 134808), and International PCT Application No. PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), the entire contents of each of which are incorporated herein by reference. In some embodiments, the supercharged protein is an engineered supercharged protein. In some embodiments, the supercharged protein is a naturally occurring supercharged protein, such as a naturally occurring supercharged protein disclosed in International PCT Application PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), each of which is incorporated herein by reference. In some embodiments, the engineered or naturally occurring supercharged protein exhibits a charge:molecular weight ratio of greater than 0.8 (e.g., >0.85, >0.9, >0.95, >1, >1.1, >1.2, >1.3, >1.4, >1.5, >1.6, >1.7, >1.8, >1.9, >2, >2.5, >3, >4, >5, >6, >7, >8, or >10).

[0080] The supercharged protein used can be derived from any species of plant, animal, and / or microbial organism. In certain embodiments, the supercharged protein is a mammalian protein. In certain embodiments, the supercharged protein is a human protein. In certain embodiments, the protein is derived from an organism typically used in research. For example, the protein to be modified can be from a primate (e.g., ape, monkey), rodent (e.g., rabbit, hamster, gerbil), pig, dog, cat, fish (e.g., Danio rerio), nematode (e.g., C. elegans), yeast (e.g., Saccharomyces cerevisiae), or bacterium (e.g., E. coli). In certain embodiments, the protein is non-immunogenic. In certain embodiments, the protein is non-antigenic. In certain embodiments, the protein has no native biological activity or has been modified to have no biological activity. In certain embodiments, the protein is selected based on its targeting ability. In certain embodiments, the protein is green fluorescent protein. In some embodiments, the supercharged protein is a supercharged glutathione S-transferase (GST). In some embodiments, the supercharged protein is a supercharged streptavidin.

[0081] In some embodiments, supercharged proteins are used that have been modified to increase their overall net charge or to increase the total number of charged residues on the protein surface. In certain embodiments, the theoretical net charge of the supercharged protein is increased by at least +1, at least +2, at least +3, at least +4, at least +5, at least +10, at least +15, at least +20, at least +25, at least +30, at least +35, or at least +40 compared to the unmodified protein. In certain embodiments, the theoretical net charge of the supercharged protein is at least +1, at least +2, at least +3, at least +4, at least +5, at least +10, at least +15, at least +20, at least +25, at least +30, at least +35, or at least +40 at physiological pH (i.e., about 7.4).

[0082] In other embodiments (e.g., those involving the use of cationic lipids and / or cationic polymers), supercharged proteins are used that have been modified to reduce the overall net charge or to reduce the total number of charged residues on the protein surface. In certain embodiments, the theoretical net charge of the supercharged protein is reduced by at least -1, at least -2, at least -3, at least -4, at least -5, at least -10, at least -15, at least -20, at least -25, at least -30, at least -35, at least -40, at least -45, or at least -50 compared to the unmodified protein ("minus" or "negative" is represented by "-"). In certain embodiments, the theoretical net charge of the supercharged protein is at least -1, at least -2, at least -3, at least -4, at least -5, at least -10, at least -15, at least -20, at least -25, at least -30, at least -35, at least -40, at least -45, or at least -50.

[0083] While several exemplary supercharged proteins are described herein to illustrate the technology of the present invention, the disclosure is not limited in this respect. Based on the present disclosure, one of skill in the art will be able to identify additional suitable supercharged proteins for delivering functional effector proteins to cells. A number of naturally occurring proteins can be modified to generate suitable supercharged proteins. The desired modifications in such proteins can be achieved using any technique known in the art. Recombinant DNA techniques for introducing such changes in protein sequences are well known in the art. In certain embodiments, the modifications are made by site-directed mutagenesis of the polynucleotide encoding the protein. Other techniques for introducing mutations are described in Molecular Cloning: A Laboratory Manual, 2002, pp. 111-114, 1997. ndEd., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989), the treatise, Methods in Enzymology (Academic Press, Inc., NY), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999), each of which is incorporated herein by reference.

[0084] Supercharged proteins can be further modified. Proteins, including supercharged proteins, can be modified using techniques known to those skilled in the art. For example, supercharged proteins can be chemically or biologically modified. One or more amino acids can be added, deleted, or changed from the primary sequence. For example, a polyhistidine tag or other tag can be added to a supercharged protein to aid in protein purification. Other peptides or proteins can be added to a supercharged protein to change the biological, biochemical, and / or biophysical properties of the protein. For example, an endosomolytic peptide can be added to the primary sequence of a supercharged protein, or a targeting peptide can be added to the primary sequence of a supercharged protein. Other modifications of supercharged proteins include, but are not limited to, post-translational modifications (e.g., glycosylation, phosphorylation, acylation, lipid modification, farnesylation, acetylation, proteolysis, etc.). In certain embodiments, a supercharged protein is modified to reduce its immunogenicity. In certain embodiments, supercharged proteins are modified to improve their ability to deliver functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.) to cells. In certain embodiments, supercharged proteins are conjugated to polymers. For example, proteins can be PEGylated by conjugating the protein to a polyethylene glycol (PEG) polymer. Other methods can be used to generate supercharged proteins without modifying the protein sequence. For example, net charge-altering moieties can be attached to the protein (e.g., by chemical or enzymatic reaction) to provide a surface charge to achieve supercharging. In certain embodiments, the protein modification methods described in Shaw et al., Protein Science 17: 1446, 2008, are used to supercharge proteins used in the inventive technology of this disclosure.

[0085] The design and production of several different supercharged protein variants suitable for use with the disclosed technology are described in International PCT Patent Application No. PCT / US07 / 70254 (filed June 1, 2007, published December 13, 2007 as WO2007 / 143574), International PCT Application No. PCT / US09 / 041984 (filed April 28, 2009, published November 5, 2009 as WO2009 / 134808), and International PCT Application No. PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), the entire contents of each of which are incorporated herein by reference. Some of the disclosed supercharged proteins described therein have been shown to be more stable and to retain their biological function (e.g., their fluorescence in the case of fluorescent proteins). For example, the green fluorescent protein (GFP) from Aequorea victoria is set forth in GenBank Accession No. P42212, incorporated herein by reference. The amino acid sequence of this wild-type GFP is as follows: MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO: 14)

[0086] Wild-type GFP has a theoretical net charge of -7. Variants with theoretical net charges of -29, -30, -25, +15, +25, +36, +48, ​​and +49 have been reported, for example, in International PCT Application No. PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), the entire contents of which are incorporated herein by reference. Even after heating +36 GFP to 95°C, 100% of the variant protein is soluble, and the protein retains ≥70% of its fluorescence.

[0087] Some aspects of the present disclosure are based on the discovery that +36 GFP efficiently delivers functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.) to target cells, and that the effector proteins so delivered retain their biological function. Therefore, GFP or other proteins having a net charge of at least +15, at least +25, at least +30, at least +35, or at least +40 are believed to be particularly useful for introducing functional effector proteins into cells.

[0088] In some embodiments, particularly useful supercharged proteins are those that allow for a charge distribution or surface charge density similar to that of +36 GFP. Additionally, in some embodiments, particularly useful supercharged proteins are those that exhibit a stable folded structure that is not easily perturbed by supercharge, thus allowing the supercharged protein to fold well. In some embodiments, particularly useful supercharged proteins are proteins that share structural features (e.g., a globular structure or a beta-barrel structure) with the supercharged proteins described herein or in International PCT Patent Application No. PCT / US07 / 70254 (filed June 1, 2007, published December 13, 2007 as WO2007 / 143574), International PCT Application No. PCT / US09 / 041984 (filed April 28, 2009, published November 5, 2009 as WO2009 / 134808), and International PCT Application No. PCT / US10 / 001250 (filed April 28, 2010, published November 11, 2010 as WO2010 / 129023), the entire contents of each of which are incorporated herein by reference. Protein folding, protein fold structural stability, and perturbations of protein folding by substitution of specific amino acids with differently charged amino acids, charge distribution, and surface charge density can be modeled in silico by the methods and algorithms provided herein and others known to those skilled in the art. Thus, whether a given supercharged protein folds well will be apparent to one skilled in the art through simple experimentation. Therefore, one skilled in the art will be able to identify, from a given amino acid sequence, whether a given supercharged protein is useful for intracellular delivery of a functional effector protein according to the techniques described herein.

[0089] Some exemplary suitable variants of GFP include, without limitation: +15GFP: MGHHHHHHGGASKGERLFTGVVPILVELDGDVNGHKFSVRGEGEGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPEGYVQERTISFKKDGTYKTRAEVKFEGRTLVNRIELKGRDFKEKGNILGHKLEYNFNSHNVYITADKRKNGIKANFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSALSKDPKEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO: 15) +25GFP: MGHHHHHHGGASKGERLFTGVVPILVELDGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGTYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHNVYITADKRKNGIKANFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSALSKDPKEKRDHMVLLEFVTAAGITHGMDELYK (SEQ ID NO: 16) +36GFP: MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYK (SEQ ID NO: 17) +42GFP: MGHHHHHHGGRSKGKRLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRKERYK (SEQ ID NO: 18) +48GFP: MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRKERYK (SEQ ID NO: 19) +49GFP: MGHHHHHHGGRSKGKRLFRGKVPILVKLKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFKGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLAKHYQQNTPIGRGPVLLPRKHYLSTRSKLSKDPKEKRDHMVLKEFVTAAGIKHGRKERYK (SEQ ID NO: 20) (-)30GFP: MGHHHHHHGGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYK (SEQ ID NO: 21) It will be clear to one skilled in the art that the above sequences include an N-terminal His6 tag, and that sequences without such a tag or with a different tag are also suitable.

[0090] To facilitate the biological function of a functional effector protein (e.g., a nuclease, transcription activator / repressor, recombinase, Cas9 protein (including variants and fusions thereof), etc.) after delivery to a cell, it may be desirable to enhance endosomal escape of the functional effector protein after cellular uptake. The supercharged protein or functional effector protein can be fused or linked to a protein, peptide, or other entity known to enhance endosomal degradation or lysis of endosomes. In certain embodiments, the peptide is a hemagglutinin 2 (HA2) peptide, which is known to enhance endosomal degradation. In certain specific embodiments, the HA2 peptide is fused to a supercharged GFP (e.g., +36GFP). In certain specific embodiments, the fused protein has the following sequence: +36GFP-HA2 MGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGKYKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAKFKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEFVTAAGIKHGRDERYKGSAGSAAGSGEFGLFGAIAGFIENGWEGMIDG (SEQ ID NO: 22) In certain embodiments, the endosomolytic peptide is the melittin peptide (GIGAVLKVLTTGLPALISWIKRKRQQ; SEQ ID NO: 23) (Meyer et al., JACS 130(11): 3272-3273, 2008, which is incorporated herein by reference). In certain embodiments, the melittin peptide is modified by one, two, three, four, or five amino acid substitutions, deletions, and / or additions. In certain embodiments, the melittin peptide has the sequence CIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 24). In certain specific embodiments, the melittin peptide is fused to a supercharged GFP (e.g., +36 GFP).

[0091] In certain embodiments, the endosomolytic peptide is penetratin peptide (RQIKIWFQNRRMKWKK-amide; SEQ ID NO: 25), bovine PrP(1-30) peptide (MVKSKIGSWILVLFVAMWSDVGLCKKRPKP-amide; SEQ ID NO: 26), MPGΔ NLSpeptide (lacking a functional nuclear localization sequence due to a K->S substitution) (GALFLGWLGAAGSTMGAPKSKRKV; SEQ ID NO: 27), TP-10 peptide (AGYLLGKINLKALAALAKKIL-amide; SEQ ID NO: 28), and / or EB1 peptide (LIRLWSHLIHIWFQNRRLKWKKK-amide; SEQ ID NO: 29) (Lundberg et al., 2007, FASEB J. 21:2664, incorporated herein by reference). In certain embodiments, the penetratin, PrP(1-30), MPG, TP-10, and / or EB1 peptides are modified by one, two, three, four, or five amino acid substitutions, deletions, and / or additions. In certain specific embodiments, the PrP(1-30), MPG, TP-10, and / or EB1 peptides are fused to supercharged GFP (e.g., +36GFP). In some embodiments, the Aurein peptide is fused to a supercharged protein.

[0092] Other peptides or proteins can also be fused to supercharged proteins or fusion proteins comprising supercharged proteins and functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.). For example, a targeting peptide can be fused to a supercharged protein to selectively deliver the functional effector protein to a specific cell type. Peptides or proteins that improve the cellular uptake of the functional effector protein can also be used. In certain embodiments, the peptide fused to the supercharged protein is a peptide hormone. In certain embodiments, the peptide fused to the supercharged protein is a peptide ligand.

[0093] The exemplary supercharged proteins detailed herein are not meant to limit the present disclosure. Those skilled in the art will recognize that other supercharged proteins can be used for intracellular delivery of functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.), including, but not limited to, other GFP-like fluorescent proteins. In certain embodiments, the supercharged protein is a supercharged version of blue fluorescent protein. In certain embodiments, the supercharged protein is a supercharged version of cyan fluorescent protein. In certain embodiments, the supercharged protein is a supercharged version of yellow fluorescent protein. Exemplary suitable fluorescent proteins include enhanced green fluorescent protein (EGFP), AcGFP, TurboGFP, Emerald, Azami-Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyan1, Midori-Ishi-Cyan, mTFP1 (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, and ZsYel. These include, but are not limited to, low1, mBanana, Kusabira-Orange, mOrange, dTomato, dTomato-Tandem, DsRed, DsRed2, DsRed-Express(T1), DsRed-Monomer, mTangerine, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, HcRed1, HcRed-Tandem, mPlum, and AQ143.

[0094] Still other proteins that are supercharged and can be used to deliver functional effector proteins disclosed herein (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof)), include histone constituents or histone-like proteins, high mobility group proteins (HMG), enzymes (e.g., amylases, pectinases, hydrolases, proteases, glucose isomerases, lipases, phytases, alglucerases, imiglucerases, agalsidase beta, α-1-iduronidase, acid α-glucosidase, and iduronate-2-sulfatase, N-acetylgalactosamine-4-sulfatase). Charged polymers other than proteins can also be used to deliver functional effector proteins. In addition, as described in more detail herein, cationic lipids and lipid-like materials, as well as cationic polymers, can also be used to deliver functional effector proteins. Suitable cationic lipids, lipid-like materials, and cationic polymers are disclosed herein, and additional suitable lipids and lipid-like materials are known to those skilled in the art (see, for example, those described in Akinc et al., Nature Biotechnology 26, 561-569 (2008) (the entire contents of which are incorporated herein by reference)).

[0095] Delivery of functional effector proteins using supercharged proteins The present invention provides systems and methods for delivery of functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.) to cells in vivo, ex vivo, or in vitro. Such systems and methods typically involve association of the functional effector protein with a supercharged protein to form a complex or fusion protein, and delivery of the complex or fusion protein to the cell. In some embodiments, the functional effector protein to be delivered by the supercharged protein has a therapeutic activity. In some embodiments, delivery of the complex or fusion protein to the cell involves administering to a subject in need thereof a complex or fusion protein comprising the supercharged protein associated with the functional effector protein.

[0096] In some embodiments, a functional effector protein (e.g., a nuclease, transcriptional activator / repressor, recombinase, Cas9 protein (including variants and fusions thereof), etc.) may not be capable of entering a cell by itself, but is capable of entering a cell when associated with a supercharged protein, e.g., via a covalent or non-covalent interaction. In some embodiments, a composition is provided that includes a functional effector protein covalently linked to a supercharged protein. In some embodiments, a composition includes a functional effector protein fused to a supercharged protein via a peptide bond (e.g., via direct fusion or via a peptide linker). In some embodiments, a composition includes a functional effector protein bound to a supercharged protein by a non-covalent interaction. In some embodiments, a supercharged protein is utilized to enable the functional effector protein to enter a cell. In some embodiments, the functional effector protein to be delivered to a cell that is linked to the supercharged protein is separated from the supercharged protein after delivery to the cell, for example, by cleavage of the linker peptide by an intracellular protease (e.g., an endosomal protease) or by dissociation of the functional effector protein from the supercharged protein within a specific intracellular microenvironment (e.g., within an endosome). In some embodiments, the functional effector protein to be delivered by a system or method provided by the present disclosure has therapeutic activity.

[0097] In some embodiments, functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.) are delivered to cells in vivo, ex vivo, or in vitro by the systems, compositions, or methods provided herein. In some embodiments, the functional effector protein is a protein capable of performing a biological function within a target cell, e.g., an enzyme capable of binding to its substrate and catalyzing an enzymatic reaction within the target cell, e.g., a nuclease capable of binding to and cleaving a nucleic acid molecule within the target cell, or a transcription factor capable of interacting with the genome of the target cell to activate or inhibit transcription of a target gene within the cell. In some embodiments, methods for generating a fusion of a functional effector protein and a supercharged protein include generating an expression nucleic acid construct containing in-frame coding sequences for the functional protein and the supercharged protein, and optionally a peptide linker, expressing such recombinant fusion protein in prokaryotic or eukaryotic cells in culture, and extracting and purifying the fusion protein. In some embodiments, the nucleic acid construct is generated in the form of an expression vector (e.g., a vector suitable for propagation in a bacterial host and expression in prokaryotic or eukaryotic cells).

[0098] In some embodiments, vectors suitable for fusion protein expression are generated by cloning a nucleotide sequence encoding a functional effector protein to be delivered into a cloning vector comprising a nucleotide sequence encoding a supercharged protein under the control of a eukaryotic and / or prokaryotic promoter by a cloning procedure that results in both coding sequences being in-frame with each other. In some embodiments, the cloning vector comprises a nucleotide sequence encoding a peptide linker between the nucleotide sequence encoding the supercharged protein and a restriction site useful for inserting the linker and the nucleotide sequence encoding a protein in-frame with the supercharged protein. In some embodiments, the cloning vector further comprises additional sequences that enhance expression of the fusion protein in prokaryotic or eukaryotic cells or facilitate purification of the expressed fusion protein from such cells (e.g., a sequence that stabilizes the transcript encoding the fusion protein, such as a polyA signal, a spliceable intron, a sequence encoding an in-frame peptide or protein domain tag (e.g., an Arg tag, calmodulin-binding peptide tag, cellulose-binding domain tag, DsbA tag, c-myc tag, glutathione S-transferase tag, FLAG tag, HAT tag, His tag, maltose-binding protein tag, NusA tag, S tag, SBP tag, Strep tag, or thioredoxin tag), or a selectable marker or reporter cassette that allows identification of cells harboring and expressing the expression construct and / or quantitating the level of expression in such cells). Methods for cloning and expressing fusion proteins are well known to those of skill in the art. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Volume 1-3, CSHL Press (1989), Gellissen et al., Production of recombinant proteins, Wiley-VCH, 2005. In some embodiments, the functional effector protein is coupled to supercharged GFP (e.g., +36 GFP or -30 GFP) for delivery to target cells. The benefits of endosome disruption in the delivery of macromolecules by supercharged proteins have previously been demonstrated (Wadia et al., Nat. Med. 10, 310-315, 2004), and in some embodiments, additional steps to achieve improved endosomal escape are performed as provided herein or known in the art. Highly efficient protein internalization, when coupled with effective endosomal release, has the potential to minimize the required dose of exogenous protein drugs, improving their potential as research tools and leads for therapeutic drug development.

[0099] In some embodiments, compositions containing functional effector proteins linked to supercharged proteins are administered to target cells after isolation and / or purification. Protein isolation methods and techniques are well known to those skilled in the art and include, for example, affinity chromatography or immunoprecipitation. The preferred method for isolating and / or purifying a particular functional effector protein, supercharged protein, and / or fusion protein will depend on the properties of the respective protein. For example, His-tagged fusion proteins can be readily isolated and purified by Ni or Co ion chromatography, while fusion proteins tagged with other peptides or domains or untagged fusion proteins can be purified by other well-established methods. Suitable functional effector proteins for delivery to target cells in vivo, ex vivo, or in vitro using the systems or methods provided herein will be apparent to those of skill in the art and include, for example, DNA-binding proteins (e.g., transcription factors and nucleases), as well as Cas9 proteins (including variants and fusions thereof). In some embodiments, the methods, compositions, or systems provided herein are used to deliver therapeutic functional effector proteins to cells. Examples of therapeutic proteins include, but are not limited to, nucleases and Cas9 proteins (including variants and fusions thereof) that target genomic alleles associated with diseases or disorders, and transcription factors that activate beneficial genes or repress pathogenic genes.

[0100] In some embodiments, Cas9 is fused to a supercharged protein for delivery to cells. In some embodiments, the supercharged protein is positively charged. In some embodiments, the supercharged protein fused to Cas9 is (+36)GFP. In some embodiments, the fusion of Cas9 and (+36)GFP comprises the amino acid sequence of SEQ ID NO:30 (e.g., with or without a nuclear localization signal (NLS) and with or without a 6xHis tag) or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:30 (e.g., with or without a nuclear localization signal (NLS) and with or without a 6xHis tag). In some embodiments, the supercharged protein fused to Cas9 is (-30)GFP. In some embodiments, the fusion of Cas9 and (-30)GFP comprises the amino acid sequence of SEQ ID NO: 31 (e.g., with or without a nuclear localization signal (NLS) and with or without a 6xHis tag), or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 31 (e.g., with or without a nuclear localization signal (NLS) and with or without a 6xHis tag).

