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Definitions

• the present invention relates to the field of CRISPR based transcriptional repression.
• the basic CRISPR/Cas9 system comprises a Cas9 protein and a guide RNA (“gRNA”).
• a spacer sequence also referred to as a targeting sequence
• dCas9 a deactivated Cas9
• the basic CRISPR/Cas9 system comprises a Cas9 protein and a guide RNA (“gRNA”).
• a spacer sequence also referred to as a targeting sequence
• dCas9 can be used for sequence -specific targeting and bringing other effectors with different functionalities.
• CRISPR-based technologies for transcriptional regulation include CRISPR interference (CRISPRi) for transcriptional repression and CRISPR activation (CRISPRa) for transcriptional upregulation (Qi, L.S., et al., “Repurposing CRISPR as an RNA-guided platform for sequence- specific control of gene expression,” Cell, 152(5): p. 1173-83 (2013); A.W. Cheng, et al., “Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system,” Cell Res., 23(10): p. 1163-71 (2013)).
• CRISPR interference CRISPR interference
• CRISPRa CRISPR activation
• KRAB Kriippel associated box
• K0X1 zinc finger protein 10
• L.A.Gilbert, et al. “CRISPR-mediated modular RNA- guided regulation of transcription in eukaryotes,” Cell, 154(2): p. 442-51 (2013)
• L. A. Gilbert et al. “Genome-scale CRISPR-mediated control of gene repression and activation,” Cell, 159: p. 647-661 (2014).
• this approach has its limitations. researchers have shown that it does not provide sufficient repression in all applications, and use of it can result in less robust repression of the target gene(s), L.
• the present invention provides novel fusion proteins, nucleic acid sequences that encode those proteins, and methods of gene repression by using those proteins and/or nucleic acids. Through the use of various embodiments of the present invention, one may efficiently and effectively regulate gene expression.
• the present invention provides a Cas fusion protein comprising a Cas protein and one or both of a SALL1 repressor domain and a SUDS3 repressor domain.
• the Cas protein is deactivated, which also may be referred to as dead or attenuated.
• the present invention provides a nucleic acid encoding a Cas fusion protein of the present invention.
• the present invention provides an RNA- repressor domain complex.
• the RNA-repressor domain complex comprises: (a) a gRNA molecule, wherein the gRNA molecule contains 30 to 180 nucleotides; (b) a ligand binding moiety, wherein the ligand binding moiety is either (i) directly bound to the gRNA molecule, or (ii) bound through a ligand binding moiety linker to the gRNA molecule; (c) a ligand, wherein the ligand is capable of reversibly associating with the ligand binding moiety; and (d) a fusion protein, wherein the fusion protein comprises a SALL1 repressor domain and a SUDS3 repressor domain, and wherein the fusion protein is either (i) directly bound to the ligand, or (ii) bound through a linker to the ligand.
• the present invention provides a method of modulating expression of a target nucleic comprising introducing a Cas fusion protein or an RNA-repressor domain complex of the present invention or a nucleic acid of the present invention into a cell such as a eukaryotic cell or an organism such as a mammal, e.g., a human.
• introduction is in vivo, in vitro, or ex vivo.
• the present invention provides a kit comprising a Cas fusion protein of the present invention or a nucleic acid encoding a Cas fusion protein of the present invention and in some embodiments may further comprise either a gRNA or a nucleic acid that encodes for a gRNA.
• the present invention provides a kit comprising an RNA-repressor domain complex, or a nucleic acid encoding, two molecules, an RNA-ligand binding domain and ligand-repressor of the present invention.
• the present invention provides a protein that comprises, consists essentially of, or consists of a sequence at least 80% similar to SEQ ID NO: 10.
• Figure 1 is a representation of Cas fusion protein of the present invention associated with a single guide RNA (“sgRNA”) and a target DNA.
• sgRNA single guide RNA
• Figure 2 is an example of an sgRNA that may be used in various embodiments of the present invention.
• Figure 3A is a graph that depicts gene knockdown in K562 cells nucleofected with either dCas9-KRAB or dCas9-SALLl-SUDS3 mRNA.
• Figure 3B is a graph that depicts gene knockdown in Jurkat cells nucleofected with either dCas9-KRAB or dCas9-SALLl-SUDS3 mRNA.
• Figure 3C is a graph that depicts gene knockdown in U2OS cells nucleofected with either dCas9-KRAB and dCas9-SALLl-SUDS3 mRNA.
• Figure 4 is a graph that compares repression of target genes when dCas9- SALL1-SUDS3 eGFP mRNA is introduced into HCT 116 cells to repression of target genes when dCas9-KRAB eGFP mRNA is introduced into HCT 116 cells.
• the genes are targeted with a pool of three synthetic sgRNAs delivered at 25 nM. Cells were sorted at 24 hours post- transfection into two categories: GFP negative (GFP Neg), and top 10% GFP expressing (Top 10%), and after 24 hours of recovery analyzed for transcriptional repression of die targeted genes.
• Figures 5A - 5C compare repression in systems that contain dCas9-KRAB versus systems that contain dCas9-SALL1- SUDS3 in different cell lines: U2OS (figure 5A); Jurkat (figure 5B); and hiPS stable hEF1 ⁇ (figure 5C).
• Figure 6A shows gene repression by dCas9-KRAB and dCas9-SALL1- SUDS3 against BRCA1, PSMD7, SEL1L, and ST3GAL4 in K562 cells.
• Figure 6B shows gene repression by dCas9-KRAB and dCas9-SALL1-SUDS3 against BRCA1, PSMD7, SEL1L, and ST3GAL4 in A375 cells.
• Figures 7A-7D compare the repression by dCas9-KRAB to repression by dCas9-SALL1-SUDS3 over a course of six days in U2OS cells for different gene targets: BRCA1 (figure 7A); CD46 (figure 7B); HBP1 (figure 7C); and SEL1L (figure 7D).
• Figure 8A shows repression using individual sgRNAs against PPIB, SEL1L, and RAB11A and pools of sgRNAs against these targets when introduced with Cas fusion proteins of the present invention.
• Figure 8B shows the repression of BRCA1, PSMD7, SEL1L, and ST3GAL4 by either individual sgRNAs or pools of sgRNAs against these targets when introduced with Cas fusion proteins of the present invention.
• Figure 9 shows expression of the following genes: PPIB, RAB11A, and SEL1, in hiPSC cells in the presence of gRNAs and dCas9-SALLl-SUDS3 when multiplexing, i.e., using sgRNAs against multiple genes.
• Figure 10 is a graph that shows functional phenotype of the repression of PSMD3, PSMD8, and PSMD11 genes in U2OS-Ubi (G76V)-EGFP reporter cell line in the presence of gRNAs and dCas9 fused to KRAB or SALL1-SUDS3 at the N terminal amino acid of the dCas9 or the C terminal amino acid of dCas9.
• Figure 11 is a graph of transcriptional repression in systems with a plasmid expressing gRNA and a plasmid expressing a fusion protein co-transfected in A375 cells.
• Figure 12 is a graph of transcriptional repression in systems with a plasmid expressing gRNA and a plasmid expressing a fusion protein co-transfected in U2OS cells.
• Figure 13 is a graph that shows the effect of combining SALL1 or SUDS3 each with an additional repressor domain.
• Figure 14A is a representation of repression by dMAD7-SALLl-SUDS3 as compared to dMAD7 in U2OS cells.
• Figure 14B is a representation of repression by dCasPhi8-SALLl-SUDS3 as compared to dCasPhi8 in U2OS cells.
• Figure 15A is a diagram of the effect of using sgRNAs of different crRNA- targeting sizes with Cas9 that is not deactivated for simultaneous repression and gene editing.
• Figure 15B is a graph that depicts the measurement of repression of MRElla while LBR is simultaneously edited.
• Figure 15C is a graph that depicts the measurement of repression of MRE1 la while PPIB is simultaneously edited.
• Figure 15D is a graph that depicts the measurement of repression of SEL1L while LBR is simultaneously edited.
• Figure 15E is a graph that depicts the measurement of repression of SEL1L while PPIB is simultaneously edited.
• Figure 16 is a graph of repression effects of systems that contain single repressor dCas9 fusion proteins in the U2OS-Ubi (G76V)-EGFP reporter cell line.
• Figure 17 is a graph that compares the transcriptional repression in U2OS cells stably expressing dCas9-KRAB, dCas9-KRAB MeCP2, or dCas9-SUDS3 that were transfected with synthetic guide RNAs.