[0101]

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[0102]

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[0103] Functional effector protein and cationic lipid compositions Certain aspects of the present disclosure relate to the use of cationic lipids for delivery of effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.), as opposed to, for example, delivering "naked" protein preparations. Surprisingly, it has been discovered that existing liposomal delivery reagents engineered for delivery of nucleic acids such as DNA and RNA effectively deliver certain effector proteins (e.g., Cas9 proteins, including variants and fusions thereof) both in vitro and in vivo, as described herein. Nucleic acid delivery has benefited greatly from the development of liposomal reagents over the past two decades. Cationic liposomal formulations have enabled DNA and RNA transfection to become routine techniques in basic research and are even being used in clinical trials. The lipid bilayer of the liposome may protect the encapsulated nucleic acid from degradation and prevent specific neutralization by antibodies that may bind to naked preparations of nucleic acid. Importantly, liposome fusion with the endosomal membrane during endosome maturation can enable highly efficient endosomal escape of cargo delivered by cationic lipids. Other non-cationic but reversibly ionizable lipid nanoparticle formulations have enabled efficient encapsulation and delivery of nucleic acids while avoiding nonspecific electrostatic interactions and consequent sequestration. However, proteins are chemically diverse, and therefore, unlike highly anionic nucleic acids, liposome formulations have not been as successful in efficiently delivering proteins. For example, while proteins can be nonspecifically encapsulated and delivered by rehydrated lipids in vitro, encapsulation efficacy is dependent on protein concentration and is generally inefficient, thus preventing widespread application. Aspects of the present disclosure relate to the recognition that anionic proteins or protein complexes (including proteins associated with nucleic acids) may be capable of utilizing the same electrostatically driven encapsulation used by cationic liposome reagents for nucleic acid delivery.While few proteins naturally possess the negative charge density found in the phosphate backbone of nucleic acids, translational fusion or noncovalent association with anionic carriers such as the negatively supercharged proteins or nucleic acids described herein renders the resulting effector protein or protein complex sufficiently anionic to drive efficient encapsulation of such protein cargo by cationic liposome reagents.

[0104] In some embodiments, conjugation or fusion with engineered, supernegatively charged GFP can drive efficient encapsulation and delivery of proteins into cultured mammalian cells using cationic lipids commonly used to transfect nucleic acids. This approach is effective even at low nanomolar protein concentrations and in the presence of serum, resulting in functional protein delivery that is up to 1,000-fold more efficient than protein delivery methods using fusions to cationic peptides or proteins. As shown in the examples, delivery efficacy in some embodiments depends, for example, on the theoretical net charge of the fusion tag, and popular naturally anionic peptide tags (e.g., 3xFLAG and VP64) can similarly enable liposomal protein delivery.

[0105] The examples further demonstrate that Cas9 nuclease proteins associated with polyanionic guide RNAs (gRNAs) can be efficiently delivered in a functional form into mammalian cells by their common cationic liposome formulations. Without wishing to be bound by any particular theory, it is believed that the gRNA acts as a polyanionic intermediary between the otherwise cationic Cas9 protein and the cationic lipids. Delivery of Cas9:gRNA complexes is not only highly efficient (e.g., up to 80% modification from a single treatment), but also results in significantly higher genome modification specificity compared to plasmid transfection, typically resulting in a >10-fold higher on-target:off-target modification ratio. This is likely due to the transient nature of the delivered Cas9:gRNA activity. In some embodiments, delivery of the Cas9:gRNA complex results in an on-target:off-target modification ratio that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, or 25-fold or more. The examples also demonstrate that this protein delivery approach can be effective in vivo, for example, by delivering a functional Cre recombinase and a functional Cas9:gRNA complex to hair cells of the inner ear of a mouse.

[0106] Accordingly, some aspects of the present invention provide compositions comprising a Cas9 protein (e.g., as described herein; see, e.g., Cas9 effector proteins, below) and a cationic lipid capable of delivering the Cas9 protein to the interior of a cell. In some embodiments, the Cas9 protein is associated with a gRNA, which, for example, provides an anionic charge to the complex, thereby allowing the Cas9:gRNA complex to be encapsulated by the cationic lipid. In some embodiments, the Cas9 protein need not be associated with a gRNA for effective encapsulation by the cationic lipid, but instead is associated with a negatively supercharged protein, as described herein. In some embodiments in which the Cas9 protein is associated with a negatively supercharged protein, the Cas9 protein is also associated with a gRNA. In some embodiments, the Cas9 protein is a wild-type Cas9 protein, a fragment of a wild-type Cas9 protein, or a variant of a wild-type Cas9 protein. In some embodiments, the Cas9 protein comprises a dCas9 domain (e.g., as described herein). In some embodiments, the Cas9 protein is a fusion protein comprising a dCas9 domain (e.g., as described herein). In some embodiments, the Cas9 protein is a Cas9 nickase.

[0107] In other embodiments, compositions are provided that include an effector protein (e.g., other than a Cas9 protein) and a cationic lipid, which can deliver the effector protein to the interior of a cell (e.g., to the nucleus of a cell). The effector protein is either naturally negatively charged, modified to have a net negative overall charge, or associated with a negative supercharged protein described herein. In some embodiments, the effector protein is any of the effector proteins described herein. In some embodiments, the effector protein is a recombinase, e.g., any of the recombinases described herein. In some embodiments, the recombinase is Cre recombinase. In some embodiments, the Cre recombinase comprises the amino acid sequence of SEQ ID NO: 32 (e.g., with or without a 6xHis tag). In some embodiments, the Cre recombinase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 32 (e.g., with or without a 6xHis tag). In some embodiments, the Cre recombinase is fused to a supercharged protein (e.g., +36GFP or -30GFP). In some embodiments, the Cre recombinase fused to the supercharged protein comprises the amino acid sequence of SEQ ID NO:33 (e.g., with or without a 6xHis tag) or SEQ ID NO:34 (e.g., with or without a 6xHis tag), or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:34 (e.g., with or without a 6xHis tag). In some embodiments, the effector protein is a TALE protein (e.g., as described herein, including those provided in the examples). In some embodiments, the TALE protein comprises one or more VP64 transcription activator domains (e.g., SEQ ID NO:35).In some embodiments, the TALE protein having a VP64 transcription activator domain further comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-39 (e.g., with or without a 6xHis tag). In some embodiments, the TALE protein having a VP64 transcription activator domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-39 (e.g., with or without a 6xHis tag). In some embodiments, the TALE effector protein comprises a (-30) GFP domain (e.g., SEQ ID NO: 21 or SEQ ID NO: 40), an N-terminal region of a TALE domain (e.g., SEQ ID NO: 41), a variable repeat domain (e.g., the 18.5mer repeat domain provided in Maeder et al., "Robust, synergistic regulation of human gene expression using TALE activators." Nat. Methods. 2013; 10, 243-245), a C-terminal TALE domain (e.g., SEQ ID NO: 42), a VP64 activation domain (e.g., SEQ ID NO: 35), and optionally one or more linkers (e.g., GGS(9), SEQ ID NO: 252) between either domain and optionally a sequence tag (e.g., 6xHis, SEQ ID NO: 253).

[0108] Liposomal delivery of cargoes such as DNA and RNA is known to induce toxicity in targeted cells. In contrast, the compositions of the invention described herein have surprisingly been found to deliver these cargoes both in vitro and in vivo with no or low toxicity. For example, in some embodiments, compositions comprising the Cas9 protein or other effector proteins described herein exhibit low toxicity when administered to a population of cells (e.g., in vitro or in vivo). In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the population are viable after administration of a composition of the invention comprising the Cas9 protein or other effector protein and a cationic lipid. Methods for assessing the toxicity of a composition when administered to a population of cells are well known in the art and include those described in the Examples.

[0109] Cre-6×His (6×His tag is underlined): MASNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLL RIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGDGGS HHHHHH (SEQ ID NO: 32)

[0110]

number

[0111]

number

[0112]

number

[0113]

number

[0114]

number

[0115]

number

[0116] (-30)GFP: MGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKFICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAEFEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEFVTAAGIDHGMDELYK (SEQ ID NO: 40)

[0117] N-terminal TALE domain: APKKKRKVGIHRGVPMVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNL (SEQ ID NO: 41)

[0118] C-terminal TALE domain: LESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSGGGS (SEQ ID NO: 42)

[0119] VP64 activation domain: GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID NO: 35)

[0120] Functional effector protein and cationic polymer compositions Certain aspects of the present disclosure relate to the use of cationic polymers for delivery of effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.), as opposed to, for example, delivering "naked" protein preparations. Similar to cationic lipids, aspects of the present disclosure relate to the recognition that anionic proteins or protein complexes (including proteins associated with nucleic acids) can utilize electrostatically driven encapsulation and / or association with cationic polymers for delivery of functional effector proteins. While few proteins naturally possess the negative charge density found in the phosphate backbone of nucleic acids, translational fusion or non-covalent association with anionic carriers, such as the negatively supercharged proteins or nucleic acids described herein, renders the resulting effector protein or protein complex sufficiently anionic to drive efficient encapsulation / association of such protein cargo by cationic polymers.

[0121] In some embodiments, conjugation or fusion with engineered, supernegatively charged GFP can drive efficient encapsulation / conjugation and delivery of the protein into cultured mammalian cells by cationic polymers. In some embodiments, Cas9 proteins conjugated to polyanionic guide RNAs (gRNAs) can be efficiently delivered in a functional form into mammalian cells using cationic polymers. Thus, in some embodiments, compositions comprising a Cas9 protein and a cationic polymer are provided, wherein the Cas9 protein is conjugated to a gRNA and the composition can deliver the Cas9 protein to the interior of cells. In some embodiments, delivery of the Cas9:gRNA complex using a cationic polymer results in at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 20-fold, or 25-fold or more higher on-target:off-target modification ratios compared to plasmid transfection of the Cas9 protein.

[0122] Accordingly, some aspects of the present disclosure provide compositions comprising a Cas9 protein (e.g., as described herein; see, e.g., Cas9 effector proteins, below) and a cationic polymer capable of delivering the Cas9 protein to the interior of a cell. In some embodiments, the Cas9 protein is associated with a gRNA, which, for example, provides an anionic charge to the complex, thereby allowing the Cas9:gRNA complex to be encapsulated and / or associated with the cationic polymer. In some embodiments, the Cas9 protein does not need to be associated with a gRNA for effective encapsulation and / or association with the cationic lipid, but instead is associated with a negatively supercharged protein described herein. In some embodiments in which the Cas9 protein is associated with a negatively supercharged protein, the Cas9 protein is also associated with a gRNA. In some embodiments, the Cas9 protein is a wild-type Cas9 protein, a fragment of a wild-type Cas9 protein, or a variant of a wild-type Cas9 protein. In some embodiments, the Cas9 protein comprises a dCas9 domain (e.g., as described herein). In some embodiments, the Cas9 protein is a fusion protein comprising a dCas9 domain (e.g., as described herein). In some embodiments, the Cas9 protein is a Cas9 nickase.

[0123] In other embodiments, compositions are provided that include an effector protein (e.g., other than a Cas9 protein) and a cationic polymer, which can deliver the effector protein to the interior of a cell (e.g., to the nucleus of a cell). The effector protein is either naturally negatively charged, modified to have a net negative overall charge, or associated with a negative supercharged protein described herein. In some embodiments, the effector protein is any effector protein described herein. In some embodiments, the effector protein is a recombinase (e.g., any recombinase described herein). In some embodiments, the recombinase is Cre recombinase. In some embodiments, the Cre recombinase comprises the amino acid sequence of SEQ ID NO: 32 (e.g., with or without a 6xHis tag). In some embodiments, the Cre recombinase comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 32 (e.g., with or without a 6xHis tag). In some embodiments, the Cre recombinase is fused to a supercharged protein (e.g., +36GFP or -30GFP). In some embodiments, the Cre recombinase fused to the supercharged protein comprises the amino acid sequence of SEQ ID NO:33 (e.g., with or without a 6xHis tag) or SEQ ID NO:34 (e.g., with or without a 6xHis tag), or comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:34 (e.g., with or without a 6xHis tag). In some embodiments, the effector protein is a TALE protein (e.g., as described herein, including those provided in the examples). In some embodiments, the TALE protein comprises one or more of the VP64 transcription activator domains (e.g., SEQ ID NO:35).In some embodiments, the TALE protein having a VP64 transcription activator domain further comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-39 (e.g., with or without a 6xHis tag). In some embodiments, the TALE protein having a VP64 transcription activator domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-39 (e.g., with or without a 6xHis tag). In some embodiments, the TALE effector protein comprises a (-30) GFP domain (e.g., SEQ ID NO: 21 or SEQ ID NO: 40), an N-terminal region of a TALE domain (e.g., SEQ ID NO: 41), a variable repeat domain (e.g., the 18.5mer repeat domain provided in Maeder et al., "Robust, synergistic regulation of human gene expression using TALE activators." Nat. Methods. 2013; 10, 243-245), a C-terminal TALE domain (e.g., SEQ ID NO: 42), a VP64 activation domain (e.g., SEQ ID NO: 35), and optionally one or more linkers (e.g., GGS(9); SEQ ID NO: 252) between either domain and optionally a sequence tag (e.g., 6xHis; SEQ ID NO: 253).

[0124] In some embodiments, compositions comprising a Cas9 protein or other effector protein and a cationic polymer described herein exhibit low toxicity when administered to a population of cells (e.g., in vitro or in vivo). In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the population are viable after administration of a composition of the invention comprising a Cas9 protein or other effector protein and a cationic polymer. Methods for assessing the toxicity of a composition when administered to a population of cells are well known in the art and include those described in the Examples.

[0125] Cas9 effector proteins In some embodiments, an effector protein comprising an RNA-programmable protein (or a fragment or variant thereof) is delivered to a target cell by a system or method provided herein. In some embodiments, an RNA-guided or RNA-programmable nuclease is delivered to a target cell by a system or method provided herein. In some embodiments, the RNA-programmable protein is a Cas9 nuclease, a Cas9 variant, or a fusion of a Cas9 protein, which is delivered to a target cell by a system or method provided herein.

[0126] In this case, the RNA-induced catalytic conjugation was performed (CR ISPR solvent) Cas9 strain strain Streptococcus Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Complete genome sequence of an M1 strain of Streptococcus pyogenes. Suvorov AN, Kenton S, Lai HS, Qian Y, Jia HG, Najar FZ, Ren Q, Zhu L. expand / collapse author list McLaughlin RE, Proc Natl small RNA and host factor RNase III.Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E, Nature 471:602-607(2011). endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012) and the specifics of this study are described in detail.Because RNA-programmable nucleases (e.g., Cas9) use RNA:DNA hybridization to determine target DNA cleavage sites, these proteins are, in principle, capable of cleaving any sequence specified by the guide RNA. Methods using RNA-programmable nucleases such as Cas9 for site-specific cleavage (e.g., to modify a genome) are known in the art (e.g., Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013), Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013), the entire contents of each of which are incorporated herein by reference.

[0127] Cas9 nuclease is sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides protection from mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain spacers (sequences complementary to the preceding mobile element) and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR-RNA (crRNA). In type II CRISPR systems, correct processing of pre-crRNA requires tracrRNA (trans-encoded small RNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA acts as a guide for ribonuclease 3-assisted processing of pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleated and then exonucleolytically trimmed from 3' to 5'. In nature, DNA binding and cleavage typically require a protein and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gRNAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence, helping it distinguish self from non-self.The complete genome sequence of an M1 strain of Streptococcus is readily available from Cas9 pyogenes.」Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Suvorov AN, Kenton S, Lai HS, Lin SP, Qian Y, Jia HG, Najar FZ, Ren Q, Zhu H, Song L. expand / collapse author list McLaughlin RE. Proc. Natl E., Nature 471:602-607(2011)、および「A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.」Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821(2012) and the specifics of this study are presented in a suitable manner.

[0128] Cas9 orthologs have been described in various species, including, but not limited to, S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on the present disclosure. Such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, proteins comprising a Cas9 protein or a fragment thereof are referred to as "Cas9 variants." Cas9 variants share homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain, an N-terminal domain or a C-terminal domain, etc.), such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, SEQ ID NO:43 (nucleotide), SEQ ID NO:44 (amino acid)). In some embodiments, the Cas9 protein has an inactive (e.g., inactivated) DNA cleavage domain. A nuclease-inactive Cas9 protein may be interchangeably referred to as a "dCas9" protein (as a nuclease "dead" Cas9).In some embodiments, dCas9 corresponds, in part or in whole, to or comprises the amino acid sequence set forth below as SEQ ID NO:45. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO:45) are provided. For example, in some embodiments, variants having mutations other than D10A and H840A are provided, which result in nuclease-inactive Cas9 (dCas9). Such mutations include, by way of example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions within the HNH nuclease subdomain and / or RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 (e.g., variants of SEQ ID NO:45) are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO:45. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 45) are provided, having an amino acid sequence that is shorter or longer than SEQ ID NO: 45 by about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or 101 amino acids or more. In some embodiments, Cas9 "nickases" are provided, which contain a mutation that inactivates a single nuclease domain in Cas9. Such nickases induce a single-stranded break in the target nucleic acid, as opposed to a double-stranded break.

[0129] Cas9

number

[0130]

number

[0131] Cas9 Nickase (D10A):

[0132] In some embodiments, a fusion protein comprising a Cas9 protein is provided for use in any of the compositions and methods described herein. In some embodiments, the fusion protein comprises a dCas9 protein (e.g., as described herein). In some embodiments, the fusion protein comprises a linker (e.g., as described herein) between dCas9 and one or more domains (e.g., enzyme domains). In some embodiments, the fusion protein comprises dCas9 and a transcriptional activator domain, a transcriptional repressor domain, a recombinase domain, a gene editing domain (e.g., a deaminase domain), or an epigenetic modifier domain.

[0133] In some embodiments, the general structure of exemplary fusion proteins provided herein is [NH2]-[enzyme domain]-[dCas9]-[COOH], or [NH2]-[dCas9]-[enzyme domain]-[COOH] wherein NH2 is the N-terminus of the fusion protein and COOH is the C-terminus of the fusion protein, and the enzymatic domain comprises a nuclease domain (e.g., FokI), a recombinase catalytic domain (e.g., Hin, Gin, or Tn3 recombinase domain), a nucleic acid editing domain (e.g., a deaminase domain), a transcriptional activator domain (e.g., VP64, p65), a transcriptional repressor domain (e.g., KRAB, SID), or an epigenetic modifier (e.g., LSD1 histone demethylase, TET1 hydroxylase).

[0134] Additional features may be present, such as one or more linker sequences between certain domains. Other exemplary features that may be present are localization sequences, such as a nuclear localization sequence (NLS, e.g., MAPKKKRKVGIHRGVP (SEQ ID NO: 47)), a cytoplasmic localization sequence, an export sequence, e.g., a nuclear export sequence, or other localization sequence, as well as sequence tags that are useful for solubilizing, purifying, or detecting the fusion protein. Suitable localization signal sequences and protein tag sequences are provided herein and are known in the art, including, but not limited to, biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags (e.g., 3xFLAG tags, MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 48)), hemagglutinin (HA) tags, polyhistidine tags (also referred to as histidine tags or His tags), maltose binding protein (MBP) tags, nus tags, glutathione-S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag1, Softag3), strep tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP tags. Additional suitable sequences will be apparent to those skilled in the art.

[0135] In some embodiments, the enzymatic domain comprises a nuclease or its catalytic domain. For example, in some embodiments, the general structure of an exemplary ligand-dependent dCas9 fusion protein having a nuclease domain is [NH2]-[NLS]-[dCas9]-[nuclease]-[COOH], [NH2]-[NLS]-[nuclease]-[dCas9]-[COOH], [NH2]-[dCas9]-[nuclease]-[COOH], or [NH2]-[nuclease]-[dCas9]-[COOH] wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, a linker is inserted between the dCas9 and the nuclease domain. In some embodiments, a linker is inserted between the NLS and the nuclease and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the nuclease and / or dCas9 domain. In some embodiments, the NLS is located between the nuclease and the dCas9 domain. Additional features, such as a sequence tag, may also be present. In some aspects, the nuclease domain is a nuclease that requires dimerization (e.g., the joining of two nuclease monomers) to cleave a target nucleic acid (e.g., DNA). In some embodiments, the nuclease domain is a monomer of the DNA cleavage domain of FokI. The DNA cleavage domain of FokI is known and, in some aspects, corresponds to amino acids 388-583 of FokI (NCBI accession number J04623). In some embodiments, the DNA cleavage domain of FokI corresponds to amino acids 300 to 583, 320 to 583, 340 to 583, or 360 to 583 of FokI. See also Wah et al., "Structure of FokI has implications for DNA cleavage," Proc. Natl. Acad. Sci. USA. 1998; 1 ;95(18):10564-9; Li et al., "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and the FokI DNA-cleavage domain," Nucleic Acids Res. 2011; 39(1):359-72; Kim et al., "Hybrid restriction enzymes: zinc finger fusions to the FokI cleavage domain," Proc. Natl Acad. Sci. USA. 1996; 93:1156-1160. the entire contents of each of which are incorporated herein by reference).In some embodiments, the DNA cleavage domain of FokI corresponds to, or comprises in part or in whole, the amino acid sequence set forth as SEQ ID NO: 49. In some embodiments, the DNA cleavage domain of FokI is a variant of FokI described herein (e.g., a variant of SEQ ID NO: 49). Other exemplary compositions and methods using dCas9-nuclease fusion proteins can be found in U.S. Patent Application No. USSN 14 / 320,498, filed June 30, 2014, entitled "Cas9-FokI Fusion Proteins and Uses Thereof," the entire contents of which are incorporated herein by reference.

[0136] FokI nuclease domain: GSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF (SEQ ID NO: 49)

[0137] fCas9 (e.g., dCas9-NLS-GGS3 linker-FokI): GTCAGACGGAAATTTAATAACGGCGAGATAAACTTT (SEQ ID NO: 50)

[0138] fCas9 (e.g., NLS-dCas9-GGS3 linker-FokI):

[0139] fCas9 (e.g., FokI-GGS3 linker-dCas9-NLS):

[0140] fCas9 (e.g., NLS-FokI-GGS3 linker-dCas9): TCCATCACGGGATTATATGAAACTCGGATAGATTTGTCACAGCTTGGGGGTGAC (SEQ ID NO: 53)

[0141] fCas9:

[0142] In some embodiments, the enzymatic domain comprises a recombinase or its catalytic domain. For example, in some embodiments, the general structure of an exemplary ligand-dependent dCas9 fusion protein having a recombinase domain is [NH2]-[NLS]-[dCas9]-[recombinase]-[COOH], [NH2]-[NLS]-[recombinase]-[dCas9]-[COOH], [NH2]-[dCas9]-[recombinase]-[COOH], or [NH2]-[recombinase]-[dCas9]-[COOH], wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, a linker is inserted between the dCas9 and the recombinase domain. In some embodiments, a linker is inserted between the NLS and the recombinase and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the recombinase domain and / or dCas9 domain. In some embodiments, the NLS is located between the recombinase domain and the dCas9 domain. Additional features, such as a sequence tag, can also be present. By "catalytic domain of a recombinase," it is meant that the fusion protein includes a domain that includes the amino acid sequence of (e.g., derived from) a recombinase, such that the domain is sufficient to induce recombination when contacted with a target nucleic acid (either alone or together with additional factors, including other recombinase catalytic domains, which may or may not form part of the fusion protein). In some embodiments, the catalytic domain of a recombinase does not include the DNA-binding domain of the recombinase. In some embodiments, the catalytic domain of a recombinase comprises part or all of a recombinase. For example, the catalytic domain can comprise a recombinase domain and a DNA-binding domain or a portion thereof, or the catalytic domain can comprise a recombinase domain and a DNA-binding domain that has been mutated or truncated to abolish DNA-binding activity. Recombinases and catalytic domains of recombinases are known to those skilled in the art and include, for example, those described herein. In some embodiments, the catalytic domain is derived from any recombinase. In some embodiments, the recombinase catalytic domain is the catalytic domain of a Tn3 resolvase, a Hin recombinase, or a Gin recombinase. In some embodiments, the catalytic domain comprises a Tn3 resolvase (e.g., Stark Tn3 recombinase) encoded by a nucleotide sequence comprising, in part or in whole, SEQ ID NO: 55, as provided below. In some embodiments, the Tn3 catalytic domain is encoded by a variant of SEQ ID NO: 55.In some embodiments, the Tn3 catalytic domain is encoded by a polynucleotide (or a variant thereof) encoding a polypeptide corresponding to SEQ ID NO: 56. In some embodiments, the catalytic domain comprises a Hin recombinase encoded by a nucleotide sequence comprising, in part or in whole, SEQ ID NO: 57, as provided below. In some embodiments, the Hin catalytic domain is encoded by a variant of SEQ ID NO: 57. In some embodiments, the Hin catalytic domain is encoded by a polynucleotide (or a variant thereof) encoding a polypeptide corresponding to SEQ ID NO: 58. In some embodiments, the catalytic domain comprises a Gin recombinase (e.g., Gin beta recombinase) encoded by a nucleotide sequence comprising, in part or in whole, SEQ ID NO: 59, as provided below. In some embodiments, the Gin catalytic domain is encoded by a variant of SEQ ID NO: 59. In some embodiments, the Gin catalytic domain is encoded by a polynucleotide (or a variant thereof) encoding a polypeptide corresponding to SEQ ID NO: 60. Other exemplary compositions and methods of using dCas9-recombinase fusion proteins can be found in U.S. patent application Ser. No. 14 / 320,467, entitled "Cas9 Variants and Uses Thereof," filed June 30, 2014, the entire contents of which are incorporated herein by reference.

[0143] Stark's Tn3 recombinase (nucleotide: SEQ ID NO: 55, amino acid: SEQ ID NO: 56): ATGGCCCTGTTTGGCTACGCACGCGTGTCTACCAGTCAACAGTCACTCGATTTGCAAGTGAGGGCTCTTAAAGATGCCGGAGTGAAGGCAAACAGAATTTTTACTGATAAGGCCAGCGGAAGCAGCACAGACAGAGAGGGGCTGGATCTCCTGAGAATGAAGGTAAAGGAGGGTGATGTGATCTTGGTCAAAAAATTGGATCGACTGGGGAGAGACACAG CTGATATGCTTCAGCTTATTAAAGAGTTTGACGCTCAGGGTGTTGCCGTGAGGTTTATCGATGACGGCATCTCAACCGACTCCTACATTGGTCTTATGTTTGTGACAATTTTGTCCGCTGTGGCTCAGGCTGAGCGGAGAAGGATTCTCGAAAGGACGAATGAGGGACGGCAAGCAGCTAAGTTGAAAGGTATCAAATTTGGCAGACGAAGG (SEQ ID NO: 55) MALFGYARVSTSQQSLDLQVRALKDAGVKANRIFTDKASGSSTDREGLDLLRMKVKEGDVILVKKLDRLGRDTADMLQLIKEFDAQGVAVRFIDDGISTDSYIGLMFVTILSAVAQAERRRILERTNEGRQAAKLKGIKFGRRR (SEQ ID NO: 56)

[0144] Hin recombinase (nucleotide: SEQ ID NO: 57, amino acid: SEQ ID NO: 58): ATGGCAACCATTGGCTACATAAGGGTGTCTACCATCGACCAAAATATCGACCTGCAGCGCAACGCTCTGACATCCGCCAACTGCGATCGGATCTTCGAGGATAGGATCAGTGGCAAGATCGCCAACCGGCCCGGTCTGAAGCGGGCTCTGAAGTACGTGAATAAGGGCGATACTCTGGTTGTTGGAAGTTGGATCGCTTGGGTAGATCAG TGAAGAATCTCGTAGCCCTGATAAGCGAGCTGCACGAGAGGGGTGCACATTTCCATTCTCTGACCGATTCCATCGATACGTCTAGCGCCATGGGCCGATTCTTCTTTTACGTCATGTCCGCCCTCGCTGAAATGGAGCGCGAACTTATTGTTGAACGGACTTTGGCTGGACTGGCAGCGGCTAGAGCACAGGGCCGACTTGGA (SEQ ID NO: 57) MATIGYIRVSTIDQNIDLQRNALTSANCDRIFEDRISGKIANRPGLKRALKYVNKGDTLVVWKLDRLGRSVKNLVALISELHERGAHFHSLTDSIDTSSAMGRFFFYVMSALAEMERELIVERTLAGLAAARAQGRLG (SEQ ID NO: 58)

[0145] Gin beta recombinase (nucleotide: SEQ ID NO: 59, amino acid: SEQ ID NO: 60): ATGCTCATTGGCTATGTAAGGGTCAGCACCAATGACCAAAACACAGACTTGCAACGCAATGCTTTGGTTTGCGCCGGATGTGAACAGATATTTGAAGATAAACTGAGCGGCACTCGGACAGACAGACCTGGGCTTAAGAGAGCACTGAAAAGACTGCAGAAGGGGGACACCCTGGTCGTCTGGAAACTGGATCGCCTCGGACGCAGCATGAAACATCTGA TTAGCCTGGTTGGTGAGCTTAGGGAGAGGAATCAACTTCAGAAGCCTGACCGACTCCATCGACACCAGTAGCCCCATGGGACGATTCTTCTTCTATGTGATGGAGCACTTGCTGAGATGGAAAGAGAGCTTATTATCGAAAGAACTATGGCTGGTATCGCTGCTGCCCGGAACAAAGGCAGACGGTTCGGCAGACCGCCGAAGAGCGGC (SEQ ID NO: 59) MLIGYVRVSTNDQNTDLQRNALVCAGCEQIFEDKLSGTRTDRPGLKRALKRLQKGDTLVVWKLDRLGRSMKHLISLVGELRERGINFRSLTDSIDTSSPMGRFFFYVMGALAEMERELIIERTMAGIAAARNKGRRFGRPPKSG (SEQ ID NO: 60)

[0146] In some embodiments, the enzymatic domain comprises a deaminase or its catalytic domain. For example, in some embodiments, the general structure of an exemplary dCas9 fusion protein having a deaminase enzyme or domain is [NH2]-[NLS]-[Cas9]-[deaminase]-[COOH], [NH2]-[NLS]-[deaminase]-[Cas9]-[COOH], [NH2]-[Cas9]-[deaminase]-[COOH], or [NH2]-[deaminase]-[Cas9]-[COOH] wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, a linker is inserted between the dCas9 and the deaminase domain. In some embodiments, a linker is inserted between the NLS and the deaminase and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the deaminase and / or dCas9 domain. In some embodiments, the NLS is located between the deaminase domain and the dCas9 domain. Additional features, such as a sequence tag, may also be present. One exemplary suitable type of nucleic acid editing enzyme and domain is, for example, a cytosine deaminase from the apolipoprotein B mRNA editing complex (APOBEC) family of cytosine deaminase enzymes, including activation-induced cytidine deaminase (AID) and apolipoprotein B editing complex 3 (APOBEC3) enzymes. Another exemplary suitable type of nucleic acid editing enzyme and domain thereof suitable for use in the present invention includes adenosine deaminase. For example, an ADAT-family adenosine deaminase can be fused to a dCas9 domain. Some exemplary suitable nucleic acid editing enzymes and domains (e.g., deaminases and deaminase domains) that can be fused to a dCas9 domain in accordance with aspects of the present disclosure are provided below. It will be understood that in some embodiments, the active domain of each sequence can be used, for example, a domain without a localization signal (nuclear localization signal, no nuclear export signal, cytoplasmic localization signal). Other exemplary compositions and methods using dCas9-nuclease fusion proteins can be found in U.S. Patent Application No. USSN 14 / 325,815, filed July 8, 2014, entitled "Fusions of Cas9 Domains and Nucleic Acid-Editing Domains," the entire contents of which are incorporated herein by reference.

[0147]

number

[0148]

number

[0149]

number

number

[0150] Mouse APOBEC-3: MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLGYAKGRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQIVRFLATHHNLSLDIFSSRLYNVQDPETQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKRLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVEGRRMDPLSEEEFYSQFYNQRVKHLCYYHRMKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSQVTITCYLTWSPCPNC AWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLRRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 65) (underlined: nucleic acid editing domain) Rat APOBEC-3: MGPFCLGCSHRKCYSPIRNLISQETFKFHFKNLRYAIDRKDTFLCYEVTRKDCDSPVSLHHGVFKNKDNI HAEICFLYWFHDKVLKVLSPREEFKITWYMSWSPCFEC AEQVLRFLATHHNLSLDIFSSRLYNIRDPENQQNLCRLVQEGAQVAAMDLYEFKKCWKKFVDNGGRRFRPWKKLLTNFRYQDSKLQEILRPCYIPVPSSSSSTLSNICLTKGLPETRFCVERRRVHLLSEEEFYSQFYNQRVKHLCYYHGVKPYLCYQLEQFNGQAPLKGCLLSEKGKQ HAEILFLDKIRSMELSQVIITCYLTWSPCPNCAWQLAAFKRDRPDLILHIYTSRLYFHWKRPFQKGLCSLWQSGILVDVMDLPQFTDCWTNFVNPKRPFWPWKGLEIISRRTQRRLHRIKESWGLQDLVNDFGNLQLGPPMS (SEQ ID NO: 66) (underlined: nucleic acid editing domain)

[0151]

number

[0152]

number

number

[0153]

number

[0154] Human APOBEC-3B: MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGQVYFKPQY HAEMCFLSWFCGNQLPAYKCFQITWFVSWTPCPDCVAKLAEFLSEHPNVTLTISAARLYYYWERDYRRALCRLSQAGARVTIMDYEEFAYCWENFVYNEGQQFMPWYKFDENYAFLHRTLKEILRYLMDPDTFTFNFNNDPLVLRRRQTYLCYEVERLDNGTWVLMDQHMGFLCNEAKNLLCGFY GRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGC AGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFEYCWDTFVYRQGCPFQPWDGLEEHSQALSGRLRAILQNQGN (SEQ ID NO: 72) (underlined: nucleic acid editing domain) Human APOBEC-3C: MNPQIRNPMKAMYPGTFYFQFKNLWEANDRNETWLCFTVEGIKRRSVVSWKTGVFRNQVDSETH CHAERCFLSWFCDDILSPNTKYQVTWYTSWSPCPDC AGEVAEFLARHSNVNLTIFTARLYYFQYPCYQEGLRSLSQEGVAVEIMDYEDFKYCWENFVYNDNEPFKPWKGLKTNFRLLKRRLRESLQ (SEQ ID NO: 73) (underlined: nucleic acid editing domain)

[0155] Human APOBEC-3A: MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGR HAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGC AGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (SEQ ID NO: 74) (underlined: nucleic acid editing domain) Human APOBEC-3H: MALLTAETFRLQFNNKRRLRRPYYPRKALLCYQLTPQNGSTPTRGYFENKKKC HAEICFINEIKSMGLDETQCYQVTCYLTWSPCSSC AWELVDFIKAHDHLNLGIFASRLYYHWCKPQQKGLRLLCGSQVPVEVMGFPKFADCWENFVDHEKPLSFNPYKMLEELDKNSRAIKRRLERIKIPGVRAQGRYMDILCDAEV (SEQ ID NO: 75) (underlined: nucleic acid editing domain)

[0156] Human APOBEC-3D: MNPQIRNPMERMYRDTFYDNFENEPILYGRSYTWLCYEVKIKRGRSNLLWDTGVFRGPVLPKRQSNHRQEVYFRFEN HAEMCFLSWFCGNRLPANRRFQITWFVSWNPCLPC VVKVTKFLAEHPNVTLTISAARLYYYRDRDWRWVLLRLHKAGARVKIMDYEDFAYCWENFVCNEGQPFMPWYKFDDNYASLHRTLKEILRNPMEAMYPHIFYFHFKNLLKACGRNESWLCFTMEVTKHHSAVFRKRGVFRNQVDPETH HAERCFLSWFCDDILSPNTNYEVTWYTSWSPCPEC AGEVAEFLARHSNVNLTIFTARLCYFWDTDYQEGLCSLSQEGASVKIMGYKDFVSCWKNFVYSDDEPFKPWKGLQTNFRLLKRRLREILQ (SEQ ID NO: 76) (underlined: nucleic acid editing domain) Human APOBEC-1: MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR (SEQ ID NO: 77)

[0157] Mouse APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK (SEQ ID NO: 78) Rat APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 79)

[0158] Human ADAT-2: MEAKAAPKPAASGACSVSAEETEKWMEEAMHMAKEALENTEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCRQSGKSPSEVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVEMLKTFYKQENPNAPKSKVRKKECQKS (SEQ ID NO: 80) Mouse ADAT-2: MEEKVESTTTPDGPCVVSVQETEKWMEEAMRMAKEALENIEVPVGCLMVYNNEVVGKGRNEVNQTKNATRHAEMVAIDQVLDWCHQHGQSPSTVFEHTVLYVTVEPCIMCAAALRLMKIPLVVYGCQNERFGGCGSVLNIASADLPNTGRPFQCIPGYRAEEAVELLKTFYKQENPNAPKSKVRKKDCQKS (SEQ ID NO: 81)

[0159] Mouse ADAT-1: MWTADEIAQLCYAHYNVRLPKQGKPEPNREWTLLAAVVKIQASANQACDIPEKEVQVTKEVV SMGTGTKCIGQSKMRESGDILNDSHAEIIARRSFQRYLLHQLHLAAVLKEDSIFVPGTQRGLWRLRPDLSFVFFSSHTPCGDASIIPMLEFEEQPCCPVIRSWANNSPVQETENLEDSKDKRNCEDPASPVAKKMRLGTPARSLSNCVAHHGTQESGPVKPDVSSSDLTKEEPDAANGIASGSFRVVDVYRTGAKCVPGETGDLREPGAAYHQVGLLR VKPGRGDRTCSMSCSDKMARWNVLGCQGALLMHFLEKPIYLSAVVIGGKCPYSQEAMRRALTGRCEETLVLPRGGFVQELEIQQSGLLFEQSRCAVHRKRGDSPGRLVPCGAAISWSAVPQQPLDVTANGFPQGTTKKEIGSPRARSRISKVELFRSFQKLLSSIADDEQPDSIRVTKKLDTYQEYKDAASAYQEAWGALRRIQPFASWIRNPPDYHQF K (SEQ ID NO: 82) (underlined: nucleic acid editing domain) Human ADAT-1: MWTADEIAQLCYEHYGIRLPKKGKPEPNHEWTLLAAVVKIQSPADKACDTPDKPVQVTKEVV SMGTGTKCIGQSKMRKNGDILNDSHAEVIARRSFQRYLLHQLQLAATLKEDSIFVPGTQKGVWKLRRDLIFVFFSSHTPCGDASIIPMLEFEDQPCCPVFRNWANHSSVEASSNLEAPGNERCKCEDPDSPVTKKMRLEPGTAAREVTNGAAHHQSFGKQKSGPISPGIHSCDLTVEGLATVRIAPGSAKVIDVYRTGAKCVPGEAGDSGKPGAAFHQV GLLRVKPGRGDRTRSMSCSDKMARWNVLGCQGALLMHLLEEPIYLSAVVIGGKCPYSQEAMQRALIGRCQNVSALPKGFGVQELKILQSDLLFEQSRSAVQAKRADSPGRLVPCGAAISWSAVPEQPLDVTANGFPQGTTKKTIGSLQARSQISKVELFRSFQKLLSRIARDKWPHSLRVQKLDTYQEYKEAASSYQEAWSTLRKQVFGSWIRNPPDYHQF K (SEQ ID NO: 83) (Underlined: nucleic acid editing domain)

[0160] In some embodiments, the enzymatic domain comprises one or more transcriptional activators. For example, in some embodiments, the general structure of an exemplary dCas9 fusion protein having a transcriptional activator domain is [NH2]-[NLS]-[Cas9]-[(transcriptional activator) n ]-[COOH], [NH2]-[NLS]-[(transcriptional activator) n ]-[Cas9]--[COOH], [NH2]-[Cas9]-[(transcriptional activator) n ]-[COOH], or [NH2]-[(transcriptional activator) n ]-[Cas9]-[COOH] wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the fusion protein includes one or more repeats of a transcription activator, e.g., n=1-10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n=1-20. In some embodiments, a linker is inserted between the dCas9 and transcription activator domain. In some embodiments, a linker is inserted between the NLS and the transcription activator and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the transcription activator and / or dCas9 domain. In some embodiments, the NLS is located between the transcription activator domain and the dCas9 domain. Additional features, such as a sequence tag, may also be present. In some embodiments, the transcription activator is selected from the group consisting of VP64 (SEQ ID NO: 84 or SEQ ID NO: 35), VP16 (SEQ ID NO: 85), and p65 (SEQ ID NO: 86). In some embodiments, the dCas9-VP64 fusion protein comprises the amino acid sequence of SEQ ID NO: 87 (e.g., with or without a 6xHis tag) or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 87 (e.g., with or without a 6xHis tag).