• Figure 18A is a representation of the phenotypic effects of gene knockdown in U2OS Ubi[G76V]-EGFP reporter cells expressing either dCas9-KRAB or dCas9- SALL1-SUDS3 and transfected with synthetic guides targeting proteasome genes.
• Figure 18B depicts the corresponding transcriptional repression of the targeted proteasome genes.
• Figure 19A shows the transcriptional repression of PPIB and SEL1L in U2OS cells stably expressing either dCas9-SALLl-SUDS3 and a guide RNA from a single lenti viral vector or from two separate vectors.
• Figure 19B shows the transcriptional repression of PPIB and SEL1L in HCT 116 cells stably expressing either dCas9- SALL1-SUDS3 and a guide RNA from a single lentiviral vector or from two separate vectors.
• Figure 20A shows the transcriptional repression of BRCA1, PSMD7, SEL1L, and ST3GAL4 by either synthetic or plasmid sgRNAs in U2OS cells stably expressing dCas9-SALLl-SUDS3.
• Figure 20B shows the transcriptional repression of BRCA1, PSMD7, SEL1L, and ST3GAL4 by either synthetic or plasmid sgRNAs in A375 cells stably expressing dCas9-SALLl-SUDS3.
• Figure 21 shows the transcriptional repression of CD151, SEL1L, SETD3, and TFRC by either synthetic sgRNAs or synthetic crRNA:tracrRNA complexes in U2OS cells stably expressing dCas9-SALLl-SUDS3.
• Figure 22 shows the transcriptional repression of LBR, MRE1 la, XRCC4, and SEL1L by synthetic sgRNAs with 5’ truncated 14 mer targeting regions or full length 20 mer targeting regions in U2OS cells stably expressing dCas9-SALLl- SUDS3.
• Figure 23A is a representation of the phenotypic effects of gene knockdown of PSMD7 and PSMD11 by synthetic sgRNAs containing various combinations of two 2'-O-methyl and phosphorothioate linkages (2x MS) and two locked nucleic acid (LNA) modifications at the 5’ and 3 ‘ end of the sgRNA in U2OS Ubi[G76V]-EGFP reporter cells expressing dCas9-SALLl-SUDS3.
• 2x MS 2'-O-methyl and phosphorothioate linkages
• LNA locked nucleic acid
• Figure 23B is a representation of the phenotypic effects of gene knockdown of PSMD7 and PSMD11 by synthetic sgRNAs end stabilized with two 2 -O-methyl and phosphorothioate linkages (2x MS) and containing various locked nucleic acids (LNA) at different positions in the targeting region in U2OS Ubi[G76V]-EGFP reporter cells expressing dCas9-SALLl-SUDS3.
• 2x MS 2 -O-methyl and phosphorothioate linkages
• LNA locked nucleic acids
• Figure 24A shows the transcriptional repression of BRCA1, CD 151, and SETD3 by synthetic crRNA:tracrRNA complexes in which the tracrRNA contains an MS2 stem loop at various positions (in stem loop 2 or 3’ end of the tracrRNA) to recruit MCP-SALL1-SUDS3 to dCas9.
• Figure 24B shows the transcriptional repression of BRCA1, CD151, and SETD3 by synthetic crRNA:tracrRNA complexes in which the tracrRNA contains various MS2 stem loop sequences to recruit MCP- SALL1-SUDS3 to dCas9.
• Figure 25A is a graph that shows the transcriptional repression and protein level knockdown of CXCR3 in primary human CD4+ T cells nucleofected with dCas9-SALLl-SUDS3 and either a synthetic non-targeting control or a pool of three guides targeting the gene of interest one and three days post-nucleofection.
• Figure 25B provides representations of CXCR3 and CD4 protein expression in the aforementioned populations of T cells.
• the phrase “2 modification” refers to a nucleotide unit having a sugar moiety that is modified at the 2' position of the sugar moiety.
• An example of a 2' modification is a 2'-O-alkyl modification that forms a 2'-O-alkyl modified nucleotide or a 2' halogen modification that forms a 2' halogen modified nucleotide.
• 2'-O-alkyl modified nucleotide refers to a nucleotide unit having a sugar moiety, for example, a deoxyribosyl or ribosyl moiety that is modified at the 2' position such that an oxygen atom is attached both to the carbon atom located at the 2' position of the sugar and to an alkyl group.
• the alkyl moiety consists of or consists essentially of carbon(s) and hydrogens.
• O-alkyl group e.g., -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-isobutyl, -O-ethyl-O-methyl (-OCH2CH2OCH3), and -O-ethyl-OH (-OCH2CH2OH).
• a 2'-O-alkyl modified nucleotide may be substituted or unsubstituted.
• halogen modified nucleotide refers to a nucleotide unit having a sugar moiety, for example, a deoxyribosyl moiety that is modified at the 2' position such that the carbon at that position is directly attached to a halogen species, e.g., Fl, Cl, or Br.
• a halogen species e.g., Fl, Cl, or Br.
• complementarity refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by either traditional Watson-Crick base-pairing or other non-traditional types of base pairs.
• a percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).
• Perfect complementarity means that all of the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
• substantially complementary refers to a degree of complementarity that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, over a region of, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more consecutive nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
• nucleotide sequence refers to the ability of a nucleotide sequence or an amino acid sequence to provide information that describes the sequence of nucleotides or amino acids in another sequence or in a molecule.
• a nucleotide sequence encodes a molecule that contains the same nucleotides as in the nucleotide sequence that encodes it; that contains the complementary nucleotides according to Watson- Crick base pairing rules; that contains the RNA equivalent of the nucleotides that encode it; that contains the RNA equivalent of the complement of the nucleotides that encode it; that contains the amino acid sequence that can be generated based on the consecutive codons in the sequence; and that contains the amino acid sequence that can be generated based on the complement of the consecutive codons in the sequence.
• a “gRNA” is a guide RNA.
• a gRNA comprises, consists essentially of, or consists of a CRISPR RNA (crRNA) and in some embodiments, it may also comprise a trans-activating CRISPR RNA (tracrRNA). It may be created synthetically or enzymatically, and it may be in the form of a contiguous strand of nucleotides in which case it is a “sgRNA” or in some embodiments, formed by the hybridization of a crRNA and a tracrRNA that are not covalently linked together to form a contiguous chain of nucleotides.
• each gRNA may independently be encoded by a plasmid, lentivirus, or AAV (adeno associated virus), a retrovirus, an adenovirus, a coronavirus, a Sendai virus or other vector.
• AAV adeno associated virus
• the gRNA introduces specificity into CRISPR/Cas systems. The specificity is dictated in part by base pairing between a target DNA and the sequence of a region of the gRNA that may be referred to as the spacer region or targeting region.
• a PAM protospacer-adjacent motif sequence
• a Cas-targeted site a target sequence and its corresponding PAM site/sequence may collectively be referred to as a Cas-targeted site.
• the Class 2 CRISPR system of S. pyogenes uses targeted sites having N12-20NGG, where NGG represents the PAM site from S. pyogenes, and
• N 12-20 represents the 12-20 nucleotides directly 5’ to the PAM site. Additional PAM site sequences from other species of bacteria include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. See, e.g., US 20140273233, WO 2013176772, Cong et al., Science 339 (6121): 819-823 (2012), Jinek et a/., Science 337 (6096): p. 816-821 (2012), Mali et al., Science 339 (6121): p. 823-826 (2013), Gasiunas et al., Proc Natl Acad. Sci. U S A, 109 (39): p.
• hybridization and “hybridizing” refer to a process in which completely, substantially, or partially complementary nucleic acid strands come together under specified hybridization conditions to form a double- stranded structure or region in which the two constituent strands are joined by hydrogen bonds. Unless otherwise stated, the hybridization conditions are naturally occurring or lab-designed conditions. Although hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or between cytidine and guanine (C and G), other base pairs may form (see e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).
• a “ligand binding moiety” refers to a moiety such as an aptamer e.g., oligonucleotide or peptide or another compound that binds to a specific ligand and can reversibly or irreversibly be associated with that ligand.
• an aptamer e.g., oligonucleotide or peptide or another compound that binds to a specific ligand and can reversibly or irreversibly be associated with that ligand.
• To be reversibly associated means that two molecules or complexes can retain association with each other by, for example, noncovalent forces such as hydrogen bonding, and be separated from each other without either molecule or complex losing the ability to associate with other molecules or complexes.