[0161] VP64 GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN (SEQ ID NO: 84)

[0162] VP16 APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGEFPGIRR (SEQ ID NO: 85)

[0163] p65: PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTPGPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFSSIADMDFSALLSQISSSGQ (SEQ ID NO: 86)

[0164] dCas9-VP64-6×His:

[0165] In some embodiments, the enzymatic domain comprises one or more transcriptional repressors. For example, in some embodiments, the general structure of an exemplary dCas9 fusion protein having a transcriptional repressor domain is [NH2]-[NLS]-[Cas9]-[(transcriptional repressor) n ]-[COOH], [NH2]-[NLS]-[(transcriptional repressor) n ]-[Cas9]--[COOH], [NH2]-[Cas9]-[(transcriptional repressor) n ]-[COOH], or [NH2]-[(transcriptional repressor) n ]-[Cas9]-[COOH], wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the fusion protein comprises one or more repeats of a transcriptional repressor, e.g., n=1-10 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n=1-20. In some embodiments, a linker is inserted between the dCas9 and the transcriptional repressor domain. In some embodiments, a linker is inserted between the NLS and the transcriptional repressor and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the transcriptional repressor and / or dCas9 domain. In some embodiments, the NLS is located between the transcriptional repressor domain and the dCas9 domain. Additional features, such as sequence tags, may also be present. In some embodiments, the transcriptional repressor is selected from the group consisting of the KRAB (Kruppel-associated box) domain of Kox1, the SID (mSin3-interacting domain), the CS (chromoshadow) domain of HP1α, or the WRPW domain of Hes1. These and other repressor domains are known in the art and, in some embodiments, correspond to those described in Urrutia, KRAB-containing zinc-finger repressor proteins. Genome Biol. 2003;4(10):231; Gilbert et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154, 442-451; Konermann et al., Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500, 472-476; and published U.S. patent application USSN 14 / 105,017, published as US2014 / 0186958A1, the entire contents of which are incorporated herein by reference.In some embodiments, the transcriptional repressor domain comprises one or more repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats) of a KRAB domain. In some embodiments, the KRAB domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 88-91. In some embodiments, the transcriptional repressor domain comprises one or more repeats of a SID protein. In some embodiments, the SID protein comprises the amino acid sequence set forth as SEQ ID NO: 80. In some embodiments, the repressor domain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of a SID protein (e.g., SEQ ID NO: 92). In some embodiments, the repressor domain comprises four repeats of SID (e.g., SID4x, SEQ ID NO: 93).

[0166] KRAB (human. GenBank: AAD20972.1): MNMFKEAVTFKDVAVAFTEEELGLLGPAQRKLYRDVMVENFRNLLSVGHPPFKQDVSPIERNEQLWIMTTATRRQGNLDTLPVKALLLYDLAQT (SEQ ID NO: 88) Partial KRAB protein domain (human. GenBank: CAB52478.1): EQVSFKDVCVDFTQEEWYLLDPAQKILYRDVILENYSNLVSVGYCITKPEVIFKIEQGEEPWILEKGFPSQCHP (SEQ ID NO: 89)

[0167] Partial KRABA domain (human. GenBank: AAB03530.1): EAVTFKDVAVVFTEEELGLLDPAQRKLYRDVMLENFRNLLSV (SEQ ID NO: 90) KRAB (mouse. C2H2-type domain-containing protein. GenBank: CAM27971.1): MDLVTYDDVHVNFTQDEWALLDPSQKSLYKGVMLETYKNLTAIGYIWEEHTIEDHFQTSRSHGSNKKTH (SEQ ID NO: 91)

[0168] SID repressor domain: GSGMNIQMLLEAADYLERREREAEHGYASMLP (SEQ ID NO: 92) SID4×repressor domain: GSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPSR (SEQ ID NO: 93)

[0169] In some embodiments, the enzymatic domain comprises an epigenetic modifier or its catalytic domain. For example, in some embodiments, the general structure of an exemplary dCas9 fusion protein having an epigenetic modifier or domain is [NH2]-[NLS]-[Cas9]-[epigenetic modifier]-[COOH], [NH2]-[NLS]-[epigenetic modifier]-[Cas9]-[COOH], [NH2]-[Cas9]-[epigenetic modifier]-[COOH], or [NH2]-[epigenetic modifier]-[Cas9]-[COOH], wherein the NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, a linker is inserted between the dCas9 and the epigenetic modifier domain. In some embodiments, a linker is inserted between the NLS and the epigenetic modifier and / or dCas9 domain. In some embodiments, the NLS is located at the C-terminus of the epigenetic modifier and / or dCas9 domain. In some embodiments, the NLS is located between the epigenetic modifier domain and the dCas9 domain. Additional features, such as a sequence tag, may also be present. Epigenetic modifiers are well known in the art and typically catalyze DNA methylation (and demethylation) or histone modifications (e.g., histone methylation / demethylation, acetylation / deacetylation, ubiquitination, phosphorylation, sumoylation, etc.). The presence of one, two or more epigenetic modifications can affect the transcriptional activity of one or more genes, for example, changing a gene from an "on" state to an "off" state, or vice versa. Epigenetic modifiers include, but are not limited to, histone demethylases, histone methyltransferases, hydroxylases, histone deacetylases, and histone acetyltransferases.Exemplary epigenetic modification proteins can be found in Konermann et al., Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500, 472-476; Mendenhall et al., Locus-specific editing of histone modifications at endogenous enhancers. Nat. Biotechnol. 2013; 31, 1133-1136; and Maeder et al., Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat. Biotechnol. 2013; 31, 1137-1142, the entire contents of each of which are incorporated herein by reference. In some embodiments, the epigenetic modifier domain is LSD1 (lysine (K)-specific demethylase 1A) histone demethylase, which in some embodiments comprises, in whole or in part, the amino acid sequence set forth as SEQ ID NO:94 or SEQ ID NO:95. In some embodiments, the epigenetic modifier domain is a TET1 hydroxylase catalytic domain, which in some embodiments comprises the amino acid sequence set forth as SEQ ID NO: 96. In some embodiments, the epigenetic modifier is a histone deacetylase (HDAC) effector domain. In some embodiments, the HDAC effector domain comprises, in whole or in part, an amino acid sequence corresponding to any of the HDAC effector proteins provided in Supplementary Table 2, SEQ ID NOs: 97-108 of Konermann et al., Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500, 472-476.In some embodiments, the epigenetic modifier is a histone methyltransferase (HMT) effector domain. In some embodiments, the HMT effector domain comprises, in whole or in part, an amino acid sequence corresponding to any of the HDAC effector proteins provided in Supplementary Table 3, SEQ ID NOS: 109-118, of Konermann et al., Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013; 500, 472-476.

[0170] LSD1, isoform a (human): (SEQ ID NO: 94)

[0171] LSD1, isoform b (human): (SEQ ID NO: 95)

[0172] TET1 catalytic domain: (SEQ ID NO: 96)

[0173] HDAC effector domain : HDAC8 (X. laevis): ASSPKKKRKVEASMSRVVKPKVASMEEMAAFHTDAYLQHLHKVSEEGDNDDPETLEYGLGYDCPITEGIYDYAAAVGGATLTAAEQLIEGKTRIAVNWPGGWHHAKKDEASGFCYLNDAVLGILKLREKFDRVLYVDMDLHHGDGVEDAFSFTSKVMTVSLHKFSPGFFPGTGDVSDIGLGKGRYYSINVPLQDGIQDDKYYQICEGVLKEVFTTFNPEAVVLQLGADTIAGDPMCSFNMTPEGIGKCLKYVLQWQLPTLILGGGGYHLPNTARCWTYLTALIVGRTLSSEIPDHEFFTEYGPDYVLEITPSCRPDRNDTQKVQEILQSIKGNLKRVVEF(SEQ ID NO: 97) RPD3 (S. cerevisiae): ASSPKKKRKVEASRRVAYFYDADVGNYAYGAGHPMKPHRIRMAHSLIMNYGLYKKMEIYRAKPATKQEMCQFHTDEYIDFLSRVTPDNLEMFKRESVKFNVGDDCPVFDGLYEYCSISGGGSMEGAARLNRGKCDVAVNYAGGLHHAKKSEASGFCYLNDIVLGIIELLRYHPRVLYIDIDVHHGDGVEEAFYTTDRVMTCSFHKYGEFFPGTGELRDIGVGAGKNYAVNVPLRDGIDDATYRSVFEPVIKKIMEWYQPSAVVLQCGGDSLSGDRLGCFNLSMEGHANCVNYVKSFGIPMMVVGGGGYTMRNVARTWCFETGLLNNVVLDKDLPYEF(SEQ ID NO: 98) MesoLo4 (M. loti): ASSPKKKRKVEASMPLQIVHHPDYDAGFATNHRFPMSKYPLLMEALRARGLASPDALNTTEPAPASWLKLAHAADYVDQVISCSVPEKIEREIGFPVGPRVSLRAQLATGGTILAARLALRHGIACNTAGGSHHARRAQGAGFCTFNDVAVASLVLLDEGAAQNILVVDLDVHQGDGTADILSDEPGVFTFSMHGERNYPVRKIASDLDIALPDGTGDAAYLRRLATILPELSARARWDIVFYNAGVDVHAEDRLGRLALSNGGLRARDEMVIGHFRALGIPVCGVIGGGYSTDVPALASRHAILFEVASTYAEF (SEQ ID NO: 99)

[0174] HDAC11 (human): ASSPKKKRKVEASMLHTTQLYQHVPETRWPIVYSPRYNITFMGLEKLHPFDAGKWGKVINFLKEEKLLSDSMLVEAREASEEDLLVVHTRRYLNELKWSFAVATITEIPPVIFLPNFLVQRKVLRPLRTQTGGTIMAGKLAVERGWAINVGGGFHHCSSDRGGGFCAYADITLAIKFLFERVEGISRATIIDLDAHQGNGHERDFMDDKRVYIMDVYNRHIYPGDRFAKQAIRRKVELEWGTEDDEYLDKVERNIKKSLQEHLPDVVVYNAGTDILEGDRLGGLSISPAGIVKRDELVFRMVRGRRVPILMVTSGGYQKRTARIIADSILNLFGLGLIGPESPSVSAQNSDTPLLPPAVPEF (SEQ ID NO: 100) HDT1 (A. thaliana): ASSPKKKRKVEASMEFWGIEVKSGKPVTVTPEEGILIHVSQASLGECKNKKGEFVPLHVKVGNQNLVLGTLSTENIPQLFCDLVFDKEFELSHTWGKGSVYFVGYKTPNIEPQGYSEEEEEEEEEVPAGNAAKAVAKPKAKPAEVKPAVDDEEDESDSDGMDEDDSDGEDSEEEEPTPKKPASSKKRANETTPKAPVSAKKAKVAVTPQKTDEKKKGGKAANQSEF (SEQ ID NO: 101) SIRT3 (Human): ASSPKKKRKVEASMVGAGISTPSGIPDFRSPGSGLYSNLQQYDLPYPEAIFELPFFFHNPKPFFTLAKELYPGNYKPNVTHYFLRLLHDKGLLLRLYTQNIDGLERVSGIPASKLVEAHGTFASATCTVCQRPFPGEDIRADVMADRVPRCPVCTGVVKPDIVFFGEPLPQRFLLHVVDFPMADLLLILGTSLEVEPFASLTEAVRSSVPRLLINRDLVGPLAWHPRSRDVAQLGDVVHGVESLVELLGWTEEMRDLVQRETGKLDGPDKEF (SEQ ID NO: 102)

[0175] HST2 (S. cerevisiae): ASSPKKKRKVEASTEMSVRKIAAHMKSNPNAKVIFMVGAGISTSCGIPDFRSPGTGLYHNLARLKLPYPEAVFDVDFFQSDPLPFYTLAKELYPGNFRPSKFHYLLKLFQDKDVLKRVYTQNIDTLERQAGVKDDLIIEAHGSFAHCHCIGCGKVYPPQVFKSKLAEHPIKDFVKCDVCGELVKPAIVFFGEDLPDSFSETWLNDSEWLREKITTSGKHPQQPLVIVVGTSLAVYPFASLPEEIPRKVKRVLCNLETVGDFKANKRPTDLIVHQYSDEFAEQLVEELGWQEDFEKILTAQGGMGEF (SEQ ID NO: 103) CobB (E. coli (K12)): ASSPKKKRKVEASMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMPLGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAEF (SEQ ID NO: 104) HST2 (C. albicans): ASSPKKKRKVEASMPSLDDILKPVAEAVKNGKKVTFFNGAGISTGAGIPDFRSPDTGLYANLAKLNLPFAEAVFDIDFFKEDPKPFYTLAEELYPGNFAPTKFHHFIKLLQDQGSLKRVYTQNIDTLERLAGVEDKYIVEAHGSFASNHCVDCHKEMTTETLKTYMKDKKIPSCQHCEGYVKPDIVFFGEGLPVKFFDLWEDDCEDVEVAIVAGTSLTVFPFASLPGEVNKKCLRVLVNKEKVGTFKHEPRKSDIIALHDCDIVAERLCTLLGLDDKLNEVYEKEKIKYSKAETKEIKMHEIEDKLKEEAHLKEDKHTTKVDKKEKQNDANDKELEQLIDKAKAEF (SEQ ID NO: 105)

[0176] SIRT5 (human): ASSPKKKRKVEASSSSMADFRKFFAKAKHIVIISGAGVSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELAHCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEALACHENETVSEF (SEQ ID NO: 106) Sir2A (P. falciparum): ASSPKKKRKVEASMGNLMISFLKKDTQSITLEELAKIIKKCKHVVALTGSGTSAESNIPSFRGSSNSIWSKYDPRIYGTIWGFWKYPEKIWEVIRDISSDYEIEINNGHVALSTLESLGYLKSVVTQNVDGLHEASGNTKVISLHGNVFEAVCCTCNKIVKLNKIMLQKTSHFMHQLPPECPCGGIFKPNIILFGEVVSSDLLKEAEEEIAKCDLLLVIGTSSTVSTATNLCHFACKKKKKIVEINISKTYITNKMSDYHVCAKFSELTKVANILKGSSEKNKKIMEF (SEQ ID NO: 107) SIRT6 (human): ASSPKKKRKVEASMSVNYAAGLSPYADKGKCGLPEIFDPPEELERKVWELARLVWQSSSVVFHTGAGISTASGIPDFRGPHGVWTMEERGLAPKFDTTFESARPTQTHMALVQLERVGLLRFLVSQNVDGLHVRSGFPRDKLAELHGNMFVEECAKCKTQYVRDTVVGTMGLKATGRLCTVAKARGLRACRGELRDTILDWEDSLPDRDLALADEASRNADLSITLGTSLQIRPSGNLPLATKRRGGRLVIVNLQPTKHDRHADLRIHGYVDEVMTRLMKHLGLEIPAWDGPRVLERALPPLEF (SEQ ID NO: 108)

[0177] HMT domain : NUE (C.trachomatis): ASSPKKKRKVEASMTTNSTQDTLYLSLHGGIDSAIPYPVRRVEQLLQFSFLPELQFQNAAVKQRIQRLCYREEKRLAVSSLAKWLGQLHKQRLRAPKNPPVAICWINSYVGYGVFARESIPAWSYIGEYTGILRRRQALWLDENDYCFRYPVPRYSFRYFTIDSGMQGNVTRFINHSDNPNLEAIGAFENGIFHIIIRAIKDILPGEELCYHYGPLYWKHRKKREEFVPQEEEF (SEQ ID NO: 109) vSET (P. bursaria cholera virus): ASSPKKKRKVEASMFNDRVIVKKSPLGGYGVFARKSFEKGELVEECLCIVRHNDDWGTALEDYLFSRKNMSAMALGFGAIFNHSKDPNARHELTAGLKRMRIFTIKPIAIGEEITISYGDDYWLSRPRLTQNEF (SEQ ID NO: 110) SUV39H1 (human): ASSPKKKRKVEASNLKCVRILKQFHKDLERELLRRHHRSKTPRHLDPSLANYLVQKAKQRRALRRWEQELNAKRSHLGRITVENEVDLDGPPRAFVYINEYRVGEGITLNQVAVGCECQDCLWAPTGGCCPGASLHKFAYNDQGQVRLRAGLPIYECNSRCRCGYDCPNRVVQKGIRYDLCIFRTDDGRGWGVRTLEKIRKNSFVMEYVGEIITSEEAERRGQIYDRQGATYLFDLDYVEDVYTVDAAYYGNISHFVNHSCDPNLQVYNVFIDNLDERLPRIAFFATRTIRAGEELTFDYNMQVDPVDMESTRMDSNFGLAGLPGSPKKRVRIECKCGTESCRKYLFEF (SEQ ID NO: 111) DIM5(N.crassa): ASSPKKKRKVEASMEKAFRPHFFNHGKPDANPKEKKNCHWCQIRSFATHAQLPISIVNREDDAFLNPNFRFIDHSIIGKNVPVADQSFRVGCSCASDEECMYSTCQCLDEMAPDSDEEADPYTRKKRFAYYSQGAKKGLLRDRVLQSQEPIYECHQGCACSKDCPNRVVERGRTVPLQIFRTKDRGWGVKCPVNIKRGQFVDRYLGEIITSEEADRRRAESTIARRKDVYLFALDKFSDPDSLDPLLAGQPLEVDGEYMSGPTRFINHSCDPNMAIFARVGDHADKHIHDLALFAIKDIPKGTELTFDYVNGLTGLESDAHDPSKISEMTKCLCGTAKCRGYLWEF (SEQ ID NO: 112)

[0178] KYP (A. thaliana): ASSPKKKRKVEASDISGGLEFKGIPATNRVDDSPVSPTSGFTYIKSLIIEPNVIIPKSSTGCNCRGSCTDSKKCACAKLNGGNFPYVDLNDGRLIESRDVVFECGPHCGCGPKCVNRTSQKRLRFNLEVFRSAKKGWAVRSWEYIPAGSPVCEYIGVVRRTADVDTISDNEYIFEIDCQQTMQGLGGRQRRLRDVAVPMNNGVSQSSEDENAPEFCIDAGSTGNFARFINHSCEPNLFVQCVLSSHQDIRLARVVLFAADNISPMQELTYDYGYALDSVHEF (SEQ ID NO: 113) SUVR4 (A. thaliana): ASSPKKKRKVEASQSAYLHVSLARISDEDCCANCKGNCLSADFPCTCARETSGEYAYTKEGLLKEKFLDTCLKMKKEPDSFPKVYCKDCPLERDHDKGTYGKCDGHLIRKFIKECWRKCGCDMQCGNRVVQRGIRCQLQVYFTQEGKGWGLRTLQDLPKGTFICEYIGEILTNTELYDRNVRSSSERHTYPVTLDADWGSEKDLKDEEALCLDATICGNVARFINHRCEDANMIDIPIEIETPDRHYYHIAFFTLRDVKAMDELTWDYMIDFNDKSHPVKAFRCCCGSESCRDRKIKGSQGKSIERRKIVSAKKQQGSKEVSKKRKEF(SEQ ID NO: 114) Set4(C.elegans): ASSPKKKRKVEASMQLHEQIANISVTFNDIPRSDHSMTPTELCYFDDFATTLVVDSVLNFTTHKMSKKRRYLYQDEYRTARTVMKTFREQRDWTNAIYGLLTLRSVSHFLSKLPPNKLFEFRDHIVRFLNMFILDSGYTIQECKRYSQEGHQGAKLVSTGVWSRGDKIERLSGVVCLLSSEDEDSILAQEGSDFSVMYSTRKRCSTLWLGPGAYINHDCRPTCEFVSHGSTAHIRVLRDMVPGDEITCFYGSEFFGPNNIDCECCTCEKNMNGAFSYLRGNENAEPIISEKKTKYELRSRSEF(SEQ ID NO: 115)

[0179] Set1(C.elegans): ASSPKKKRKVEASMKVAAKKLATSRMRKDRAAAASPSSDIENSENPSSLASHSSSSGRMTPSKNTRSRKGVSVKDVSNHKITEFFQVRRSNRKTSKQISDEAKHALRDTVLKGTNERLLEVYKDVVKGRGIRTKVNFEKGDFVVEYRGVMMEYSEAKVIEEQYSNDEEIGSYMYFFEHNNKKWCIDATKESPWKGRLINHSVLRPNLKTKVVEIDGSHHLILVARRQIAQGEELLYDYGDRSAETIAKNPWLVNTEF (SEQ ID NO: 116) SETD8 (human) ASSPKKKRKVEASSCDSTNAAIAKQALKKPIKGKQAPRKKAQGKTQQNRKLTDFYPVRRSSRKSKAELQSEERKRIDELIESGKEEGMKIDLIDGKGRGVIATKQFSRGDFVVEYHGDLIEITDAKKREALYAQDPSTGCYMYYFQYLSKTYCVDATRETNRLGRLINHSKCGNCQTKLHDIDGVPHLILIASRDIAAGEELLYDYGDRSKASIEAFPWLKHEF (SEQ ID NO: 117) TgSET8 (T. gondii): ASSPKKKRKVEASASRRTGEFLRDAQAPSRWLKRSKTGQDDGAFCLETWLAGAGDDAAGGERGRDREGAADKAKQREERRQKELEERFEEMKVEFEEKAQRMIARRAALTGEIYSDGKGSKKPRVPSLPENDDDALIEIIIDPEQGILKWPLSVMSIRQRTVIYQECLRRDLTACIHLTKVPGKGRAVFAADTILKDDFVVEYKGELCSEREAREREQRYNRSKVPMGSFMFYFKNGSRMMAIDATDEKQDFGPARLINHSRRNPNMTPRAITLGDFNSEPRLIFVARRNIEKGEELLVDYGERDPDVIKEHPWLNSEF (SEQ ID NO: 118)

[0180] Those skilled in the art will understand that any of the exemplary Cas9 proteins (including, for example, the exemplary Cas9 nucleases described herein, variants and fusions thereof) can be delivered to cells using the techniques of this disclosure, and that the disclosure is not limited in this respect.

[0181] Nuclease Effector Proteins TALE nucleases or TALENs are artificial nucleases that contain a transcription activator-like effector DNA binding domain linked to a DNA cleavage domain (e.g., a FokI domain). Several modular assembly schemes for generating engineered TALE constructs have been reported (Zhang, Feng; et.al. (February 2011). "Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription". Nature Biotechnology 29 (2): 149-53; Geiβler, R.; Scholze, H.; Hahn, S.; Streubel, J.; Bonas, U.; Behrens, SE; Boch, J. (2011), Shiu, Shin-Han. ed. "Transcriptional Activators of Human Genes with Programmable DNA-Specificity". PLoS ONE 6 (5): e19509; Cermak, T.; Doyle, EL; Christian, M.; Wang, L.; Zhang, Y.; Schmidt, C.; Baller, JA; Somia, NV et al. (2011). "Efficient design and Nucleic Acids Research, Morbitzer, R.; Elsaesser, J.; Hausner, J.; Lahaye, T. (2011). “Assembly of custom TALE-type DNA binding domains by modular cloning”. Nucleic Acids Research, Li, T.; Huang, S.; Zhao, X.; Wright, DA; Carpenter, S.; Spalding, M.H.; Weeks, DP; Yang, B. (2011). "Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes". Nucleic Acids Research.; Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S. (2011). Bendahmane, Mohammed, ed. "Assembly of Designer TAL Effectors by Golden Gate Cloning". PLoS ONE 6 (5): e19722, the entire contents of each of which are incorporated herein by reference. Those skilled in the art will understand that TALE nucleases can be engineered to target almost any genomic sequence with high specificity, and that such engineered nucleases can be used in embodiments of the present technology to manipulate the genome of a cell (e.g., by delivering the respective TALEN by a method or strategy disclosed herein in a suitable context for the TALEN to bind to and cleave its target sequence in the genome of the cell). In some embodiments, the delivered TALEN targets a gene or allele associated with a disease or disorder. In some embodiments, delivery of the TALEN to a subject confers a therapeutic benefit to the subject.

[0182] Zinc finger nucleases are a class of artificial nucleases that contain a DNA cleavage domain and a zinc finger DNA binding domain. In some embodiments, the DNA cleavage domain is a nonspecific DNA cleavage domain of a restriction endonuclease (e.g., FokI). In some embodiments, the DNA cleavage domain cleaves double-stranded DNA only when dimerized with a second DNA cleavage domain of the same type. In some embodiments, the DNA cleavage domain is fused to the C-terminus of the zinc finger domain via a linker (e.g., a peptide linker). In some embodiments, the zinc finger domain contains about three to about six zinc fingers and specifically recognizes and binds to a target sequence about 9 to 20 nucleotides in length. In some embodiments, multiple zinc finger nuclease molecules are delivered to a target cell by the systems or methods provided herein, and the zinc finger domain of one zinc finger nuclease molecule binds to a target sequence that is closely proximal to the target sequence of a second zinc finger nuclease molecule. In some embodiments, the zinc finger domains of the zinc finger nuclease molecules that bind to target sequences that are close to each other are different. In some embodiments, the zinc finger nuclease molecule delivered to a cell by the system or method provided herein binds to a target nucleic acid sequence that is close to the target sequence of another zinc finger nuclease molecule, so that the DNA cleavage domains of the molecules dimerize at the site between the two target sequences and cleave the DNA molecule.

[0183] In some embodiments, the genome of a target cell is edited by a nuclease (e.g., a TALEN or zinc finger nuclease, or a combination of multiple such nucleases) delivered to the cell by the strategies or methods disclosed herein. In some embodiments, a single- or double-strand break is introduced by the nuclease at a specific site in the genome of the target cell, resulting in the destruction of the targeted genomic sequence. In some embodiments, the targeted genomic sequence is a nucleic acid sequence within the coding region of a gene. In some embodiments, the strand break introduced by the nuclease leads to a mutation in the target gene that impairs the expression of the encoded gene product. In some embodiments, a nucleic acid is co-delivered to the cell together with the nuclease. In some embodiments, the nucleic acid comprises a sequence identical or homologous to the sequence adjacent to the nuclease target site. In some such embodiments, the strand break achieved by the nuclease is repaired by intracellular DNA repair mechanisms, introducing all or part of the co-delivered nucleic acid into the intracellular DNA at the break site, resulting in the targeted insertion of the co-delivered nucleic acid or part thereof. In some embodiments, the insertion results in the destruction or repair of a pathogenic allele. In some embodiments, the insertion is detected by a suitable assay, such as a DNA sequencing assay, a Southern blot assay, or an assay for a reporter gene (e.g., a fluorescent protein or resistance to an antibiotic) encoded by the co-delivered nucleic acid. In some embodiments, the nucleic acid is co-delivered by attachment to a supercharged protein. In some embodiments, the supercharged protein is also attached to a functional effector protein (e.g., a nuclease). In some embodiments, delivery of the nuclease to the target cell results in clinically or therapeutically beneficial disruption of gene function.

[0184] In some embodiments, cells from a subject are obtained and a nuclease or other effector protein is delivered to the cells ex vivo using the systems or methods provided herein. In some embodiments, the treated cells are selected for cells in which the desired nuclease-mediated genome editing event has occurred. In some embodiments, the treated cells bearing the desired genomic mutation or alteration are returned to the subject from which they were obtained.

[0185] Methods for engineering, producing, and isolating nucleases (e.g., TALEs, or zinc finger nucleases) that target specific sequences and editing the genome of cells at specific target sequences are well known in the art (e.g., Mani et al., Biochemical and Biophysical Research Communications 335:447-457, 2005; Perez et al., Nature Biotechnology 26:808-16, 2008; Kim et al., Genome Research, 19: 1279-88, 2009; Urnov et al., Nature 435:646-51, 2005; Carroll et al., Gene Therapy 15: 1463-68, 2005; Lombardo et al., Nature Biotechnology 25: 1298-306, 2007; Kandavelou et al., Biochemical and Biophysical Research Communications 388:56-61, 2009, and Hockemeyer et al., Nature Biotechnology 27(9):851-59, 2009, as well as the references described in each section for each nuclease.) Based on the guidance provided herein, one of skill in the art will be able to ascertain suitable methods for use in the context of the present disclosure.

[0186] TALE effector proteins In some embodiments, an effector protein comprising a TALE domain is delivered to a target cell by a system or method provided herein. In some embodiments, a TALE effector (e.g., an engineered TALE transcription factor comprising a TALE DNA-binding domain and a heterologous transcriptional activator or repressor domain) is delivered to a cell by a system or method provided by aspects of the present invention. In some embodiments, a TALE effector (e.g., a transcription factor) is delivered to a cell in an amount sufficient to activate or inhibit transcription of a target gene of the transcription factor in the cell. In some embodiments, the transcription factor is delivered in an amount and for a duration sufficient to effect a phenotypic change in the target cell (e.g., an altered cell function or an altered developmental potential). Exemplary TALE transcription factors are described herein, and one of skill in the art will be able to identify additional suitable TALE transcription factors based on the guidance provided herein and knowledge of such TALE transcription factors in the art.

[0187] In some embodiments, target cells (e.g., somatic cells) are contacted with a TALE transcription factor associated with a supercharged protein provided herein, or a combination of such factors. In some embodiments, the target cells are primary somatic cells and are contacted in vitro or ex vivo with a TALE transcription factor associated with a supercharged protein. In some embodiments, the TALE transcription factor is associated with a positively charged supercharged protein, e.g., as described herein. In some embodiments, the TALE transcription factor is associated with a negatively charged supercharged protein, e.g., as described herein. In some embodiments, the TALE transcription factor is associated with a cationic lipid and / or cationic polymer, e.g., as described herein. In some embodiments, the TALE transcription factor is associated with a negatively charged supercharged protein, e.g., as described herein.

[0188] In some embodiments, target cells are contacted or repeatedly contacted with a TALE transcription factor associated with a supercharged protein (and optionally a cationic lipid and / or cationic polymer) provided herein, and a desired change in cell phenotype or gene expression is detected. In some embodiments, target cells are repeatedly contacted with a TALE transcription factor associated with a supercharged protein (and optionally a cationic lipid and / or cationic polymer) provided herein until the formation of a desired cell phenotype is detected. Methods for detecting cell phenotype and gene expression are well known to those skilled in the art and include, for example, detection of marker gene expression and morphological analysis by well-established methods (e.g., immunohistochemistry, fluorescence-activated cell sorting (FACS), or fluorescence microscopy). In some embodiments, target cells are contacted with a TALE transcription factor associated with a supercharged protein provided herein for a period of at least 3 hours, at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10-12 days, at least 12-15 days, at least 15-20 days, at least 20-25 days, at least 25-30 days, at least 30-40 days, at least 40-50 days, at least 50-60 days, at least 60-70, or at least 70-100 days.

[0189] In some embodiments, target cells are contacted with a TALE transcription factor associated with a supercharged protein (and optionally a cationic lipid and / or cationic polymer) provided herein in an amount and for a duration effective to program the cell toward a different cellular state. As will be apparent to one skilled in the art, the amount required to program or reprogram a cell will depend on various factors (e.g., cell type and treatment schedule). Generally, delivery of a TALE transcription factor to a target somatic cell by a system or method provided herein will be at a concentration below that at which significant toxicity can be observed. The critical concentration will depend, for example, on the particular TALE transcription factor, the supercharged protein to which it is associated, the type of association, and the type of cell to be treated.

[0190] Useful concentrations of functional effector protein associated with supercharged protein (and optionally cationic lipid and / or cationic polymer) for delivery to a particular cell type can be determined by one of skill in the art by routine experimentation. In some embodiments, target cells are contacted in vitro or ex vivo with functional effector protein associated with supercharged protein (and optionally cationic lipid and / or cationic polymer) at a concentration of about 1 pM to about 1 μM. In some embodiments, the target cells are administered at a concentration of about 1 pM, about 2.5 pM, about 5 pM, about 7.5 pM, about 10 pM, about 20 pM, about 25 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 75 pM, about 80 pM, about 90 pM, about 100 pM, about 200 pM, about 250 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 750 pM, about 800 pM, about 900 pM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, The supercharged protein may be contacted in vitro or ex vivo with a functional effector protein associated with the supercharged protein at a concentration of about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 20 nM, about 25 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 75 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 750 nM, about 800 nM, about 900 nM, or about 1 μM. Useful exposure of target cells to functional effector protein, and, if necessary, the time of incubation after administration in the absence of functional effector protein, as well as the number of administration / incubation cycles useful to achieve a desired biological effect (e.g., alteration of gene transcription, cleavage of a target site by a delivered nuclease, etc.) or a desired cellular phenotype, can also be determined by one of skill in the art by routine experimentation.

[0191] In some embodiments, target cells for delivery of functional effector proteins by the systems or methods provided herein are primary cells obtained by biopsy from a subject. In some embodiments, the subject is diagnosed with a disease. In some embodiments, the disease is a degenerative disease characterized by impaired function of a specific cell type (e.g., neuronal cells). In some embodiments, cells treated with functional effector proteins according to the strategies or methods disclosed herein, or the progeny of such cells, are used in a cell replacement therapy procedure. In some embodiments, the treated cells are administered to the subject from whom the somatic cells were obtained in an autologous cell replacement therapy procedure.

[0192] In some embodiments, functional effector proteins (e.g., TALE transcription factors capable of converting cells from one differentiation state to another) are delivered to target cells in vitro or in vivo by the systems or methods provided herein. Transcription factors that achieve transdifferentiation are known in the art (see, e.g., Zhou et al., Nature 455:627-33, 2008). In some embodiments, TALE transcription factors that regulate the expression of PPARγ or PRDM16 are delivered to fibroblasts by the systems or methods provided by the present inventors. It is known in the art that the expression of these transcription factors is a pivotal step in programming fibroblasts toward brown fat or white adipocyte states. In some embodiments, programmed brown adipocytes are generated from fibroblasts obtained from a subject in need of brown adipocytes and administered to the subject (e.g., for use in a cell replacement therapy procedure involving the subject).

[0193] Complex formation The present invention provides complexes comprising a supercharged protein associated with one or more functional effector proteins to be delivered. In some embodiments, the supercharged protein is associated with one or more functional effector proteins to be delivered via non-covalent interactions. In some embodiments, the supercharged protein is associated with one or more functional effector proteins via electrostatic interactions. In certain embodiments, the supercharged protein has a net positive overall charge, and the functional effector proteins to be delivered have a net negative overall charge. In some embodiments, the complex further comprises a cationic lipid and / or a cationic polymer. For example, in some embodiments, the supercharged protein of the complex is supernegatively charged, allowing it to associate with the cationic lipid and / or polymer.

[0194] In certain embodiments, the supercharged protein is covalently linked to one or more functional effector proteins to be delivered. For example, the supercharged protein can be fused to the functional effector protein to be delivered. The covalent attachment can be direct or indirect (e.g., via a linker). In some embodiments, the covalent attachment is mediated through one or more linkers. In some embodiments, the linker is a cleavable linker. In certain embodiments, the cleavable linker comprises an amide, ester, or disulfide bond. For example, the linker can be an amino acid sequence cleavable by an intracellular enzyme. In certain embodiments, the enzyme is a protease. In other embodiments, the enzyme is an esterase. In some embodiments, the enzyme is one that is more highly expressed in certain cell types than other cell types. For example, the enzyme can be one that is more highly expressed in tumor cells than non-tumor cells. Exemplary linkers and enzymes that cleave those linkers are provided below. [Table 1]

[0195] To take just one specific example, +36GFP can be linked to a functional effector protein to be delivered by a cleavable linker such as ALAL (SEQ ID NO: 254) to generate +36GFP-(GGS)-ALAL-(GGS)-[functional effector protein X] (SEQ ID NO: 255).

[0196] In certain embodiments, the functional effector protein to be delivered is contacted with the supercharged protein to form a complex. In some embodiments, complex formation is carried out at or about pH 7. In some embodiments, complex formation is carried out at about pH 5, about pH 6, about pH 7, about pH 8, or about pH 9. Complex formation is typically carried out at a pH that does not negatively affect the function of the supercharged protein and / or the functional effector protein. In some embodiments, complex formation is carried out at room temperature. In some embodiments, complex formation is carried out at or about 37°C. In some embodiments, complex formation is carried out at temperatures below 4°C, about 4°C, about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 37°C, about 40°C, or above 40°C. Complex formation is typically carried out at a temperature that does not negatively affect the function of the supercharged protein and / or the functional effector protein. In some embodiments, complex formation is carried out in serum-free medium. In some embodiments, complex formation is carried out in the presence of CO2 (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6% or more).

[0197] In some embodiments, complex formation is carried out using a functional effector protein concentration of about 100 nM. In some embodiments, complex formation is carried out using a functional effector protein concentration of about 25 nM, about 50 nM, about 75 nM, about 90 nM, about 100 nM, about 110 nM, about 125 nM, about 150 nM, about 175 nM, or about 200 nM. In some embodiments, complex formation is carried out using a supercharged protein concentration of about 40 nM. In some embodiments, complex formation is carried out using a supercharged protein concentration of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM.

[0198] In some embodiments, complex formation is carried out under conditions of excess functional effector protein. In some embodiments, complex formation is carried out at a ratio of functional effector protein:supercharged protein of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1. In some embodiments, complex formation is carried out at a ratio of functional effector protein:supercharged protein of about 3:1. In some embodiments, complex formation is carried out at a ratio of supercharged protein:functional effector protein of about 20:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0199] In some embodiments, complex formation is carried out by mixing the functional effector protein and the supercharged protein and agitating the mixture (e.g., by inversion). In some embodiments, complex formation is carried out by mixing the functional effector protein and the supercharged protein and allowing the mixture to stand. In some embodiments, complex formation is carried out in the presence of a pharmaceutically acceptable carrier or excipient. In some embodiments, the complex is further combined with a pharmaceutically acceptable carrier or excipient. Exemplary excipients or carriers include water, solvents, lipids, proteins, peptides, endosomolytic agents (e.g., chloroquine, pyrenebutyric acid), small molecules, carbohydrates, buffers, natural polymers, synthetic polymers (e.g., PLGA, polyurethane, polyester, polycaprolactone, polyphosphazene), pharmaceutical agents, and the like.

[0200] In some embodiments, a complex comprising a supercharged protein and a functional effector protein may migrate slower in a gel electrophoresis assay than either the supercharged protein alone or the functional effector protein alone.

[0201] application The present invention provides compositions comprising naturally occurring or engineered supercharged proteins linked to functional effector proteins (e.g., nucleases, transcriptional activators / repressors, recombinases, Cas9 proteins (including variants and fusions thereof), etc.) to be delivered to cells, as well as methods and uses of such compositions. In certain embodiments, compositions comprising a Cas9 protein (e.g., a Cas9 protein linked to a gRNA) and a cationic lipid are provided. In certain embodiments, compositions comprising a Cas9 protein (e.g., a Cas9 protein linked to a gRNA) and a cationic polymer are provided. The compositions of the invention can be used, for example, to treat or prevent any disease that would benefit from the delivery of a drug to cells. The compositions of the invention can also be used to transfect or treat cells for research purposes.

[0202] In some embodiments, compositions in accordance with the invention can be used for research purposes (e.g., to efficiently deliver functional effector proteins to cells in a research context). In some embodiments, compositions in accordance with the invention can be used for therapeutic purposes. In some embodiments, compositions in accordance with the invention can be used for the treatment of any of a variety of diseases, disorders, and / or conditions, including, but not limited to, one or more of the following: autoimmune diseases (e.g., diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis), inflammatory diseases (e.g., arthritis, pelvic inflammatory disease), infectious diseases (e.g., viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis), neurological diseases (e.g., Alzheimer's disease, Huntington's disease, autism, Duchenne muscular dystrophy), Cardiovascular disorders (e.g., atherosclerosis, hypercholesterolemia, thrombosis, coagulation disorders, angiogenesis disorders such as macular degeneration), proliferative disorders (e.g., cancer, benign neoplasms), respiratory disorders (e.g., chronic obstructive pulmonary disease), digestive disorders (e.g., inflammatory bowel disease, ulcers), musculoskeletal disorders (e.g., fibromyalgia, arthritis), endocrine, metabolic, and nutritional disorders (e.g., diabetes, osteoporosis), urinary disorders (e.g., kidney disease), psychiatric disorders (e.g., depression, schizophrenia), skin disorders (e.g., wounds, eczema), blood and lymphatic system disorders (e.g., anemia, hemophilia), etc.

[0203] The compositions of the present invention can be used in clinical settings. For example, the supercharged protein can be linked to a functional effector protein that can be used for therapeutic applications. Such a functional effector protein can be, for example, a nuclease or a transcription activator. Other compositions containing a Cas9 protein and a cationic lipid can also be used for therapeutic applications.

[0204] In some embodiments, the supercharged protein or the functional effector protein associated with the supercharged protein includes a detectable label. These molecules can be used for detection, imaging, disease staging, diagnosis, or patient selection. Suitable labels include fluorescent, chemiluminescent, enzyme labels, colorimetric, phosphorescent, density-based labels, e.g., electron density-based labels, and generally imaging agents, and / or radioactive labels.

[0205] Pharmaceutical Composition The present invention provides compositions comprising a supercharged protein associated with at least one functional effector protein to be delivered, in some embodiments encapsulated by a cationic lipid. Other compositions comprising a Cas9 protein and a cationic lipid are provided. Accordingly, the present invention provides pharmaceutical compositions comprising one or more supercharged proteins associated with a functional effector protein and / or one or more functional effector proteins associated with a cationic lipid and / or cationic polymer, and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may optionally contain one or more additional therapeutically active substances. According to some embodiments, methods are provided for administering pharmaceutical compositions comprising one or more supercharged proteins associated with a functional effector protein to be delivered to a subject in need thereof. In some embodiments, the compositions are administered to a human. For purposes of this disclosure, the term "active ingredient" generally refers to the functional effector protein to be delivered, or the supercharged protein and / or Cas9 protein associated with a functional effector protein, as described herein.

[0206] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to all kinds of animals. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to various animals are well known, and those skilled in the art of veterinary pharmacology can design and / or implement such modifications at most through mere average experimentation. Subjects to which the pharmaceutical compositions are contemplated include, but are not limited to, humans and / or other primates, mammals (including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats), and / or birds (including commercially relevant birds such as chickens, ducks, geese, and / or turkeys).