• modified nucleotide refers to a nucleotide having at least one modification in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5- bromo-uracil or 5-iodouracil; and 2'- modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 , or CN.
• Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
• Some examples of these types of modifications include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, alone and in various combinations.
• More specific modified bases include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N, -dimethyladenine, 2- propyladenine, 2-propylguanine, 2-aminoadenine, 1 -methylinosine, 3 -methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1 -methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2- methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza- adenosine, 6-azouridine
• Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
• the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4- thioribose, and other sugars, heterocycles, or carbocycles.
• nucleotide refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof.
• Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.
• a nucleotide comprises a cytosine, uracil, thymine, adenine, or guanine moiety.
• nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.
• nucleotide also includes what are known in the art as universal bases.
• universal bases include but are not limited to 3 -nitropyrrole, 5-nitroindole, or nebularine.
• Nucleotide analogs are, for example, meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2'-methyl ribose, and non-natural phosphodiester internucleotide linkages such as methylphosphonates, phosphorothioates, phosphoroacetates and peptides.
• repressor domain refers to the amino acid sequence that form the domain of a repressor molecule that leads to inhibition of the expression of a gene.
• subject and “patient” are used interchangeably herein to refer to an organism, e.g., a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets such as dogs and cats. The tissues, cells and their progeny of an organism or other biological entity obtained in vivo or cultured in vitro are also encompassed within the terms subject and patient. Additionally, in some embodiments, a subject may be an invertebrate animal, for example, an insect or a nematode; while in others, a subject may be a plant or a fungus.
• a “terminal amino acid” is the last amino acid within a protein or within a region of a fusion protein.
• a terminal amino acid of a Cas protein may, for example, be bound not only to another amino acid within the Cas protein region of the fusion protein, but also to a repressor domain or to a linker.
• a terminal amino acid of a repressor domain may, for example, be bound not only to another amino acid within the repressor domain, but also to another repressor domain or to a Cas protein region of a fusion protein or to a linker.
• a terminal amino acid may be a C terminal amino acid or an N terminal amino acid.
• treatment As used herein, “treatment,” “treating,” “palliating,” and “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit.
• therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
• the complexes of the present invention may be administered to a subject, or a subject’s cells or tissues, or those of another subject extracorporeally before re-administration, at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom might not have yet been manifested.
• vector refers to a molecule or complex that transports another molecule and includes but is not limited to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, or that has been incorporated within the vector sequence.
• a vector can be introduced into cells and organisms to express RNA transcripts, proteins, and peptides, and may be termed an “expression vector.” Examples of vectors include, but are not limited to, plasmids, lentiviruses, alphaviruses, adenoviruses, or adeno-associated viruses.
• the vector may be single stranded, double stranded or have at least one region that is single stranded and at least one region that is double stranded.
• the nucleic acid may comprise, consist essentially of, or consist of RNA or DNA.
• the term “about” generally refers to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 20” may mean from 18-22. Other meanings of “about” may be apparent from the context, such as rounding off; for example “about 1” may also mean from 0.5 to 1.4.
• Fusion proteins are molecules that contain a portion or a complete amino sequence of each of two or more proteins.
• the components of fusion proteins may be fused directly to each other through, for example, covalent bonds or through linkers as described below. Fusion proteins may also be associated with moieties that are do not contain amino acids such as nucleotides sequences.
• a Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and one or both of a SALL1 repressor domain and a SUDS3 repressor domain or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as one of the aforementioned repressor domains.
• the Cas protein may be any CRISPR associated protein that is naturally occurring in for example, archaea or bacteria, or a modified version thereof such as a deactivated version, a truncated version thereof, or a derivative thereof.
• Cas proteins include but are not limited to: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, Cas12j, Mad7, CasX, CasY, Cas 13a, Casl4, C2cl, C2c2, C2c3, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc
• the Cas protein is a Type II Cas protein such as Cas9 or a Type V Cas protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12h, Cas12i, Cas12j, and MAD7.
• Modified versions of Cas proteins that may be used in the present invention, include but are not limited to catalytically inactive versions such as dCas9 and dCas12 or versions that have modified attenuated catalytic activity to provide a nicking function such as the nickase nCas9.
• a nicking enzyme is an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence. These enzymes cut only one strand of the DNA duplex, to produce DNA molecules that are "nicked,” rather than cleaved.
• the Cas proteins may be used with repressor domains.
• the repressor domain of SALL1 is:
• the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and the SALL1 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ
• the SALL1 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:
• the SALL1 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 1 is attached to the C terminal amino acid of the Cas protein.
• the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and the SUDS3 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 2.
• the SUDS3 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO:
• the SUDS3 repressor domain or a repressor domain that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 2 is attached to the C terminal amino acid of the Cas protein.
• the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and both the SALL1 repressor domain and the SUDS3 repressor domain.
• this Cas fusion protein is organized in one of the following ways (written N terminus to C terminus):
• the Cas fusion protein comprises a SALL1 repressor domain and a SUDS3 repressor domain
• the SALL1 repressor domain comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 1
• the SUDS3 repressor domain comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 2.
• the Cas fusion protein comprises a SALL1 repressor domain and a SUDS3 repressor domain, wherein the SALL1 repressor domain comprises, consists essentially of, or consists of a sequence is the same as SEQ ID NO: 1 and the SUDS3 repressor domain comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 2.
• the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and two or more copies of both the SALL1 repressor domain and the SUDS3 repressor domain. In some embodiments, this Cas fusion protein is organized in one of the following ways:
• the Cas fusion protein comprises a plurality of SALL1 repressor domains and a plurality of SUDS3 repressor domains, wherein each SALL1 repressor domain comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 1 and each SUDS3 repressor domain comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 2.
• the Cas fusion protein comprises a plurality of SALL1 repressor domains and a plurality of SUDS3 repressor domains, wherein each SALL1 repressor domain comprises, consists essentially of, or consists of a sequence is the same as SEQ ID NO: 1 and each SUDS3 repressor domain comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 2.
• the Cas fusion protein also comprises a domain of an additional repressor protein: [R],
• [R] is selected from the group consisting of the NIPP1 repressor domain, the KRAB repressor domain, the DNMT3A repressor domain, the BCL6 repressor domain, the CbpA repressor domain, the H-NS repressor domain, the MBD3 repressor domain, and the KRAB- Me-CP2 repressor domain.
• the NIPP1 repressor domain may be represented as follows: MVQTAVVPVKKKRVEGPGSLGLEESGSRRMQNFAFSGGLYGGLPPTHSEAGSQP HGIHGTALIGGLPMPYPNLAPDVDLTPVVPSAVNMNPAPNPAVYNPEAVNEPKK KKYAKEAWPGKKPTPSLLI (SEQ ID NO: 34) or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% that same as SEQ ID NO: 34.
• the KRAB repressor domain may be represented as follows:
• MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNL VSLGYQLTKPDVILRLEKGEEPWLV SEQ ID NO: 35
• the DNMT3A repressor domain may be represented as follows:
• SEQ ID NO: 36 or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% that same as SEQ ID NO: 36.
• the BCL6 repressor domain may be represented as follows:
• TAMYLQMEHVVDTCRKFIKASEAEM (SEQ ID: 173) or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% that same as
• the Cbp A repressor domain may be represented as follows:
• SEQ ID: 174 or a sequence that is at least 80%, at least 85%, at least 90%, or at least
• H-NS repressor domain may be represented as follows:
• the MBD3 repressor domain may be represented as follows:
• SEQ ID: 176 or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% that same as SEQ ID NO: 176.
• the KRAB-MeCP2 repressor domain may be represented as follows: MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNL VSLGYQLTKPDVILRLEKGEEPWLVSGGGSGGSGSSPKKKRKVEASVQVKRV LEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRPGRKRKAEADPQAI PKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRETVSIEVK EVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKE
• SEQ ID: 177 or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% that same as SEQ ID NO: 177.
• the Cas fusion protein comprises, consists essentially of, or consists of a Cas protein and each of the SALL1 repressor domain, the SUDS3 repressor domain, and the [R] repressor domain.
• all three repressor domains may all be on the C terminal amino acid of the Cas protein, all be on the N terminal amino acid of the Cas protein, two be on the C terminal amino acid of the Cas protein and one be on the N terminal amino acid of the Cas protein, or two be on the N terminal amino acid of the Cas protein and one be on the C terminal amino acid of the Cas protein. Examples of the orientation of these sequences may be represented as follows:
• the Cas protein in the Cas fusion protein is dCas9 or dCasl2 such as dCasl2a and the Cas fusion protein comprises, consists essentially of or consists of both the SALL1 repressor domain and the SUDS3 repressor domain.
• amino acid sequences of fusion constructs of the present invention include but are not limited to:
• SEQ ID NO: 41 may, for example be coded by nucleic acid comprises, consisting essentially of or consisting of SEQ ID NO: 170
• SEQ ID NO: 171 may, for example be coded by nucleic acid comprises, consisting essentially of or consisting of SEQ ID NO: 172:
• proteins and polypeptide sequences that are fragments of SEQ ID NO: 41 and 171 and derivatives of those sequences that can be used to perform substantially similar functions.
• the proteins or polypeptides are at least 80%, at least 85%, at least 90%, at least 95% similar to either SEQ ID NO: 41 and 171.
• nucleic acid sequences comprises, consist essentially of, or consist of SEQ ID NO: 170 or 172 or complement thereof, or sequences that are at least 80%, at least 85%, at least 90%, at least 95% similar to or complementary to either SEQ ID NO: 170 and 172.
• the fusion may be by a direct bond (e.g., a covalent bond) between the N terminal amino acid of the repressor protein and the C terminal amino acid of the Cas protein or the C terminal amino acid of the repressor protein and the N terminal amino acid of the Cas protein.
• a direct bond e.g., a covalent bond
• the linker comprises, consists essentially of, or consists of an amino acid sequence that is, e.g., 1 to 100 amino acid long or 3 to 90 amino acids long or 10 to 50 amino acids long. In some embodiments, the linker comprises, consists essentially of, or consists of a sequence that is not an amino acid sequence.
• the linker When the linker is between a Cas protein and a repressor domain, the linker may be referred to as a Cas linker.
• the Cas protein has a C terminal amino acid and the Cas fusion protein comprises a Cas linker, wherein the Cas linker is covalently bound to the C terminal amino acid of the Cas protein.
• the Cas protein has an N terminal amino acid and the Cas fusion protein comprises a Cas linker, wherein the Cas linker is covalently bound to the N terminal amino acid of the Cas protein.
• the Cas protein has a C terminal amino acid and an N terminal amino acid and the Cas fusion protein comprises two Cas linkers, wherein a first Cas linker is covalently bound to the C terminal amino acid of the Cas protein and a second Cas linker is covalently bound to the N terminal amino acid of the Cas protein.
• a first Cas linker and a second Cas linker the first Cas linker may be bound to a first repressor domain and the second Cas linker may be bound to a second repressor domain.
• the Cas linker comprises, consists essentially of, or consists of a sequence that is SEQ ID NO: 7.
• each of two Cas linkers comprises, consists essentially of, or consists of a sequence that is SEQ ID NO: 7.
• the Cas linker is covalently bound to a Cas protein and a repressor domain that comprises, consists essentially of or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95% similar to SEQ ID NO: 1.
• the Cas linker is covalently bound to a Cas protein and a repressor domain that comprises, consists essentially of or consists of a sequence that is SEQ ID NO: 1.
• the Cas linker is covalently bound to a Cas protein and a repressor domain that comprises, consists essentially of or consists of a sequence that is at least 80%, at least 85%, at least 90%, at least 95% similar to SEQ ID NO: 2.
• the Cas linker is covalently bound to a Cas protein and a repressor domain that comprises, consists essentially of or consists of a sequence that is SEQ ID NO: 2.
• each pair of repressor domains may be directly, e.g., covalently bound to each other, or they may be joined through a linker.
• a linker that joins two repressor domains may be referred to as a repressor linker.
• the repressor linker may be the same as or different from the Cas linker.
• the repressor linker comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 7: GSGGGSGGSGS. In some embodiments, the repressor linker comprises, consists essentially of, or consists of a sequence that is SEQ ID NO: 7.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a C terminal amino acid and the Cas fusion protein further comprises a Cas linker and a repressor linker, wherein the Cas linker is covalently bound to the C terminal amino acid of the Cas protein and to the N terminal amino acid of the SALL1 repressor domain and wherein the repressor linker is between the SALL1 repressor domain and the SUDS3 repressor domain.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a C terminal amino acid and the Cas fusion protein further comprises a Cas linker and a repressor linker, wherein the Cas linker is covalently bound to the C terminal amino acid of the Cas protein and to the SUDS3 repressor domain and wherein the repressor linker is bound to both the SUDS3 repressor domain and the SALL1 repressor domain.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a N terminal amino acid and the Cas fusion protein further comprises a Cas linker and a repressor linker, wherein the Cas linker is covalently bound to the N terminal amino acid of the Cas protein and to the SUDS3 repressor domain and wherein the repressor linker is bound to both the SUDS3 repressor domain and the SALL1 repressor domain.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a N terminal amino acid and the Cas fusion protein further comprises a Cas linker and a repressor linker, wherein the Cas linker is covalently bound to the N terminal amino acid of the Cas protein and to the SALL1 repressor domain and wherein the repressor linker is bound to both the SALL1 repressor domain and the SUDS3repressor domain.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a N terminal ammo acid and a C terminal ammo acid and the Cas fusion protein further comprises a first Cas linker and a second Cas linker, wherein the first Cas linker is covalently bound to the N terminal amino acid of the Cas protein and to the SUDS3 repressor domain and wherein the second Cas linker is bound to the C terminus of Cas protein and to the SALL1 repressor domain.
• the Cas protein may be a dCas9 protein or dCasl2 such as dCasl2a protein, wherein the Cas protein has a N terminal amino acid and a C terminal amino acid and the Cas fusion protein further comprises a first Cas linker and a second Cas linker, wherein the first Cas linker is covalently bound to the C terminal amino acid of the Cas protein and to the SUDS3 repressor domain and wherein the second Cas linker is bound to the N terminal amino acid of Cas protein and to the SALL1 repressor domain.
• the Cas fusion protein comprises a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% similar to SEQ ID NO: 10:
• the Cas fusion protein comprises a sequence that the same as SEQ ID NO: 10.
• the Cas fusion protein is:
• the Cas fusion protein is:
• the Cas fusion protein is: [dMAD7]-[Cas linker]- [SALL1 repressor domain] -[repressor linker]-[SUDS3 repressor domain].
• the Cas fusion protein is: [178] [dMAD7]-[Cas linker]-[SUDS3 repressor domain] -[repressor linker]- [SALL 1 repressor domain].
• the Cas fusion protein is:
• the Cas fusion protein is:
• the Cas fusion protein is:
• the Cas fusion protein is:
• the Cas-fusion proteins of the present invention may be used in conjunction with gRNAs.
• the gRNA contains 30 to 180 nucleotide or 45 to 135 nucleotides or 60 to 120 nucleotides.
• a gRNA may be chemically synthesized or enzymatically synthesized. When enzymatically synthesized, the synthesis may occur in vitro, in vivo, or ex vivo.
• the nucleotides of the gRNA may be exclusively modified ribonucleotides, exclusively unmodified ribonucleotides, or a combination or modified and unmodified ribonucleotides.
• the gRNA contains one or more modification such as 2' modifications, e.g., 2-O-alkyl such as 2'-O-methyl or 2'-O-ethyl, or 2'- halogenmodifications such as 2' Fluoro.
• the gRNA contains one or more modified intemucleotide linkages such a phosphorothioate linkages.
• the gRNA has the following modifications:
• the gRNA has the following modifications:
• the gRNA comprises, consists essentially of or consists of a crRNA. In some embodiments, the gRNA comprises, consists essentially of or consists of a crRNA sequence and a tracrRNA sequence.
• the crRNA and the tracrRNA may be part of a sgRNA or they each may be on a separate strand of nucleotides and form a crRNA molecule and a tracrRNA molecule, each of which is a polynucleotide.
• one of the tracrRNA molecule and the crRNA molecule may be referred to as a first RNA molecule and the other of the other tracrRNA molecule and the crRNA molecule may be referred to as a second RNA molecule.
• the total number of nucleotides in those two molecules combined may, for example, be the same as in the sgRNA described in various embodiments of the present invention.
• any chemical modifications to nucleotides of sgRNAs may be present in either or both of the tracrRNA molecule and crRNA molecule, and any internucleotide modifications of sgRNAs may be present in either or both of the tracrRNA molecule and crRNA molecule.
• any moieties described as being present on the 5' end or 3' end of a gRNA may in the case of a sgRNA be present on the 5' end or 3' end of the sgRNA, and in the case of separate tracrRNA molecules and crRNA molecules, each of which has a 5' end or 3' end, be present on the 5' end or 3' end of the tracrRNA molecule or crRNA molecule.