[0207] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods include bringing into association the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single- or multi-dose unit.

[0208] Pharmaceutical compositions according to the present invention can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient that would be administered to a subject and / or a convenient fraction of such a dose (e.g., 1 / 2 or 1 / 3 of such a dose).

[0209] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is to be administered. For example, the composition may contain from 0.1% to 100% (w / w) of the active ingredient.

[0210] In addition, pharmaceutical formulations may contain pharmaceutically acceptable excipients, which as used herein includes any and all solvents, dispersion media, diluents, or other liquid carriers, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional excipient vehicle is incompatible with the substance or its derivatives (e.g., by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition), its use is considered within the scope of this disclosure.

[0211] In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0212] Pharmaceutically acceptable excipients used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may be optionally included in the formulation. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and / or perfuming agents may be present in the composition according to the discretion of the formulator.

[0213] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and the like, and / or combinations thereof.

[0214] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, gelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and / or combinations thereof.

[0215] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, woolfat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitol, ... sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span® 65], glyceryl monooleate, sorbitan monooleate [Span® 80]), ... monooleate [Tween® 60], polyoxyethylene sorbitan monooleate [Tween® 80], polyoxyethylene sorbitan monooleate [Span® 80], polyoxyethylene sorbitan monolaurate [Tween® 20], polyoxyethylene sorbitan monooleate [Tween® 60], polyoxyethylene sorbitan monooleate [Span® 80], polyoxyethylene sorbitan monooleate [Span® 60], polyoxyethylene sorbitan monooleate [Span® 60], polyoxyethylene sorbitan monooleate [Span® 60], polyoxyethylene sorbitan monooleate [Span® 80], polyoxyethylene sorbitan monooleate [Span® 60] ethylene esters (e.g., polyoxyethylene monostearate [Myrj® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij® 30]), polyvinylpyrrolidone, diethylene glycol monolaurate, triethanolamine oleate,Examples of suitable anti-inflammatory agents include, but are not limited to, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F68, Poloxamer® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0216] Exemplary binders include, but are not limited to, starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrin, molasses, lactose, lactitol, mannitol), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, panwar gum, ghatti gum, isapol shell mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginic acid, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and the like, and combinations thereof.

[0217] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus®, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone™, Kathon™, and / or Euxyl®.

[0218] Exemplary buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and / or combinations thereof.

[0219] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0220] Exemplary oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, lily of the valley, Exemplary oils include, but are not limited to, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, camellia, peppermint, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0221] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavorings, and / or fragrances. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof. Injectable preparations, for example, sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents.Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, such as solutions in 1,3-butanediol.Acceptable bases and solvents that can be used include water, Ringer's solution (USP), and isotonic sodium chloride solution.Sterile, fixed oils are conventionally used as solvents or suspending media.For this purpose, any non-irritating, fixed oil can be used, including synthetic mono- or diglycerides.Fatty acids such as oleic acid can be used to prepare injectables. Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0222] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil base. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Compositions for rectal or vaginal administration are typically suppositories, which can be prepared by mixing the composition with a suitable non-irritating excipient (e.g., cocoa butter, polyethylene glycol, or a suppository wax) that is solid at ordinary temperatures but liquid at body temperature and therefore melts in the rectum or vaginal cavity to release the active ingredient. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient. These include, for example, sodium citrate or dicalcium phosphate, and / or fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarders (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), adsorbents (e.g., kaolin and bentonite clay), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain buffering agents.

[0223] Solid compositions of a similar type can be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells (such as enteric coatings and other coatings well known in the pharmaceutical formulating art). They can optionally contain opacifying agents and can be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers for soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Dosage forms for topical and / or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffers, as required. In addition, the present invention contemplates the use of transdermal patches, which often have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispensing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by either providing a rate-controlling membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0224] Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices that limit the effective needle insertion length into the skin, and their functional equivalents. Jet injection devices are suitable, delivering liquid compositions to the dermis by a liquid jet injector and / or by a needle that generates a jet that penetrates the stratum corneum and reaches the dermis. Ballistic powder / particle delivery devices are suitable, using compressed gas to accelerate the powder form of vaccines through the outer layer of the skin and into the dermis. Alternatively or additionally, conventional syringes can be used in the classic Mantoux method of intradermal administration. Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions, such as creams, ointments, and / or pastes, and / or solutions and / or suspensions. Topically administrable formulations may contain, for example, about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further include one or more of the additional ingredients described herein. Pharmaceutical compositions may be prepared, packaged, and / or sold as formulations suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles comprising the active ingredient and having diameters ranging from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir (to which a propellant stream can be directed to disperse the powder) and / or using a self-propelling solvent / powder dispenser container (e.g., a device comprising the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container). Such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0225] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional ingredients, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient). Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as optionally sterile aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any nebulization and / or atomization device. Such formulations may further contain one or more additional ingredients (including, but not limited to, flavorings, such as saccharin sodium, volatile oils, buffers, surfactants, and / or preservatives, such as methylhydroxybenzoate). The droplets provided by this administration route may have an average diameter ranging from about 0.1 nm to about 200 nm. Formulations described herein as useful for pulmonary delivery are also useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nostrils).

[0226] Formulations suitable for nasal administration may contain, for example, as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more of the additional ingredients described herein. A pharmaceutical composition may also be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges made using conventional methods and may contain, for example, 0.1% to 20% (w / w) of the active ingredient, the remainder comprising an orally dissolvable and / or disintegrable composition, and optionally one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized and / or aerosolized formulations, when dispersed, may have an average particle size and / or droplet size in the range of about 0.1 nm to about 200 nm, and may further comprise one or more of any of the additional ingredients described herein. The pharmaceutical composition may be prepared, packaged, and / or sold as a formulation suitable for ocular administration. Such a formulation may be, for example, in the form of eye drops, including, for example, a 0.1 / 1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such drops may further include buffering agents, salts, and / or one or more other of any of the additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those that contain the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or eye drops are considered within the scope of the present invention. General considerations in the formulation and / or manufacture of pharmaceutical agents are discussed, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (hereby incorporated by reference).

[0227] Administration The present invention provides methods comprising administering to a subject in need thereof a composition of a supercharged protein associated with a functional effector protein. In some embodiments, methods are provided for administering a composition comprising another functional effector protein (e.g., a Cas9 protein) and a cationic lipid and / or cationic polymer. Such compositions may be administered to a subject in any amount and using any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and / or condition. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions according to the present invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend on a variety of factors. These include the disorder to be treated and the severity of the disorder, the activity of the particular compound used, the particular composition used, the age, weight, general health, sex, and diet of the patient, the time of administration, the route of administration, and the rate of excretion of the particular compound used, the length of treatment, drugs used in combination or concomitantly with the particular compound used, and similar factors well known in the medical arts. Compositions of supercharged proteins linked to functional effector proteins to be delivered, including compositions comprising, for example, a Cas9 protein and a cationic lipid, can be administered by any route. In some embodiments, such compositions are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intraspinal, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or drops), mucosal, nasal, buccal, enteral, vitreous, intratumoral, sublingual, intratracheal instillation, intrabronchial instillation, and / or by inhalation, as an oral spray, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, the supercharged protein or complex, and / or pharmaceutical, prophylactic, diagnostic, or imaging composition thereof, is administered by systemic intravenous injection. In certain embodiments, the supercharged protein or complex and / or pharmaceutical, prophylactic, diagnostic, or imaging composition thereof may be administered intravenously and / or orally, hi certain embodiments, such compositions may be administered in a manner that allows functional effector proteins to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[0228] In certain embodiments, compositions in accordance with the invention may be administered once or twice daily at a dosage level sufficient to deliver a functional effector protein amount of about 0.0001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg of subject body weight per day to achieve the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times daily, twice daily, once daily, every other day, every third day, weekly, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more administrations). Compositions comprising supercharged proteins linked to functional effector proteins can be administered in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By "in combination with," it is not intended that the agents must be formulated for simultaneous administration and / or delivery together, although these methods of delivery are within the scope of the present invention. The compositions can be administered concurrently with, prior to, or after one or more other desired therapeutic agents or procedures. Generally, each agent will be administered at a dose and / or time schedule determined for that agent. In some embodiments, the present invention encompasses delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and / or alter their metabolism, inhibit their excretion, and / or alter their biodistribution.

[0229] kit The present invention provides various kits for conveniently and / or effectively carrying out the methods of the present invention. Typically, the kits will contain sufficient quantities and / or numbers of components to allow a user to perform multiple treatments of a subject(s) and / or conduct multiple experiments. In some embodiments, the kits include one or more instructions for formulating a composition comprising (i) a supercharged protein described herein, (ii) a functional effector protein to be delivered, (ii) cationic lipids and / or cationic polymers, and (iv) a functional protein associated with the supercharged protein. In some embodiments, the kits include a Cas9 protein and a cationic lipid. In some embodiments, the kits include a nucleic acid encoding the supercharged protein and / or the functional protein to be delivered. In some embodiments, the kits include a cloning vector encoding the supercharged protein and a cloning site (allowing in-frame cloning of a functional effector protein to generate a fusion protein). In some embodiments, the kit comprises a pharmaceutical composition provided herein comprising a supercharged protein associated with a functional effector protein, a syringe, needle, or applicator for administering the pharmaceutical composition to a subject, and instructions for administering the pharmaceutical composition to a subject. These and other aspects of the present invention will be further appreciated by consideration of the following examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, which is defined by the claims. [Example]

[0230] Example 1: Delivery of TALE activators fused to supercharged GFP A key target for reprogramming fibroblast cell fate toward brown or white adipocytes is the switch from white adipose tissue (WAT) to brown adipose tissue (BAT), which is governed by the expression of PRDM16 and PPARγ. We engineered robust TALE transcription activators fused to +36 GFP to target PPARγ and PRDM16 genomic sequences in fibroblasts. The fusion proteins were purified using a heparin column and / or SEC. The gel showed a single band at 130 kD. The modulation of expression and effects on cell phenotype after delivery of the TALE activators were compared with that after viral delivery of PPARγ cDNA followed by 7 days of treatment with an adipogenic cocktail. We observed that adipocytes formed after treatment with the +36 GFP-TALE-PRDM16 fusion. Expression of white adipose tissue marker genes was detected after delivery of the supercharged PRDM16-TALE activator.

[0231] One-time supercharged protein-mediated delivery of a TALE activator to PPARγ was found to induce white adipose gene expression and differentiate fibroblasts into white adipocytes. Supercharged protein-mediated delivery of both PPARγ and PRDM16-TALE activators induced adipocyte differentiation with increased expression of brown adipose markers (e.g., PRDM16, cox8b, elov13, and cidea), as well as smaller increases in the thermogenic gene expression markers PGC1a and UCP1.

[0232] The Aurein peptide was fused to the N-terminus of the +36GFP-TALE activator fusion protein. Delivery of the Aurein-+36GFP-TALE purified through a heparin column was monitored by detecting fluorescence in the nuclei of treated cells.

[0233] Figure 1 shows a schematic diagram of macromolecule delivery into mammalian cells. Figure 2 shows a schematic diagram of white adipose tissue (WAT) to brown adipose tissue (BAT) switching. Figure 3 shows a schematic diagram of a supercharged delivery platform for delivering TALE activators programmed to target PPARγ or PRDM16. Figure 4 shows a schematic diagram of a fusion protein containing a +36GFP fusion, an 18.5mer TALE domain, and a VP64 activation domain. Figure 5 shows the expression and purification of a +36GFP-TALE activator fusion protein. Figure 6 shows a test assay for adipocyte regulatory gene activation by delivery of +36GFP-PPARγ and PRDM16-TALE activator fusion proteins.

[0234] Figure 7 shows the delivery efficacy of +36GFP-TALE activator fusion proteins at different concentrations. Figure 8 shows a comparison of the delivery efficacy of two different +36GFP-PRDM16-TALE fusion proteins in NIH-3T3 cells. Figure 9 shows PPARγ gene expression after delivery of PPARγ-TALE activator fusions compared to various controls. Figure 10 shows PRDM16 gene expression after delivery of RDM16-TALE activator fusions compared to various controls. Figure 11 shows moderate TALE activity observed in the presence of serum.

[0235] Figure 12 shows validation of viral delivery of PPARγ followed by 7 days of treatment with an adipogenic cocktail. Figure 13 shows a schematic diagram of the assay for programming fibroblasts into WAT and BAT. Figure 14 shows adipocyte formation observed with treatment with +36GFP-TALE activator fusion protein. Figure 15 shows staining of various treatments with LipidTOX red after 7 days, demonstrating adipocyte formation after viral delivery and delivery of a supercharged PPARγ-TALE activator fusion protein. Figure 16 shows staining of cells with LipidTOX red after 7 days of treatment, demonstrating adipocyte formation after viral delivery and delivery of a supercharged PPARγ-TALE activator fusion protein. Figure 17 shows WAT biomarker gene expression after viral delivery and delivery of a supercharged PPARγ-TALE activator fusion protein.

[0236] Example 2: In vivo delivery of TALE activators fused to supercharged GFP NIH-3T3 cells were grown to 70-90% confluence and treated with 1 μM or 0.5-5 μM of +36GFP-PPARγ-TALE and / or +36GFP-PRDM16-TALE fusion proteins in serum-free DMEM. Serum-free medium was chosen because serum can reduce the efficacy of protein-based delivery. After incubation of cells with the respective fusion protein solutions for 4 hours, the medium was removed and serum-containing complete DMEM was replaced. Control cells were infected with viral constructs encoding PPARγ or PRDM16 to serve as positive controls for WAT and BAT gene expression, according to published protocols (see, e.g., Seale et al. 2008 Nature 454, 961-967, the entire contents of which are incorporated herein by reference). Once all cells reached 100% confluence, an adipogenic cocktail containing isobutylmethylxanthine, insulin, rosiglitazone, dexamethosone, T3, and indomethacin was added to the cells, and 48 hours later, this was replaced with a cocktail containing only insulin, T3, and rosiglitazone. Forty-eight hours after this second cocktail change, another dose of T3, insulin, and rosiglitazone was added to the cells. The next day (which was now 1 week from the start of the experiment), cells were harvested using TRIzol, total RNA was extracted, and qRT-PCR was performed to measure gene expression levels of PPARγ, PRDM16, and other brown fat marker genes (e.g., UCP1, PGC1a, Elov13, and Cidea).

[0237] Figure 18 shows the delivery of supercharged PRDM16-TALE activator fusion proteins to induce brown fat adipogenesis in vivo. Robust adipogenesis was observed after viral delivery of PPARγ and PRDM16, as well as after delivery of supercharged TALE activator protein fusions. Figure 19 shows a comparison of supercharged (TALE) and viral delivery of PPARγ and PRDM16 into cells. The figure shows TALE / TALE, virus / TALE, and virus / virus-induced expression of brown fat markers due to PPARγ and PRDM16 expression. Figure 20 shows RT-qPCR assessment consistent with adipocyte differentiation, which was also observed by LipidTOX staining.

[0238] Example 3: Delivery of TALE activators complexed with supercharged GFP To improve delivery efficiency, protein complexes were generated in which functional proteins were non-covalently linked to supercharged proteins and administered to cells. Figure 21 shows that delivery of functional TALE activator fusion proteins as complexes with +36GFP improved TALE activator activity after delivery. Figure 22 shows PRDM16 gene expression after delivery of TALE activator fusions either as a fusion (+36GFP-PRDM16-TALE-3) or as a complex with +36GFP (+36GFP+PRDM16-TALE-3). It was observed that delivery of the complex tended to increase TALE activator activity.

[0239] Example 4: Effect of Aurein fusion on delivery efficacy Figure 23 shows the effect of N-terminal Aurein peptide fusion to +36GFP on PRDM16 gene expression after TALE activator fusion delivery (either as a fusion or complex with +36GFP). The Aurein peptide was fused to the N-terminus of the GFP-TALE construct via a GGS(9) (SEQ ID NO: 252) linker, resulting in the Aurein peptide-GGS(9) linker-(+)36GFP protein-GGS(9) linker-PRDM16-TALE-3 fusion protein. The protein was purified using size-exclusion chromatography.

[0240] Example 5: Delivery of TALE activators complexed with supercharged GFP or cationic lipids Figure 24 shows PRDM16 gene expression after delivery of TALE-PRDM16 activator protein either as a fusion with +36GFP (+36GFP-PRDM16-TALE-3), a complex with +36GFP (+36GFP+PRDM16-TALE-3), or a complex with Lipofectamine LTX, where increased gene expression was observed.

[0241] Example 6: Delivery of Cas9 fused to supercharged GFP Supercharged delivery of Cas9 into mammalian cells will enable the application of potent RNA-programmable nuclease technology to cells without the drawbacks of previous delivery methods. To this end, we generated a Cas9 fusion with +36GFP using an ALAL linker. Figure 25 shows a schematic diagram of the supercharged fusion protein with Cas9. Figure 26 shows the purification of wild-type Cas9 protein and a Cas9 fusion protein with +36GFP and Aurein-GGS9. The fusion protein is administered to cells in the same manner as the TALE activator fusion protein above. Once delivered to cells, Cas9 binds to and cleaves its target site in the cellular genome. Nuclease activity within the target cells is detected by a suitable assay (e.g., Southern blot or sequencing assay).

[0242] Example 7: Efficient delivery of genome editing proteins in vitro and in vivo Efficient intracellular delivery of proteins to the nucleus or cytoplasm is required to fully realize the potential of protein therapeutics, including genome-editing agents. Current methods of protein delivery often suffer from poor tolerance to serum proteins, poor endosomal escape, and limited in vivo efficacy. As demonstrated in this example, common cationic lipid reagents originally developed for nucleic acid transfection can potently deliver proteins fused to negatively charged proteins, containing natural anionic domains, or that naturally bind to anionic nucleic acids. This approach mediates functional delivery of Cre recombinase, TALE and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells at low nanomolar concentrations in medium containing 10% serum. Lipid-based delivery can be >1,000-fold more potent than cationic protein delivery strategies. Delivery of the Cas9:sgRNA complex resulted in genome modification efficiencies as high as 80% with substantially higher specificity compared to standard DNA transfection. This is likely due to the transient nature of the delivered Cas9:sgRNA complex. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes to the mouse inner ear in vivo, achieving up to 90% Cre-mediated recombination and up to 20% Cas9-mediated genome modification in the targeted hair cell population.

[0243] Materials and methods Construction of Cas9, Cre, and TALE fusion and sgRNA expression plasmids The sequences of all constructs used in this paper are listed below or provided elsewhere in the specification. All protein constructs were generated from previously reported plasmids of the protein of interest cloned into the pET29a expression plasmid.

[0244] Expression and purification of Cas9 and other proteins of S. pyogenes E. coli BL21 STAR(DE3) competent cells (Life Technologies) were transfected with pMJ806, which encodes S. pyogenes Cas9 fused to an N-terminal 10xHis tag / maltose binding protein. 47 The resulting expression strain was inoculated into Luria-Bertani (LB) broth containing 100 μg / mL of ampicillin overnight at 37 °C. The cells were diluted 1:100 in the same growth medium and grown at OD 600 The cells were grown at 37°C until the RI = approximately 0.6. The culture was incubated at 20°C for 30 min, and isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at 0.5 mM to induce Cas9 expression. After approximately 16 h, the cells were harvested by centrifugation at 8,000 g and resuspended in lysis buffer (50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl (pH 8.0), 1 M NaCl, 20% glycerol, 10 mM tris(2-carboxyethyl)phosphine (TCEP)). The cells were lysed by sonication (6 W output, 1 sec pulse on, 1 sec pulse off for a total of 15 min), and the soluble lysate was obtained by centrifugation at 20,000 g for 30 min.

[0245] The cell lysate was incubated with HisPur nickel-nitriloacetic acid (nickel-NTA) resin (Thermo Scientific) at 4°C for 30 minutes to capture His-tagged Cas9. The resin was transferred to a 20 mL column and washed with 20 column volumes of lysis buffer. Cas9 was eluted in 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, 10 mM TCEP, and 300 mM imidazole and concentrated to approximately 50 mg / mL using an Amicon ultracentrifugal filter (Millipore, 100 kDa molecular weight cutoff). The 6xHis tag and maltose-binding protein were removed by TEV protease treatment for 20 hours at 4°C and captured by a second Ni-affinity purification step. The eluate containing Cas9 was injected onto a Hi-Trap SP HP column (GE Healthcare) in a purification buffer containing 50 mM Tris-HCl (pH 8), 0.1 M NaCl, 20% glycerol, and 10 mM TCEP. Cas9 was eluted with a linear NaCl gradient from 0.1 M to 1 M over five column volumes. The elution fraction containing Cas9 was concentrated to a concentration of 200 μM, which was quantified by bicinchoninic acid assay (BCA) (Pierce Biotechnology). The fractions were flash-frozen in liquid nitrogen and stored in aliquots at -80°C. All other proteins were purified by this method without the TEV cleavage step. Proteins containing (-30)GFP were purified by anion exchange using a Hi-Trap Q HP anion exchange column (GE Healthcare) using the same purification protocol.

[0246] In vitro transcription of sgRNA A linear DNA fragment containing a T7 promoter binding site followed by a 20-bp sgRNA target sequence was in vitro transcribed using the T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's instructions. The in vitro transcribed RNA was precipitated with ethanol and purified by gel electrophoresis on a Criterion 10% polyacrylamide TBE-urea gel (Bio-Rad). The excised gel fragment was extracted in 420 μL of 300 mM NaCl overnight on a shaking surface at 4°C. The gel-purified sgRNA was precipitated with ethanol, redissolved in water, and the sgRNA concentration was finally quantified by UV absorbance and flash-frozen at -80°C.

[0247] Plasmid transfection Plasmid DNA was transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol. For the TALE activator plasmids, 300 ng of DNA was transfected. For activator synergy experiments, 60 ng of each of the five plasmids was pooled and transfected. For Cas9 nuclease delivery experiments, linear DNA PCR products expressing sgRNAs targeting genomic sites in CLTA, EMX, VEGF, and GFP (sgRNAs GFPg1, GFPg3, GFPg5, and GFPg7 for nickase studies) were used in transfection experiments. The linear DNA PCR products were generated using a plasmid containing a U6 promoter as a template and a forward primer (bearing the U6 promoter upstream sequence) and a reverse primer (containing the U6 downstream sequence followed by the sgRNA sequence (a 20-bp sequence unique to each target plus the constant sgRNA backbone structural sequence)). The sgRNAs expressed from linear DNA templates contained at least two 5' guanosines matching those required for T7 transcription in vitro. Primer sequences and PCR conditions are listed below. For dCas9 activator experiments, 700 ng of Cas9 or dCas9-VP64 plasmid DNA was co-transfected with 250 ng of the appropriate sgRNA expression plasmid. For activator synergy experiments, 50 ng of DNA from each of the six sgRNAs was pooled and co-transfected with 700 ng of dCas9-VP64 plasmid.

[0248] Delivery of transcription factor proteins complexed with cationic lipids in cell culture. A more in-depth description of both in vitro and in vivo genome editing protein delivery can be found below. Briefly, cultured cells were plated in a 48-well format (250 μL volume) in Dulbecco's Modified Eagle's Medium + GlutaMAX (Life Technologies, Carlsbad, CA) with 10% FBS ("full serum medium") and antibiotics at the cell density required to reach approximately 70% confluence the next day. The full serum medium was replaced with the same medium but without antibiotics 1 h prior to delivery. Cre and TALE protein delivery was performed by combining 0.5–1.5 μL of commercially available cationic lipid with 1 nM–1 μM protein in 25 μL of OPTIMEM medium (Life Technologies, Carlsbad, CA) (in a final volume of 275 μL), following the manufacturer's protocol for normal plasmid transfection (including incubation times). For in vitro Cas9 delivery, the transcribed sgRNA was complexed with a cationic lipid reagent after a 5-minute incubation with the Cas9 protein. 25 μL of the lipid complex in OPTIMEM medium was added to cells, and unless otherwise noted, the medium was replaced with fresh medium 12–16 hours later. Cells were assayed for recombination 48 hours after delivery, for gene activation either 4 or 16 hours after delivery, and for gene modification 72 hours after delivery.

[0249] T7 endonuclease I assay for detecting genome modifications U2OS-EGFP or HEK293T cells were transfected with Cas9 and sgRNA expression plasmids or linear DNA PCR products (as described above), or treated with Cas9 protein alone, in vitro transcribed sgRNA alone, or RNAiMAX alone. Genomic DNA was isolated from cells two days after transfection using the DNAdvance kit (Agencourt) according to the manufacturer's instructions. 200 ng of genomic DNA was used as a template in PCR reactions to amplify the targeted genomic locus using the flanking survey primer pairs defined below. PCR products were purified using a QIAquick PCR Purification Kit (Qiagen) and quantified using the Quant-iT™ PicoGreen® dsDNA Kit (Life Technologies). 250 ng of purified PCR DNA was combined with 2 μL of NEBuffer 2 (NEB) to a total volume of 19 μL and denatured by thermocycling at 95°C for 5 min, 95 to 85°C at 2°C / s, and 85 to 20°C at 0.2°C / s, followed by reannealing. The reannealed DNA was incubated with 1 μL of T7 endonuclease I (10 U / μL, NEB) for 15 min at 37°C. Ten μL of 50% glycerol was added to the T7 endonuclease reaction, and 12 μL was analyzed on an 18-well Criterion PAGE gel (Bio-Rad) in 5% TBE (electrophoresis at 200 V for 30 min, followed by staining with 1× SYBR Gold (Life Technologies) for 30 min). Cas9-induced cleaved and uncleaved bands were visualized using an AlphaImager HP (Alpha Innotech) and quantified using ImageJ software. 54 The peak intensity of the cleaved band was divided by the total intensity of all bands (uncleaved + cleaved bands) to determine the cleaved fraction, which was used to estimate the genetic modification level as previously described. 46For each sample, transfection and subsequent modification measurements were performed in triplicate on different days.

[0250] Stem Cell Culture and Delivery A mouse embryonic stem cell (ES) line, Tau-GFP, containing a permanent GFP gene insertion was cultured in DMEM with 15% FBS (Gibco), 100 mM MEM non-essential amino acids (Gibco), 0.55 mM 2-mercaptoethanol, and leukemia inhibitory factor (1,000 units / ml, Chemicon). After 5 days, floating spheres exhibiting GFP fluorescence were formed. Cas9:sgRNA and RNAiMAX complexes were added to the culture containing the floating spheres for 16 hours. After Cas9:sgRNA treatment, the cells were cultured in the above medium for 3 days. The floating spheres were treated with trypsin for 5 minutes and then passed through a 70 μm filter to collect single cells. Cells were cultured on laminin-coated slides in DMEM / F12 (1:1) supplemented with 1x N2, 1x B27, penicillin-streptomycin (100 μg / mL), and 10% FBS for 2 days before labeling. Immunohistochemistry was performed using an anti-GFP antibody (#ab13970, Abcam) to assess GFP expression. To quantify the number of GFP-negative cells, we counted the total number of GFP-positive and GFP-negative cells from three representative fields at 20x magnification and calculated the average efficiency. Three independent experiments were performed for each condition.

[0251] Microinjection of proteins into the mouse inner ear P0 floxP-tdTomato mice were used for (-30)GFP-Cre injections, and P2 Atoh1-GFP mice were used for Cas9:sgRNA injections. Animals were used under a protocol approved by the Massachusetts Eye & Ear Infirmary ALCUC committee. Mice were anesthetized by lowering their temperature on ice. A cochleostomy was performed by making an incision behind the pinna to expose the otic bulla. A glass micropipette supported by a micromanipulator was used to deliver the complex to the scala media, allowing access to the inner ear hair cells. For (-30)GFP-Cre delivery, 3 μL of 45 μM protein was mixed with 3 μL of either RNAiMAX or Lipofectamine 2000 and incubated at room temperature for 30 minutes prior to injection. Four mice were injected per treatment group. For delivery of the Cas9:sgRNA complex, 1 μL of 200 μM Cas9 protein was mixed with 2 μL of 100 μM sgRNA and incubated at room temperature for 5 minutes. Then, 3 μL of either RNAiMAX or Lipofectamine 2000 was added and incubated for an additional 30 minutes prior to injection. Three mice were injected per treatment group. The total delivered volume for each injection was 0.3 μL per cochlea, and release was controlled by a micromanipulator at a rate of 32 nL / sec.

[0252] Immunohistochemistry and quantification Five to ten days after injection, mice were sacrificed and cochleae were collected according to standard protocols. 55 For immunohistochemistry, antibodies against hair cell markers (Myo7a and Esp) and supporting cell markers (Sox2) were used according to previously described protocols. 55To quantify the number of tdTomato-positive cells after (-30)GFP-Cre or GFP-negative cells after Cas9:sgRNA delivery, we counted the total number of outer hair cells within a 200 μm area around the injection site in the basal whorl of the cochlea. The efficiency of (-30)GFP-Cre-induced recombination or Cas9:sgRNA-induced genomic modification was calculated as the percentage of outer hair cells that expressed tdTomato or lost GFP expression.

[0253] High-throughput DNA sequencing of genome modifications HEK293T cells were either transfected with Cas9 and sgRNA expression plasmids or linear DNA PCR products, or treated with 50 nM Cas9 protein, 250 nM purified sgRNA, and cationic lipids (as previously described for Cas9 protein delivery into U2OS-EGFP reporter cells). For plasmid-based transfection experiments, 700 ng of Cas9 expression plasmid plus 250 ng of sgRNA plasmid or 50 ng of linear DNA PCR product (expressing sgRNAs targeting either the EMX1, CLTA2, or VEGF loci) was transfected using Lipofectamine 2000 (Life Technologies), and cells were isolated 2 days later. For in vivo protein delivery experiments, approximately 30 mg of mouse tissue was isolated from anesthetized mice as previously described. 55Genomic DNA was extracted using the Agencourt DNA Advance Genomic DNA Isolation Kit (Beckman Coulter). For cell culture experiments, genomic DNA was isolated as described above. Using 150 ng of genomic DNA as a template, on-target and off-target genomic sites were amplified by PCR using the flanking HTS primer pairs defined below. The relative amounts of crude PCR products were quantified by gel electrophoresis, and samples treated with different sgRNA pairs or Cas9 nuclease types were separately pooled at equimolar concentrations before purification with a QIAquick PCR Purification Kit (Qiagen). Approximately 150 ng of pooled DNA was electrophoresed on an 18-well Criterion PAGE gel (BioRad) in 5% TBE at 200 V for 30 min, and DNA ranging in length from approximately 125 bp to approximately 300 bp was isolated and purified with a QIAquick PCR Purification Kit (Qiagen). The purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced by a MiSeq high-throughput DNA sequencer (Illumina) as previously described. 47 .

[0254] Data analysis Illumina sequencing reads were filtered and parsed by a script written in Unix Bash, as outlined below. Sample size for sequencing experiments was maximized (within realistic experimental considerations) to ensure maximum statistical power for detecting effects. Statistical analysis of Cas9-modified genomic sites (Table 2) was performed as previously described, with multiple comparison correction using the Bonferroni method. 56 .

[0255] Below is a list of the upstream and downstream flanking sequences for each genomic target site. [Table 2]

[0256] Primers to generate a PCR product that serves as a substrate for T7 transcription of the sgRNA. T7_gRNA-Rev was used in all cases. The DNA template used was the EGFP sgRNA plasmid, as noted above. The NTF3 and VEGF sgRNAs for the dCas9-VP64 activator experiments were previously reported (Maeder et al., CRISPR RNA-guided activation of endogenous human genes. Nat. Methods. 2013;10, 977-979). [Table 3]

[0257] Primers for generating linear DNA PCR products for transfection PCR extension at (72°C, 3 min) of a plasmid containing the U6 promoter as a template with PCR_sgRNA-fwd1, PCR_sgRNA-rev2, and the appropriate PCR_sgRNA primers listed below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0258] result Highly efficient delivery of Cre recombinase fused to a highly negatively charged protein It has been speculated that imparting the highly anionic electrostatic properties of nucleic acids to genome editing proteins may enable their efficient delivery into mammalian cells using cationic lipids (Figure 27(A)). For proteins of interest that are not naturally highly negatively charged, fusion with natural or engineered ultranegatively charged proteins may be feasible. 17 It was speculated that for nucleic acid binding proteins, simple complexation with natural DNA or RNA substrates might provide sufficient anionic character to support cationic lipid-based delivery (Figure 27(A)).

[0259] Engineered hypernegatively charged GFP variants 35 We first tested whether (-30)GFP could mediate the encapsulation and delivery of fused protein cargo (Figure 27(B)). We fused (-30)GFP to Cre recombinase and tested several commercially available cationic lipids for their ability to functionally deliver the fusion into HeLa cells, which express DsRed only upon Cre-mediated recombination (Figure 28(A)). Delivery of 10 nM (-30)GFP-Cre complexed with 1.5 μL of Lipofectamine RNAiMAX (hereafter referred to as "RNAiMAX"; Life Technologies, Carlsbad, CA) in medium containing 10% fetal bovine serum (FBS) led to a strong DsRed fluorescent signal in the treated cells. Fluorescence-activated cell sorting (FACS) revealed that 52% of the cells expressed DsRed after 48 hours of treatment, consistent with Cre recombination (Figure 28(B)).

[0260] Optimization resulted in a 65% recombination efficiency using 25 nM (-30)GFP-Cre complexed with 1.5 μL of RNAiMAX in 250 μL of medium containing 10% FBS (Figure 28(C)). The efficacy of lipid-mediated anionic Cre delivery is striking compared to that of delivery mediated by cationic proteins. Only 1 nM (-30)GFP-Cre and cationic lipid were required to yield 15-20% recombined cells. In contrast, 1 μM (+36)GFP-Cre was required to achieve this degree of recombination, representing a 1,000-fold difference in delivery efficacy (Figure 28(C)). Nearly identical results were observed in a second Cre reporter cell line (BSR TdTomato) (Figure 33(A)). Increasing the amount of cationic lipid increased toxicity (Figure 33(B)), and it was found that 1.5 μL of RNAiMAX per 250 μL sample maximized recombination efficiency while inducing minimal cytotoxicity. Under these conditions, cationic lipids did not increase the delivery efficacy of neutral or cationic Cre recombinase fusions (Figure 28(C) and Figure 33(C)). This indicates that the strong negative charge of (-30)GFP-Cre is required to participate in cationic lipid-mediated delivery. It was also observed that increasing the amount of cationic lipid increased the protein concentration required for maximal recombination. This is consistent with a model in which deliverable proteins are complexed with specific cationic lipid stoichiometry (Figure 28(D)). Taken together, these observations indicate that cationic lipids can mediate potent delivery of polyanionic proteins into mammalian cells, even in the presence of serum.

[0261] To determine whether the higher efficacy of cationic lipid-mediated (-30)GFP-Cre delivery relative to delivery mediated by cationic proteins is due to greater total protein uptake by cells or a higher fraction of functional non-endosomal protein molecules entering cells, we measured cellular GFP fluorescence using flow cytometry. Cells were treated with either (+36)GFP-Cre or liposomal (-30)GFP-Cre at their respective optimal Cre delivery conditions. Comparison of intracellular fluorescence and recombination efficiency revealed that lipid-mediated functional delivery of (-30)GFP-Cre was 9,800-fold more potent per amount of endocytosed protein than delivery of (+36)GFP-Cre (Figure 34). Together, these results suggest that the unusually high efficacy of lipid-mediated delivery of anionic proteins is due not to abnormally high protein uptake by each cell, but rather to post-endocytic processes likely involving endosomal escape into the cytoplasm and avoidance of lysosomal proteolysis.

[0262] To test whether the ability to deliver polyanionic proteins is dependent on the unique composition of RNAiMAX or whether other cationic lipids can similarly mediate strong delivery, several other transfection reagents designed to deliver nucleic acids were tested (Figure 28(E)). While RNAiMAX remained the most effective functional delivery agent for (-30)GFP-Cre, other cationic lipid formulations also resulted in strong delivery. Lipofectamine 2000 and Lipofectamine LTX (Life Technologies, Carlsbad, CA), two plasmid transfection reagents based on cationic lipid formulations, were tested. 21), and SAINT-Red (Synvolux Therapeutics, Groningen, Netherlands), an siRNA delivery formulation containing a synthetic pyridinium-containing cationic lipid, all resulted in robust and functional (-30)GFP-Cre delivery over a wide range of concentrations (Figure 28(E)). In contrast, robust delivery was not observed with the cationic lipid DOTAP (Roche Diagnostics, Indianapolis, IN) or the peptide-based nucleic acid delivery agent EZ-PLEX (Ascension Bio, Tampa, FL) (Figure 28(E)). These observations, taken together, indicate that some (but not all) cationic lipids are capable of encapsulating and delivering negatively charged proteins into human cells.

[0263] It was speculated that it should be possible to use cationic lipids to deliver polyanionic proteins other than (-30)GFP. Engineered polyanionic protein domains commonly used in biomedical research include the VP64 activation domain (net theoretical charge of -22), widely used in engineered zinc finger arrays, TALE repeat arrays, or fusions with dCas9 for transcriptional activation, and 3xFLAG (net theoretical charge of -7), an epitope tag used for protein purification and visualization (Figure 28(F)). We observed that both VP64 and 3xFLAG improved functional delivery of Cre recombinase by cationic lipids, although not as effectively as (-30)GFP. This is likely due to their lower overall negative charge (Figure 33(C)). These observations demonstrate that unusually negatively charged proteins other than (-30)GFP can mediate efficient cationic lipid-based delivery into mammalian cells.

[0264] Functional delivery of TALE activator proteins Lipid-mediated delivery of the TALE-VP64 transcriptional activator (with a theoretical net charge of approximately +4, depending on the TALE variant used) into cultured human cells was tested. Although slightly more effective cleavage of endogenous genes by delivered TALEN proteins has been demonstrated in mammalian cells in the absence of serum using cationic peptides such as Arg9. 36 However, TALE-based delivery of transcription factor proteins has not yet been reported, and to the inventors' knowledge, effective delivery of TALE proteins in serum has not been previously described. Targeting the gene encoding neurotrophin-3 (NTF3), a nerve growth factor that has been linked to neurodegenerative diseases, was performed. 37 TALE-VP64, which targets NTF3, has been previously described. 38 (-30)GFP-NTF3-TALE1-VP64 (Figure 29(A)) was fused to (-30)GFP (Figure 29(A)). HEK293T cells were treated with 25 nM (-30)GFP-NTF3-TALE1-VP64 and RNAiMAX under conditions optimized for Cre delivery. After 4 hours of treatment, the gene expression level of NTF3 was 3.5-fold higher in cells treated with 25 nM (-30)GFP-NTF3-TALE1-VP64 and RNAiMAX than in untreated cells, cells treated with RNAiMAX alone, or cells treated with a TALE transcription activator targeting VEGF (Figure 29(B)). Similar levels of NTF3 expression were observed 48 hours after transfection with a plasmid encoding the same NTF3-targeting TALE-VP64 (Figure 29(B)).

[0265] Synergistic expression of multiple TALE activators targeting different sites on the same gene has been shown to enhance gene activation. 38Five distinct NTF3-targeting TALE activators fused to (-30)GFP were delivered simultaneously using RNAiMAX. Protein-lipid complexes were prepared as above by adding five (-30)GFP-NTF3-TALE-VP64 proteins at 5 nM each for a total of 25 nM protein. A 6.5-fold increase in NTF3 expression was observed after 4 hours of incubation (Figure 29(B) and Figure 35), while plasmid cotransfection of all five NTF3-TALE activators followed by 48 hours of incubation resulted in a 10-fold increase in NTF3 expression levels (Figure 29(B)). These findings demonstrate that TALE activator proteins can be delivered using cationic lipids to transiently activate gene expression in human cells. Delivery of programmable transcription activator proteins can enable one-time activation of target genes while avoiding chronic gene expression (a common challenge with DNA-based delivery of programmable transcription factors). This ability may be particularly valuable for proteins that, when transiently expressed, effect a one-time, permanent change in cell state or cell fate. 39 .

[0266] Highly efficient delivery of Cas9:sgRNA protein:RNA complexes into human cells The robust lipid-mediated delivery of polyanionic Cre and TALE activator protein variants in full serum medium suggests that CRISPR-Cas9:sgRNA complexes, either as fusions with (-30)GFP or as native polyanionic Cas9:guide RNA complexes, may also be delivered to human cells using this approach. Well-established Cas9-induced gene disruption assays 40was used to target a specific site in the genomic EGFP reporter gene in human U2OS cells (Figure 36(A)). On-target Cas9 cleavage induces non-homologous end joining (NHEJ) within EGFP and loss of cellular fluorescence. To avoid interference from the fluorescence of (-30)GFP, a Y67S mutation was introduced into (-30)GFP to eliminate its fluorescence, and this non-fluorescent variant was named (-30)dGFP.

[0267] Treatment of U2OS reporter cells by RNAiMAX with 25 nM (-30)dGFP-NLS-Cas9 and 50 nM sgRNA targeting EGFP in medium containing 10% FBS showed loss of EGFP expression in 48% of the cells (Figure 30(A)). Co-transfection of plasmids expressing Cas9 or sgRNA resulted in similar EGFP loss in 37% of the cells (Figure 30(A)). Significant EGFP destruction was not observed by transfection of a plasmid encoding an sgRNA for EGFP alone, Cas9 alone, or co-transfection of a plasmid encoding Cas9 with an sgRNA designed to target the VEGF locus (Figure 30(A), Figure 36(B)). It was confirmed that the robust destruction of EGFP was not the result of cytotoxicity (Figures 36(C)-(D)). It was also observed that treatment of cells with (+36)dGFP-NLS-Cas9 and sgRNA in the presence of 10% FBS serum did not lead to efficient gene disruption (Figure 30(A)). This suggests that the cationic peptide-based method of delivery for Cas9 and sgRNA is ineffective, possibly due to interference of gRNA:Cas9 complex formation or nuclease function by highly positively charged proteins. 41 Together, these results establish that cationic lipid-mediated delivery of the (-30)dGFP-NLS-Cas9:sgRNA complex can result in efficient sgRNA-dependent targeted gene disruption in human cells.