• the crRNA comprises, consists essentially of or consists of a Cas association region and a spacer region (also referred to as a targeting region).
• the targeting region is sufficiently complementary to and capable of hybridizing to a pre-selected target site of interest.
• the target specifying component of the guide sequence can comprise from about 10 nucleotides to more than about 25 nucleotides, for example up to 36 nucleotides.
• the region of base pairing between the guide sequence and the corresponding target site sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length.
• the targeting region is 12 to 30 nucleotides long, or 14 to 25 nucleotides long or about 17 to 20 nucleotides long or about 14 nucleotides long or about 20 nucleotides long.
• the targeting region may be at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% complementary to a region of the target dsDNA over at least 14 contiguous nucleotides, at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, at least 20 contiguous nucleotides, or 14 to 20 contiguous nucleotides.
• the targeting region is about 20 nucleotides long and used with an active Cas protein that is capable of cleaving both DNA strands, a double- strand break will be generated on the targeted DNA that can lead to insertions and/or deletions (indel) in the genome.
• an inactive Cas protein such as a deactivated Cas9 protein.
• an active Cas protein that is generally capable of cleaving both DNA strands or a Cas nickase variant that is generally capable of cleaving one strand of the targeted DNA one may use a gRNA that has a shorter targeting region, such as about 14 nucleotides long for gene repression. Guides with a 20nt targeting region can lead the active Cas9-repressor to another genomic site for DNA cleaving and subsequent editing.
• the Cas association region which may for example, be about 18 - 36 nucleotides long is the portion of the crRNA that allows the crRNA (and thus the gRNA to retain association with the Cas protein).
• association with the Cas protein is possible in the absence of a tracrRNA. In other embodiments, association requires the presence of a tracrRNA.
• the Cas association region hybridizes with an anti-repeat region within the tracrRNA.
• the tracrRNA may also contain a distal region that is 3' of the anti-repeat region and is not complementary to any region of the crRNA.
• the repeat: anti-repeat region of the gRNA scaffold can be split into 3 parts: the lower stem, bulge, and upper stem.
• the single strand may contain regions that are complementary and that when the complementary regions hybridize allow association with a Cas protein such as Type II Cas enzymes, including but not limited to Cas9 in active or deactivated form, and Type V Cas enzymes such as Cas12c, Cas12d, Cas12e, and Cas12f in active or deactivated form.
• a Cas protein such as Type II Cas enzymes, including but not limited to Cas9 in active or deactivated form, and Type V Cas enzymes such as Cas12c, Cas12d, Cas12e, and Cas12f in active or deactivated form.
• the gRNA when the gRNA is a sgRNA, there are no regions that are complementary, but the sgRNA is capable of association with a Cas enzyme, such as certain Type V Cas enzymes such as Cas12a, MAD7 (an engineered variant of ErCas12a), Cas12h, Cas12i, and Cas12j (Cas ⁇ ) in active or deactivated form.
• a Cas enzyme such as certain Type V Cas enzymes such as Cas12a, MAD7 (an engineered variant of ErCas12a), Cas12h, Cas12i, and Cas12j (Cas ⁇ ) in active or deactivated form.
• Cas enzyme such as Cas12a, MAD7 (an engineered variant of ErCas12a), Cas12h, Cas12i, and Cas12j (Cas ⁇ ) in active or deactivated form.
• the sgRNA of figure 2 has the following sequence: (SEQ ID NO: 11): 5'mN*mN*NNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAG CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUmU*mU*U3' m signifies a 2'O-methyl group; * signifies a phosphorothioate linkage; and N signifies any of A, C, G, or U. [200] As shown in figure 2, the crRNA region and the tracrRNA regions are joined by a tetra loop and the tracrRNA region has three stem loop regions. This example of an sgRNA has 100 nucleotides.
• the sgRNA is 60 to 120 nucleotides long or 90 to 110 nucleotides long.
• the N region as shown is 20 nucleotides long. In some embodiments, the N region is 10 to 36 nucleotides long or 14 to 26 nucleotides long or 18 to 22 nucleotides long.
• Various tracrRNA sequences are known in the art and examples include SEQ ID Nos: 27-34, as well as active portions thereof.
• an active portion of a tracrRNA retains the ability to form a complex with a Cas protein, such as Cas9 or dCas9 or nCas9.
• the gRNA can be a hybrid RNA molecule where the above-described crRNA comprises a programmable gRNA fused to a tracrRNA to mimic the natural crRNA:tracrRNA duplex.
• crRNA:tracrRNA gRNA sequence: 5'-(20 nt guide)- GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAG UCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU- 3 ' (SEQ ID NO: 27).
• crRNA-tracrRNA hybrid RNAs also known as sgRNAs
• the two components are linked together via a tetra stem loop.
• the repeat anti -repeat region is extended. There may, for example, be an extension of 2, 3, 4, 5, 6, 7 bases or more than 7 bases at either side of the repeat: anti-repeat region.
• the repeat: antirepeat region has an extension of 7 nucleotides at either side of the stem. The extension of 7 bases at either side results in a region that is 14 base pairs longer. In other embodiments, the extension may be more than 7 bases. See e.g., WG2014099750, US 20140179006, and US 20140273226 for additional disclosure of tracrRNAs. The contents of these documents are incorporated herein by reference in their entireties.
• the tracrRNA is from or derived from S. pyogenes.
• the target site resides on DNA.
• the target nucleic acid strand can be either of the two strands and e.g., be in genomic DNA within a host cell.
• genomic dsDNA include, but are not necessarily limited to, a host cell chromosome, mitochondrial DNA and a stably maintained plasmid.
• the present method can be practiced on other dsDNA present in a host cell, such as non-stable plasmid DNA, viral DNA, and phagemid DNA, as long as there is Cas-targeted site.
• the fusion proteins of the present invention may be used in a system or as part of a complex that has: (a) a crRNA, wherein the crRNA is 30 to 60 nucleotides long and the crRNA comprises a Cas association region and a targeting region, wherein the Cas association region is 15 to 30 nucleotides long and the targeting region is 15 to 30 nucleotides long; (b) a scoutRNA, wherein the scoutRNA is 20 to 100 nucleotides long and wherein the scoutRNA comprises an anti-repeat region, wherein the anti-repeat region is 3 to 10 nucleotides long, and the anti -repeat region is complementary to at least 3 consecutive nucleotides within the Cas association region, and the anti -repeat region is capable of hybridizing with
• RNA-repressor domain complex comprises, consists essentially of, or consists of: a gRNA such as a gRNA described above or a scoutRNA and/or a crRNA capable of associating with a scoutRNA as described above, a ligand binding moiety, a ligand, and one or more repressor domains.
• the RNA-repressor domain complexes may be used in conjunction with the Cas-fusion proteins of the present invention or with other Cas proteins that are not fusion proteins.
• the gRNA or scoutRNA or crRNA capable of associating with a scoutRNA may be fused directly to a ligand binding moiety or associated with a ligand binding moiety through a ligand binding moiety linker.
• the ligand binding moiety is capable of reversibly associating with a ligand.
• the ligand is directly or through a ligand linker fused to a repressor domain.
• the repressor domain may be any effector.
• Each of the ligand binding moiety linker and the ligand linker if either or both are present may comprise, consist essentially of or consist of nucleotide(s), amino acids and other organic and inorganic moieties and combinations thereof.
• PCP PP7 coat protein
• a complex is formed that comprises, consists essentially of, or consists of a Cas-fusion protein of the present invention and RNA- repressor domain complex of the present invention.
• the Cas-fusion protein comprises a Cas protein fused to the repressor domain SUDS 3
• the ligand may be fused to SALL1 or to any other repressor domain that is now known or that comes to be known.
• the Cas-fusion protein comprises a Cas protein fused to the repressor domain SALL1
• the ligand may be fused to SUDS3 or to any other repressor domain that is now known or that comes to be known.
• the Cas-fusion protein comprises, consists essentially of, or consists or a Cas protein, a SALL1 repressor domain and a SUDS3 repressor domain
• the RNA-repressor domain complex comprises a gRNA, a ligand binding moiety, a ligand and one or more repressor domains other that SALL1 or SUDS3.
• the one or more repressor domains may be selected from the group consisting of NIPP1, KRAB and DNMT3A.
• the RNA-repressor domain complex may comprise a gRNA, a ligand-binding moiety and one or both of the SUDS3 repressor domain and the SALL1 repressor domain as defined above.
• a repressor linker as defined above may be present between the SUDS3 repressor domain and the SALL1 repressor domain, and the ligand may be attached directly or through a ligand linker to either one of the SALL1 repressor domain and the SUDS3 repressor domain.
• the present invention provides a nucleic acid that encodes for a fusion protein of the present invention.
• the nucleic acid may be single stranded, double stranded or have at least one region that is single stranded and at least one region that is double stranded. Further, the nucleic acid may comprise, consist essentially of, or consist of RNA or DNA.
• the nucleic acid that encodes the fusion protein only contains nucleotides for the fusion protein and any linkers that are present. In other embodiments, the nucleic acid that encodes the fusion protein is part of a larger nucleic acid or a vector.
• the present invention is directed to a vector that comprises a nucleic acid that encodes a fusion protein of the present invention.
• the vector is a plasmid or a viral vector.
• the viral vector is a lenti viral vector.
• the present invention is directed to an mRNA that encodes a Cas fusion protein of the present invention.
• the nucleic acid comprises a sequence that encodes a Cas protein and at least one repressor domain such as SUDS3 or SALL1. In some embodiments, the nucleic acid comprises a sequence that encodes a Cas protein and at least two repressor domains, such as SUDS3 and SALL1. In some embodiments, the nucleic acid comprises a sequence that encodes a Cas protein and at least three repressor domains such as SUDS3, SALL1, and one or more of NIPP1, KRAB, and DNMT3A.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 4, which encodes the SALL1 repressor domain:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 5, which encode the SUDS3 repressor domain:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 5.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 6, which encodes the NIPP1 repressor domain:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 6.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 37, which encodes the KRAB repressor domain:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 37.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 38, which encodes the DNMT3A repressor domain:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 38.
• the nucleic acid sequence comprises a sequence that encodes at least one a linker sequence and is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 8:
• the nucleic acid sequence comprises a sequence that is the same as SEQ ID NO: 8.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 184, which encodes for both the SALL1 and SUDS3 repressor domains:
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 183, which encodes deactivated Cas9 (dCas9): [235] ATGGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAG CGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCA TCGGCCTGGCCATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGAC GAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACC
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 178, which encodes deactivated MAD7 (dMAD7):
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 178.
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is the same as SEQ ID NO: 179, which encodes deactivated CasPhi8 (dCasPhi8):
• the nucleic acid sequence comprises, consists essentially of, or consists of a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% the same as or complementary to SEQ ID NO: 179.
• the fusion protein of the present invention may be linked to nuclear localization signals (NLS), epitope tags, or reporter gene sequences.
• nuclear localization signals include, but are not limited to, those of the SV40 Large T-antigen, nucleoplasmin, EGL-13, and TUS-protein.
• epitope tags include, but are not limited to, FLAG tags, V5 tags, histidine (His) tags, and influenza hemagglutinin (HA) tags.
• reporter genes include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), small ubiquitin-like modifier (SUMO), ubiquitin, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), and luciferase.
• GFP green fluorescent protein
• RFP red fluorescent protein
• SUMO small ubiquitin-like modifier
• ubiquitin glutathione-S-transferase
• HRP horseradish peroxidase
• CAT chloramphenicol acetyltransferase
• luciferase include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), small ubiquitin-like modifier (SUMO), ubiquitin, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acet
• the nucleic acid or vector that encodes the fusion proteins of present invention will also encode various regulatory elements or selection markers.
• Regulatory elements include, but are not limited to promoters such as the cytomegalovirus (CMV) promoter or human EFla promoter, enhancers such as the woodchuck hepatitis post- transcriptional regulatory element (WPRE) or HIV-1 Rev response element (RRE), polyadenylation signals, self-cleaving peptides such as T2A, and internal ribosomal entry sites (IRES).
• selection markers include, but are not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), puromycin A-acetyl-transferase (PAC) conferring resistance to puromycin, the hygromycin resistance gene, and blasticidin-S deaminase (BSD).
• GFP green fluorescent protein
• RFP red fluorescent protein
• PAC puromycin A-acetyl-transferase
• BSD blasticidin-S deaminase
• the present invention is directed to a method of modulating expression of a target nucleic acid in a eukaryotic cell.
• the method comprises providing to the cell a gRNA and a Cas fusion protein of any of the embodiments of the present invention.
• the Cas protein shown as a dCas protein
• SALL1 120 which is fused to SUDS3 130 may act upon a target region of genomic DNA 150.
• the method comprises introduce a plurality of gRNAs with the Cas fusion protein.
• the plurality of gRNAs may be two or more, e.g., 2 - 10 or 4 - 8 gRNAs.
• Two or more or all of the gRNAs may target the same gene or the same locus within a gene. If two or more gRNAs target the same locus, they may have the same or overlapping spacer sequences or non-overlapping sequences. In some embodiments, two or more or all of the gRNAs may target the different genes or the different loci within a gene.
• one or more gRNAs are provided to a cell by introducing to the cell a nucleic acid encoding the gRNA, and the Cas fusion protein is provided to the cell by introducing to the cell a nucleic acid encoding the Cas fusion protein.
• the cell may be placed under conditions in which the cell expresses the gRNA and the Cas fusion protein.
• the present invention is directed to a method of modulating expression of a target nucleic acid in a eukaryotic cell by introduce a Cas fusion protein and an RNA-repressor domain complex. In some embodiments, the present invention is directed to a method of modulating expression of a target nucleic acid in a eukaryotic cell by introduce a Cas protein that is not a fusion protein and an RNA-repressor domain complex.
• the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell such as a human or murine cell.
• the cell is part of a cell line, e.g., HEK293, K562, Jurkat, or US2OS.
• the fusion protein may be synthesized outside of a cell or an organism. Alternatively, one may introduce an mRNA that encodes the fusion protein.
• a gRNA is synthetically made outside of the cell and a Cas fusion protein is provided to the cell by introducing to the cell a nucleic acid encoding the Cas fusion protein.
• the Cas fusion proteins, RNA-repressor domain complexes and/or gRNAs may be delivered to target cells and organisms via other various methods and various formats (DNA, RNA or protein) or combination of these different formats.
• different components may be delivered as: (a) DNA polynucleotides that encode the relevant sequence for the Cas fusion protein or the gRNAs; (b) RNA encoding the sequence for the Cas fusion protein (messenger RNA) and synthetic gRNAs; (c) purified protein for the Cas fusion protein; (d) RNA that encode gRNA; and (e) purified RNA-repressor domain complexes.
• the Cas protein can be assembled with the applicable gRNA to form a ribonucleoprotein complex (RNP) for delivery into target cells, organisms and subjects.
• RNP ribonucleoprotein complex
• the components or complexes ([Cas fusion protein]-[gRNA]) as assembled may be delivered together or separately by electroporation, by nucleofection, by transfection, via nanoparticles, via viral mediated RNA delivery, via non-viral mediated delivery, via extracellular vesicles (for example, exosome and microvesicles), via eukaryotic cell transfer (for example, by recombinant yeast) and other methods that can package molecules such that they can be delivered to a target viable cell without changes to the genomic landscape.
• DNA-only vehicles for example, plasmids, MiniCircles, MiniVectors, MiniStrings, Protelomerase generated DNA molecules (for example, Doggybones), artificial chromosome (for example HAC), and cosmids
• DNA vehicles by nanoparticles, extracellular vesicles (for example, exosome and microvesicles), via eukaryotic cell transfer (for example, by recombinant yeast), transient viral transfer by AAV, non-integrating viral particles (for example, lentivirus and retrovirus based systems), cell penetrating peptides and other technology that can mediate the introduction of DNA into a cell without direct integration into the genomic landscape.
• RNA components include the use of integrative gene transfer technology for stable introduction of the machinery for RNA transcription into the genome of the target cells. These methods can be controlled via constitutive or promoter inducible systems to attenuate the RNA expression and this can also be designed so that the system can be removed after the utility has been met (for example, introducing a Cre-Lox recombination system), such technology for stable gene transfer includes, but is not limited to, integrating viral particles (for example lentivirus, adenovirus and retrovirus based systems), transposase mediate transfer (for example, Sleeping Beauty and Piggybac), exploitation of the non-homologous repair pathways introduced by DNA breaks (for example, utilizing CRISPR and TALEN) technology and a surrogate DNA molecule, and other technology that encourages integration of the target DNA into a cell of interest.