[0268] Polyanionic sgRNAs are necessary and sufficient for efficient lipid-mediated Cas9 delivery Because the complex between the native Cas9 protein (net theoretical charge of +22) and sgRNA (approximately 10 anionic phosphate groups) should be highly anionic overall, we next tested whether the native Cas9:sgRNA complex, without fusion to a polyanionic protein, could be delivered into human cells using cationic lipids. Treatment of U2OS EGFP reporter cells with 100 nM Cas9, 100 nM EGFP sgRNA, and 0.8 μL RNAiMAX resulted in 65% disruption of the EGFP reporter gene (Figure 30(A)). Treatment of cells with Cas9 protein and sgRNA, but without RNAiMAX, did not result in loss of GFP fluorescence (Figure 30(A)). These observations suggest that sgRNA alone, even in the absence of a highly negatively charged fusion protein, can provide the highly anionic character required to mediate cationic lipid-based delivery of Cas9.

[0269] Comparison of the gene disruption efficiency resulting from cationic lipid-mediated delivery of (-30)dGFP-NLS-Cas9:sgRNA versus Cas9:sgRNA revealed that at low doses, (-30)dGFP-NLS-Cas9 resulted in more efficient gene disruption than native Cas9 (Figure 37(A)), while outperforming native Cas9 at higher concentrations, even at the respective optimal protein:sgRNA doses for both proteins (Figures 37(B)-37(C)). These results further establish that sgRNA can provide sufficient negative charge to support cationic lipid-based delivery of complexed Cas9 protein.

[0270] Although optimal delivery of (-30)dGFP-NLS-Cas9 requires less protein overall than Cas9, we also observed that maximal (-30)dGFP-NLS-Cas9-mediated EGFP gene disruption requires a higher sgRNA:protein ratio than native Cas9-mediated gene disruption (Figure 37(D)). We speculate that a greater equivalent of sgRNA is required to complex with (-30)dGFP-NLS-Cas9 because the fused (-30)dGFP may electrostatically interfere with Cas9:sgRNA complexation. Because the ideal protein dose for EGFP gene disruption mediated by (-30)dGFP-NLS-Cas9 is 10-fold lower than that of wild-type Cas9, the results also suggest that (-30)dGFP-Cas9 is better encapsulated by cationic liposomes than Cas9:sgRNA due to its higher overall negative charge. However, this charge magnitude may interfere with the Cas9:sgRNA interaction, requiring more sgRNA per protein and potentially reducing the total delivered Cas9 activity. In addition, NLS-Cas9 and Cas9-NLS proteins were generated and tested. While the presence of the NLS in (-30)dGFP-NLS-Cas9 may at least partially explain the difference in delivery efficiency at very low concentrations, we observed that Cas9, NLS-Cas9, and Cas9-NLS all resulted in higher efficiency of EGFP destruction than (-30)dGFP-NLS-Cas9 at concentrations of 25 nM or higher (Figure 38(A)-(C)).

[0271] Cas9:sgRNA delivery with cationic lipid formulations other than RNAiMAX was also tested. Delivery with Lipofectamine 2000 was significantly more efficient than RNAiMAX, resulting in up to 80% Cas9-mediated gene disruption (Figure 39(A)), and maintained high efficiency (60% gene disruption) even with 1 nM protein (Figure 39(A)). However, due to the slightly higher toxicity of Lipofectamine 2000 compared to RNAiMAX in cell culture conditions (Figures 33(B)-(C)), RNAiMAX was used for all subsequent cell culture studies.

[0272] To verify that EGFP disruption is due to genome modification rather than Cas9 binding alone 42 , T7 endonuclease I (T7EI) assay 43 We used the T7EI assay to detect and quantitate the frequency of Cas9-mediated genomic insertion / deletion mutations (indels) at the target EGFP locus (Figure 30(B)). Results from the T7EI assay indicated that only cells treated with both Cas9 and EGFP sgRNA plasmids, or Cas9 protein and purified EGFP sgRNA, contained indels at the target site. Together, these findings establish that active Cas9:sgRNA complexes can be potently delivered into human cells with cationic lipids in a manner dependent on the negative charge provided by the sgRNA.

[0273] U2OS EGFP reporter cells were also treated with a single lipid-mediated delivery of Cas9 complexed with a mixture of four gRNAs targeting EGFP, CLTA, EMX, and VEGF. This treatment resulted in efficient destruction of all four targets with cleavage efficiencies of 58%, 28%, 16%, and 40%, respectively (measured by T7EI cleavage assay). These high gene disruption efficiencies from a single delivery of 50 nM Cas9 and 25 nM of each sgRNA (100 nM total sgRNA) demonstrate that lipid-mediated Cas9:sgRNA delivery can support efficient multiplex genome editing (Figure 30(C)).

[0274] Functional delivery of Cas9 nickase and dCas9 activator Next, cationic lipid-based protein delivery will be extended to other Cas9-derived genome engineering tools, such as Cas9 nickase. 44 and Cas9-based transcriptional activators) could be delivered to the target cells. 45 Gene disruption efficiency in U2OS EGFP reporter cells resulting from delivery of Cas9D10A nickase was measured either by co-transfection of the nickase with the appropriate pair of EGFP-targeting sgRNA plasmids or as purified protein complexed with the EGFP sgRNA pair using RNAiMAX (Figure 30(D)). Both dual Cas9 nickase plasmid and cationic lipid-mediated protein:RNA delivery resulted in EGFP disruption with similar efficiency only in the presence of sgRNA pairs targeting opposite strands (sgRNA pairs g1 + g5 and g3 + g7) (Figure 30(D)), but not in sgRNA pairs targeting the same strand (sgRNA pair g5 + g7) (Figure 30(D)). This is consistent with previous reports of the requirement for Cas9 nickase cleavage. 46 .

[0275] We also compared the efficiency of NTF3 transcriptional activation in HEK293T cells resulting from either plasmid transfection of dCas9 fused to the VP64 activation domain or direct protein:sgRNA complex delivery. 45 Delivery of the dCas9-VP64 activator by either plasmid transfection or RNAiMAX-mediated protein delivery resulted in strong (>10-fold) activation of NTF3 transcription (Figure 30(E) and Figure 40). The level of transcriptional activation resulting from plasmid transfection was stronger than that resulting from protein delivery at the optimal assay time for each delivery method (Figure 30(E)). This is likely due to sustained expression of both the Cas9 activator protein and sgRNA from the plasmid compared with transient single doses of purified protein and RNA. The above results indicate that such factors do not limit the efficacy of irreversible genome modification by delivered Cas9 nuclease and nickase proteins (Figures 40(A) and 40(D)). However, the low dose and transient nature of delivered proteins may more strongly limit the efficacy of dynamic processes such as transcriptional activation. Nevertheless, these results collectively demonstrate that both the Cas9 nickase and the Cas9 transcriptional activator can be effectively delivered by cationic lipid-mediated protein:RNA complex delivery.

[0276] Cas9:sgRNA delivery alters genomes with higher specificity than DNA transfection DNA-free delivery of functional Cas9:sgRNA complexes avoids the risks associated with viral or other gene delivery methods and has the potential to improve the specificity of genome modification by avoiding unnecessary expression of the genome editing agent after the target locus has been modified. To test whether the described approach can disrupt endogenous genes in human cells, genomic loci within the EMX1, CLTA2, and VEGF genes were targeted. This is due to their potential biomedical relevance and their use in previous studies of Cas9 off-target cleavage activity. 40、46、47 Cationic lipid-mediated delivery of Cas9:sgRNA complexes into HEK293T cells resulted in robust cleavage of all three human genes, with efficiencies comparable to or higher than those of the plasmid transfection method. This was demonstrated by a T7EI assay using the same Cas9:sgRNA delivery conditions previously optimized for U2OS cells (Figure 31(A)).

[0277] To compare the endogenous gene modification specificity of plasmid versus protein:RNA delivery methods for Cas9, we amplified the on-target locus, as well as several known off-target sites, from genomic DNA isolated from HEK293 cells (Figure 44). Cells were treated with either transfection of Cas9 and sgRNA expression plasmids or RNAiMAX-mediated delivery of Cas9:sgRNA complexes under conditions that resulted in comparable on-target modification efficiencies. Indel frequencies at three on-target and 11 off-target sites were assayed by high-throughput DNA sequencing (Figure 45). For all three target genes, the frequency of on-target DNA modifications resulting from either plasmid or protein:sgRNA delivery was approximately 10% (Figure 41(A)-(C)). This allows for comparison of off-target modifications between the two technologies under treatment conditions that resulted in very similar on-target genome modification efficiencies. Importantly, for all 11 off-target sites, the frequency of off-target genomic modifications was lower with protein:sgRNA delivery compared to plasmid delivery; as a result, the ratio of on-target to off-target modifications for all sites tested was up to 19-fold higher with protein:sgRNA delivery than with plasmid delivery (Figure 31(B)-(D)).

[0278] DNA modification specificity was higher with protein:sgRNA delivery than with plasmid delivery at loci with high levels of off-target modification (e.g., four VEGF off-target sites, where plasmid delivery yielded a 4- to 20-fold average on-target:off-target modification ratio, whereas protein:RNA delivery yielded a 9- to 400-fold average on-target:off-target modification ratio), as well as at loci with lower levels of off-target modification (e.g., three EMX off-target loci, where plasmid delivery yielded a 64-fold lower average on-target:off-target modification ratio, whereas protein:RNA delivery yielded a 500- to 2,000-fold average on-target:off-target modification ratio). Taken together, these results demonstrate that delivery of Cas9:sgRNA complexes using cationic lipids can achieve targeted gene modification with high efficiency and substantially higher specificity than delivery of DNA expressing Cas9 and sgRNA.

[0279] Delivery of Cas9:sgRNA into mouse embryonic stem cells Potent, transient cationic lipid-mediated delivery of Cas9:sgRNA to achieve efficient, permanent, and highly specific gene editing may be particularly useful in stem cells. To test this possibility, we used mouse embryonic stem cells expressing Tau-EGFP. 48The cells were treated with Cas9 and an sgRNA targeting EGFP. Under standard stem cell culture conditions, EGFP-positive floating spheres were formed. The floating spheres were treated with Cas9:sgRNA complexed with RNAiMAX or with RNAiMAX and Cas9 without sgRNA as a control. Three days after treatment, reduced GFP fluorescence was observed in the Cas9:sgRNA-treated spheres compared to the control samples (Figure 42(A)). The treated spheres were dissociated, and the cells were allowed to adhere to gelatin-coated dishes and differentiate into progenitor cells. Immunohistochemistry using an anti-GFP antibody confirmed knockdown of EGFP expression in cells from the Cas9:sgRNA-treated sample; many nuclei lacked any visible EGFP. In contrast, all cells derived from the control spheres were EGFP-positive (Figure 42(B)). Genomic DNA collected from cells treated with Cas9:sgRNA was subjected to a T7EI assay, which provided clear evidence of indels at the Tau-EGFP locus (Figure 42(C)). From this assay, an indel frequency of 42% was calculated from both cationic lipid-mediated Cas9:sgRNA delivery and transfection of Cas9 and sgRNA DNA. Target modification was not detected in control samples lacking Cas9:sgRNA or containing Cas9 and an unassociated gRNA. These findings demonstrate that cationic lipid-mediated Cas9:sgRNA delivery can achieve highly efficient gene disruption in mouse embryonic stem cells.

[0280] In vivo cationic lipid-mediated delivery of Cre recombinase and Cas9:sgRNA Highly efficient delivery of functional genome-editing proteins in vivo will enable a wide range of applications, including non-viral therapeutic genome editing to correct genetic diseases. To evaluate the protein delivery methods described above in living mammals, we chose delivery to the mouse inner ear. This is due to its limited space, well-characterized inner ear cell types, and the existence of a genetically deaf mouse model that may enable future hearing restoration studies. We attempted in vivo delivery of two types of proteins to the mouse inner ear. First, we tested delivery of (-30)GFP-Cre protein to evaluate the efficiency of inner ear cell type targeting and functional protein delivery. Second, we evaluated delivery of Cas9:sgRNA complexes to the inner ear to determine whether cationic ...

Claims

1. A composition comprising 1) an RNA-programmable protein, 2) a gRNA, and 3) a cationic lipid, a cationic polymer, or both a cationic lipid and a cationic polymer, wherein the RNA-programmable protein is associated with the gRNA.

2. The composition of claim 1 , wherein the composition is capable of delivering an RNA programmable protein to the interior of a cell.

3. 3. The composition of claim 1 or 2, comprising an RNA programmable protein associated with a gRNA and a cationic lipid.

4. The composition of claim 1 or 2, comprising an RNA programmable protein associated with a gRNA and a cationic polymer.

5. The composition of any one of claims 1 to 4, comprising an RNA programmable protein associated with a gRNA, a cationic lipid, and a cationic polymer.

6. The composition of any one of claims 1 to 5, wherein the composition exhibits low toxicity when administered to a population of cells.

7. 7. The composition of claim 6, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells are viable following administration of the composition.

8. 8. The composition of any one of claims 1 to 7, wherein the cationic lipid is Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® RNAiMAX, Lipofectamine® LTX, or any combination thereof.

9. The composition of claim 1, comprising a cationic polymer, and wherein the RNA-programmable protein comprises a wild-type Cas9 protein or a variant thereof, a Cas9 nickase or a variant thereof, or a nuclease-inactive Cas9 (dCas9) or a variant thereof.

10. The composition of any one of claims 1 to 9, wherein the RNA programmable protein is fused to a functional effector protein.

11. The composition of claim 10 , wherein the RNA programmable protein is fused to a functional effector protein via a linker.

12. The linker is selected from the group consisting of SGSETPGTSESATPES (SEQ ID NO:3), SGSETPGTSESA (SEQ ID NO:4), SGSETPGTSESATPEGGSGGS (SEQ ID NO:5), VPFLLEPDNINGKTC (SEQ ID NO:6), GSAGSAAGSGEF (SEQ ID NO:7), SIVAQLSRPDPA (SEQ ID NO:8), MKIIEQLPSA (SEQ ID NO:9), VRHKLKRVGS (SEQ ID NO:10), GHGTGSTGSGSS (SEQ ID NO:11), MSRPDPA (SEQ ID NO:12), GGSM (SEQ ID NO:13), GGS, and (GGS). ｎ and n is any integer from 1 to 10.

13. The RNA programmable protein fused to a functional effector protein has the structure: [NH ２ ]-[RNA programmable protein]-[functional effector protein]-[COOH], or [NH ２ ]-[functional effector protein]-[RNA programmable protein]-[COOH], optionally wherein a linker is inserted between the RNA programmable protein and the functional effector protein.

14. The composition according to any one of claims 10 to 13, wherein the functional effector protein is a transcription activator or a transcription repressor.

15. The composition of claim 14, wherein the transcriptional activator is selected from the group consisting of VP16, VP64 and p65.

16. The composition according to claim 14, wherein the transcriptional repressor is a KRAB protein or a SID protein.

17. The composition of any one of claims 1 to 13, wherein the functional effector protein is a nuclease domain.

18. The composition of claim 17 , wherein the nuclease domain comprises a FokI nuclease domain, a TALE nuclease domain, or a zinc finger nuclease domain.

19. The composition of any one of claims 10 to 13, wherein the functional effector protein is a recombinase catalytic domain.

20. 20. The composition of claim 19, wherein the recombinase catalytic domain comprises a Hin recombinase catalytic domain, a Gin recombinase catalytic domain, or a Tn3 recombinase catalytic domain.

21. The composition of any one of claims 10 to 13, wherein the functional effector protein is a deaminase.

22. 22. The composition of claim 21 , wherein the deaminase comprises a cytidine deaminase.

23. 23. The composition of claim 21 or 22, wherein the deaminase comprises an APOBEC family deaminase.

24. 23. The composition of claim 21 or 22, wherein the deaminase is selected from the group consisting of APOBEC1, AID and ACF1 / ASE deaminase.

25. 22. The composition of claim 21 , wherein the deaminase comprises adenosine deaminase.

26. 26. The composition of claim 25, wherein the adenosine deaminase is an ADAT family deaminase.

27. The composition according to any one of claims 10 to 13, wherein the functional effector protein is an epigenetic modifier.

28. 28. The composition of claim 27, wherein the epigenetic modifier is selected from the group consisting of histone demethylases, histone methyltransferases, hydroxylases, histone deacetylases, and histone acetyltransferases.

29. 29. The composition of claim 27 or 28, wherein the epigenetic modifier comprises LSD1 histone demethylase or TET1 hydroxylase.

30. 30. The composition of any one of claims 1 to 29, further comprising a nuclear localization signal (NLS) fused to the RNA programmable protein or to the functional effector protein.

31. 31. The composition of claim 30, wherein the NLS comprises the amino acid sequence MAPKKKRKVGIHRGVP (SEQ ID NO:47).

32. 32. The composition of claim 30 or 31, wherein the NLS is at the C-terminus of the functional effector protein.

33. 32. The composition of claim 30 or 31, wherein the NLS is C-terminal to the RNA programmable protein.

34. The RNA programmable protein fused to a functional effector protein has the structure: [NH ２ ]-[NLS]-[RNA programmable protein]-[functional effector protein]-[COOH] or [NH ２ ]-[NLS]-[functional effector protein]-[RNA programmable protein]-[COOH], optionally wherein a linker is inserted between the RNA programmable protein and the functional effector protein, or a linker is inserted between the NLS and the RNA programmable protein or the functional effector protein, or both.

35. The composition of any one of claims 1 to 34, wherein the RNA programmable protein is associated with a supercharged protein having a net negative charge.

36. The composition of claim 35, wherein the RNA programmable protein is associated with the supernegatively charged protein via a non-covalent interaction or a covalent bond, directly or indirectly, via a linker, and the associated RNA programmable protein and the supernegatively charged protein have a negative charge, and optionally the supernegatively charged protein has a net negative charge of -5 to -50.

37. 37. The composition of claim 35 or 36, wherein the supercharged protein is a fluorescent protein.

38. The composition according to any one of claims 35 to 37, wherein the supercharged protein is a supercharged GFP.

39. The composition of any one of claims 1 to 38, comprising a cationic polymer, and wherein the RNA programmable protein comprises an amino acid sequence that is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to the amino acid sequence of a wild-type Cas9 of SEQ ID NO:

44.

40. The composition of any one of claims 1 to 38, comprising a cationic polymer, and wherein the RNA programmable protein is dCas9 or nCas9 comprising an amino acid sequence that is at least 70% identical, at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, or at least 99.9% identical to the amino acid sequence of dCas9 of SEQ ID NO:45, and comprises D10A and H840A substitutions compared to the amino acid sequence of wild-type Cas9 of SEQ ID NO:

44.

41. The composition of any one of claims 1 to 38, comprising a cationic polymer, and wherein the Cas9 protein is a Cas9 nickase that includes a D10A substitution compared to the wild-type Cas9 amino acid sequence of SEQ ID NO:

44.

42. A pharmaceutical composition comprising the composition of any one of claims 1 to 41 and a pharma- ceutically acceptable excipient.

43. A method for introducing an RNA-programmable protein into a cell in vitro or ex vivo, comprising contacting the cell in vitro or ex vivo with a composition according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 under suitable conditions for the RNA-programmable protein to enter the cell.

44. 44. The method of claim 43, wherein the cell is a somatic cell.

45. 45. The method of claim 43 or 44, wherein the cell is contacted with the composition or pharmaceutical composition in an amount, for a time, and under conditions sufficient to induce programming of the cell towards a desired cell fate.

46. The method of any one of claims 43 to 45, further comprising confirming that the RNA programmable protein has penetrated the cell.

47. The method of any one of claims 43 to 46, wherein the contacting step is performed in vitro.

48. Use of a composition according to any one of claims 1 to 41, or a pharmaceutical composition according to claim 42, in the manufacture of a medicament for treating a disease in a subject.

49. 49. The use of claim 48, wherein the treatment of a disease in a subject is by introducing an RNA programmable protein into a cell.

50. 50. The use according to claim 49, wherein the cell is a somatic cell.

51. 51. The use of claim 49 or 50, wherein the cell is contacted with the composition or pharmaceutical composition in an amount, for a time, and under conditions sufficient to induce programming of the cell towards a desired cell fate.

52. A kit comprising a composition according to any one of claims 1 to 41, or a pharmaceutical composition according to claim 42.

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