• integrative gene transfer technology for stable introduction of the machinery for RNA transcription into the genome of the target cells.
• kits comprising, consists essentially of, or consists of a Cas fusion protein of the present invention or a polynucleotide with a nucleic acid sequence that encodes a protein of the present invention.
• the kit further comprises a gRNA or a nucleic acid that encodes a gRNA or a plurality of gRNAs or a library of gRNAs, and optionally reagents for transfection and/or other delivery into a cell or to a subject.
• the kit comprises a nucleic acid that is capable of expressing both a gRNA and a Cas fusion protein of the present invention.
• the kit comprises a cell line that has been engineered to express a Cas fusion protein of the present invention and optionally further comprises a gRNA or a nucleic acid that encodes a gRNA.
• the present invention is directed to a kit that comprises, consists essentially of, or consists of an RNA-repressor domain complex of the present invention.
• the kit further comprises a Cas protein or a Cas fusion protein or a nucleic acid that encodes a Cas protein or a Cas fusion protein, and optionally reagents for transfection and/or other delivery into a cell or to a subject.
• the present invention provides a kit, wherein the kit comprises: (1) a lentiviral particle, wherein the lentiviral particle comprises a first polynucleotide that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl-SUDS3; and (2) a second polynucleotide, wherein the second polynucleotide is an sgRNA.
• the present invention provides a kit, wherein the kit comprises: (1) a first lentiviral particle, wherein the first lentiviral particle comprises a first polynucleotide that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl-SUDS3; and (2) a second lentiviral particle, wherein the second lentiviral particle comprises a second polynucleotide, wherein the second polynucleotide codes for an sgRNA.
• a first lentiviral particle wherein the first lentiviral particle comprises a first polynucleotide that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl-SUDS3
• a second lentiviral particle wherein the second lentiviral particle comprises a second polynucleotide, wherein the second polynucleotide codes for an sgRNA.
• the present invention provides a kit, wherein the kit comprises: (1) a lentiviral particle, wherein the lentiviral particle comprises a first polynucleotide that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl-SUDS3; and (2) a second polynucleotide, wherein the second polynucleotide is a plasmid, wherein the plasmid encodes a second polynucleotide and the second polynucleotide is an sgRNA.
• a lentiviral particle comprises a first polynucleotide that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl-SUDS3
• a second polynucleotide wherein the second polynucleotide is a plasmid, wherein the plasmid encodes a second polynucleotide and the second polynucleotide is
• the present invention provides a kit, wherein the kit comprises: (1) a first polynucleotide, wherein the first polynucleotide is an mRNA that encodes a Cas fusion protein of the present invention, such as dCas9-SALLl- SUDS3; and (2) a second polynucleotide, wherein the second polynucleotide is an sgRNA.
• the sgRNAs in the kits may be designed to associate with the Cas fusion protein that is encoded by the polynucleotides described above.
• the kits may be one or more of the following: target cells, and one or more a selection chemicals and/or media (e.g., blasticidin, puromycin).
• a selection chemicals and/or media e.g., blasticidin, puromycin.
• the present invention provides method for simultaneous repression of multiple genes. In some of these methods one may deliver the same dCas9-repressor Cas fusion protein with different gRNAs that target different gene promoters or different transcriptional start sites of the same gene. In another embodiment, the present invention provides a method for simultaneous repression and gene editing. In these methods one may deliver the Cas9-repressor Cas fusion protein with regular gRNAs (20 nucleotide targeting region) to cause gene editing and truncated gRNAs (14 nucleotide targeting region) to cause gene repression.
• These methods may be used to repress an inflammatory response such as the myeloid differentiation primary response 88 (MyD88) while performing gene editing, or to repress various genes involved in non-homologous end-joining thereby increasing the likelihood of a homology-directed DNA repair event (HDR) or to modulate host genes that are involved in the regulation of repair of double-stranded DNA breaks, leading to different outcomes.
• MyD88 myeloid differentiation primary response 88
• HDR homology-directed DNA repair event
• These methods may be used to effect synthetic lethality whereby a gene target can be edited and a secondary gene target can be repressed to cause a cytotoxic response not present in cells containing only one of the genomic perturbations.
• Figure 15 illustrates the effect of using different sized crRNA regions with an active Cas9 that is fused to SALL1 and SUDS3.
• a 14-mer crRNA targeting region When a 14-mer crRNA targeting region is used, there is transcriptional repression of the target (left y-axis of figure).
• a 20-mer crRNA targeting region When a 20-mer crRNA targeting region is used, there is gene-editing of the target (right y-axis of figure).
• the various embodiments of the present invention may also be used in arrayed screening applications. For example, one may use a library of arrayed gRNAs for systematic loss-of-function studies. In some embodiments 2-5 synthetic guide RNAs can be pooled for arrayed screening applications.
• the various embodiments of the present invention may also be used in pooled lentiviral screening applications.
• These gRNAs can be delivered in cells expressing the Cas fusion constructs of the present invention, or via a lentiviral construct that expresses both the Cas fusion protein and a gRNA.
• one may combine different CRISPR Cas systems with different effectors in the same cells to cause transcriptional repression with one system and another effect (activation, gene editing, base editing, or epigenetic modification) with the other Cas system.
• These methods may, for example, be used to cause specific gene repression of an immune cell selected from a T cell (including a primary T cell), Natural Killer (NK cell), B cell, or CD34+ hematopoietic stem progenitor cell (HSPC).
• the immune cell may be an engineered immune cell, such as T-cell comprising a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).
• CAR chimeric antigen receptor
• TCR engineered T cell receptor
• stem cells include, but are not limited to, mammalian stem cells such as human stem cells, including, but not limited to, hematopoietic, neural, embryonic, iPSC, mesenchymal, mesodermal, liver, pancreatic, muscle, and retinal stem cells.
• Other stems cells include, but are not limited to, mammalian stem cells such as mouse stem cells, e.g., mouse embryonic stem cells.
• the methods provided herein may be useful for targeted gene expression modulation in mammalian cells including primary human T cells, NK cells, CD34+ HSPCs, such as HSPCs isolated from umbilical cord blood or bone marrow and cells differentiated from them.
• T cells Also provided herein are genetically engineered cells arising from haematopoietic stem cells, such as T cells, that have been modified according to the methods described herein.
• the various embodiments of the present invention may be used for the following applications, base editing, genome editing, genome screening, generation of therapeutic cells, genome tagging, epigenome editing, karyotype engineering, chromatin imaging, transcriptome and metabolic pathway engineering, genetic circuits engineering, cell signaling sensing, cellular events recording, lineage information reconstruction, gene drive, DNA genotyping, miRNA quantification, in vivo cloning, site-directed mutagenesis, genomic diversification, and proteomic analysis in situ.
• a cell or a population of cells are exposed to a fusion protein of the present invention and the cell or cells are introduced to a subject by infusion.
• Applications also include research of human diseases such as cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnosis, pathogen screening, microbiota remodeling, stem-cell reprogramming, immunogenomic engineering, vaccine development, and antibody production.
• human diseases such as cancer immunotherapy, antiviral therapy, bacteriophage therapy, cancer diagnosis, pathogen screening, microbiota remodeling, stem-cell reprogramming, immunogenomic engineering, vaccine development, and antibody production.
• one or more molecules or complexes descried herein, including a Cas fusion protein, a fusion protein, a Cas protein, a gRNA, and a nucleic acid that encodes any of the foregoing is introduced to a subject.
• Introduction may, for example, be in the form of a medicament.
• sgRNA, crRNA and tracrRNA were synthesized at Horizon Discovery (formerly Dharmacon). sgRNA and crRNA molecules were designed based on the CRISPRi version 2.1 (v2.1) guide RNA prediction algorithm developed in 2016, M. A. Horlbeck et al., “Compact and highly active next-generation libraries for CRISPR- mediated gene repression and activation,” eLife. 5, el9760 (2016). Unless otherwise stated, experiments utilized modified sgRNAs delivered as an equimolar pool of the top three algorithmically ranked sgRNAs, labeled gl-g3 in table 2 below. The same targeting sequences were used for the sgRNA, crRNA, and expressed sgRNA with the exception that the first base in the expressed sgRNAs is always G.
• U2OS or A375 cells were seeded in 96-well plates at 10,000 or 20,000 cells per well, respectively, one day prior to transfection.
• Cells were transfected with synthetic guide RNAs targeting specific genes at a final concentration of 25 nM.
• Synthetic guide RNAs were complexed with DharmaFECT 4 Transfection Reagent (Horizon Discovery, cat # T-2005-01) for each experiment in serum-free medium (GE Healthcare HyClone, cat #SH30564.01) for 20 minutes. Medium on the plated cells was removed and replaced with the transfection mixture. The cells were incubated at 37° C with 5% CO2 for 24-144 hours until the assays were performed.
• U2OS cells were seeded at 10,000 cells per well in clear 96-well plates one day prior to transfection; HCT 116 cells were seeded at 200,000 cells per well in clear 6-well plates one day prior to transfection.
• U2OS cells were co-transfected with 0.2 pg/well of dCas9-SALLl-SUDS3 or dCas9-KRAB mRNA and 25 nM synthetic sgRNA;
• HCT 116 cells were co-transfected with 2.5 pg/well of dCas9-SALLl- SUDS3 or dCas9-KRAB mRNA and 25 nM synthetic sgRNA.
• dCas9 mRNA and sgRNAs were complexed with DharmaFECT Duo Transfection Reagent (Horizon Discovery, cat #T-2010) in serum-free medium (GE Healthcare HyClone, cat #SH30564.01) for 20 minutes. Medium on the plated cells was removed and replaced with the transfection mixture. The cells were incubated at 37° C with 5% CO2.
• K562 J562, Jurkat, WTC-11 human induced pluripotent stem cells (hiPS cells), and primary human CD4+ T cells were electroporated per well using the Amaxa 96-well Shuttle System.200,000 K562 cells per replicate were resuspended in SF buffer (Lonza, cat #V4SC-2096) and nucleofected using the FF-120 program; 200,000 Jurkat cells were resuspended in SE buffer (Lonza, cat #V4SC-1960) and nucleofected using program Cl-120; 80,000 hiPS cells were resuspended in P3 buffer (Lonza, cat #V4SP- 3096) and nucleofected using program DC-100; 250,000 primary human CD4+ T cells were resuspended in P3 buffer and nucleofected using program E0-115.
• Synthetic guide RNAs were delivered at cell-line-dependent final concentrations between 2.5 and 9 ⁇ M. In cases where the cells were not stably expressing a dCas9 CRISPRi construct, dCas9-SALL1-SUDS3 or dCas9-KRAB mRNA was delivered at cell-line-dependent concentrations ranging from 1-2.5 ⁇ g per nucleofection.
• Transfections with plasmid sgRNA [288] U2OS and A375 cells were seeded in 96-well plates at 10,000 or 20,000 cells per well one day prior to transfection with CRISPRi sgRNA plasmids.
• Plasmids were complexed with DharmaFECT kb Transfection Reagent (Horizon Discovery, Cat #T- 2006) in serum-free medium (GE Healthcare HyClone, #SH30564.01) for 10 minutes. Medium on the plated cells was removed and replaced with the transfection mixture. The cells were incubated at 37° C with 5% CO2 for 72 hours until the assays were performed. [289] Lentiviral transduction [290] U2OS and HCT 116 cells were seeded at 10,000 cells per well and transduced with CRISPRi sgRNA lentiviral particles at a multiplicity of infection (MOI) of 0.3 to obtain cells with a single integrant.
• MOI multiplicity of infection
• NTC non-targeting control
• the proteasome assay utilizes a recombinant U2OS cell line that stably expresses a mutant Ubiquitin fused to enhanced green fluorescent protein (Ubi[G76V]-EGFP).
• Ubi[G76V]-EGFP enhanced green fluorescent protein
• cell media was replaced with Dulbecco's Phosphate Buffered Saline (Cytivia, cat # SH30028.02) and EGFP fluorescence was measured using an EnVision® plate reader. Fluorescent values of cell populations transfected with guide RNAs targeting critical proteasome genes were normalized to fluorescent values of the untreated cell populations.
• TIDE quantifies the frequency and types of small insertions and deletions (indels) at a target locus using quantitative sequence trace data from a targeted sample that is normalized to sequence trace data of a control sample.
• Example 1 Comparison of silencing of dCas9-KRAB and dCas9-SALLl- SUDS3 delivered as mRNA
• Figure 3B shows the results of a similar study, except that the target cells were Jurkat cells and the expression was measured 72 hours after nucleofection.
• the target cells were Jurkat cells and the expression was measured 72 hours after nucleofection.
• silencing of gene expression by dCas9-SALLl-SUDS3 was comparable to, if not better than, silencing by dCas9- KRAB.
• Figure 3C shows the results of another similar study, except that the target cells were U2OS cells, reagents were delivered via lipid transfection using a 25 nM mixture of a pool of three pooled synthetic sgRNAs targeting the respective gene, and the expression was measured 72 hours after transfection.
• the target cells were U2OS cells
• reagents were delivered via lipid transfection using a 25 nM mixture of a pool of three pooled synthetic sgRNAs targeting the respective gene, and the expression was measured 72 hours after transfection.
• silencing of gene expression by dCas9-SALLl-SUDS3 was comparable to, if not better than, silencing by dCas9-KRAB.
• Example 2 Comparison of Effectiveness of Cas fusion protein Repressor to dCas9-KRAB
• HCT116 cells were plated at 400,000 cells per well. Twenty-four hours later, the cells were co-transfected with dCas9-SALLl-SUDS3 eGFP mRNA or dCas9- KRAB eGFP mRNA and a 25 nM mixture of a pool of three synthetic sgRNAs targeting each of the following genes: PPIB, PSMD7, and SEL1L, as well as a nontargeting control (NTC), using DharmaFECT® Duo Transfection reagent. At 24 hours post-transfection, cells were trypsinized, and FACS was performed. Cells were sorted into two categories: Negative, and Top 10%, then plated in 6-well dishes and allowed to recover.
• NTC nontargeting control
• the relative expression of each gene was calculated with the AACq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
• dCas9-SALLl-SUDS3 eGFP mRNA can be used for FACS enrichment and provides greater repression of target genes than dCas9-KRAB eGFP mRNA in both selected and unselected populations.
• Example 3 Comparison repression of dCas9-KRAB to dCas9-SALLl- SUDS3 in Different Cell Lines
• U2OS, Jurkat, and hiPS cells stably expressing dCas9-SALLl-SUDS3 or dCas9-KRAB were transfected or nucleofected with pools of three synthetic sgRNAs targeting the listed genes, as well as NTCs. Cells were harvested 72 hours later. In each cell line dCas9-KRAB or dCas9-SALLl-SUDS3 were under control of the hEFla promoter. The total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the AACq method using GAPDH as the housekeeping gene and normalized to a nontargeting control.
• FIG. 5A shows, in the U2OS stable hEFla cell line, dCas9-SALLl- SUDS3 demonstrated greater gene repression against BRCA1, PSMD7, SEL1L, and ST3GAL4.
• FIG. 5B shows, in the Jurkat stable hEFla cell line, dCas9-SALLl- SUDS3 also demonstrated greater gene repression against BRCA1, PSMD7, SEL1L, and ST3GAL4.
• FIG. 5C shows, in the USOS stable hEFla cell line, dCas9- SALL1-SUDS3 demonstrated greater or similar gene repression against RAB11A, PPB, and SEL1L.
• dCas9- SALL1-SUDS3 also demonstrated greater gene repression against BRCA1, PSMD7, SEL1L, and ST3GAL4.
• dCas9-SALLl-SUDS3 demonstrated greater or similar gene repression against BRCA1, PSMD7, SEL1L, and ST3GAL4.
• Example 4 dCas9-KRAB versus dCas9-SALLl-SUDS3 over course of 6 days
• U2OS cell lines stably expressing dCas9-SALL1-SUDS3 or dCas9-KRAB under the control of the hEF1 ⁇ promoter were transfected with the pools of three synthetic sgRNAs targeting each of the following genes : BRCA1, CD46, HBP1, and SEL1L. Repression was measured over six days with samples harvested every 24 hours post-transfection. Total RNA was isolated, and gene expression was assessed via RT-qPCR.
• FIG. 7A shows that dCas9-SALL1-SUDS3 caused greater repression than dCas9-KRAB did against BRCA1 at all timepoints.
• Figure 7B shows that dCas9- SALL1-SUDS3 caused greater repression than dCas9-KRAB did against CD46 at all timepoints.
• Figure 7C shows that dCas9-SALL1-SUDS3 caused greater repression than dCas9-KRAB did against HBP1 at all timepoints.
• Figure 7D shows that dCas9- SALL1-SUDS3 caused greater repression than dCas9-KRAB did against SEL1L at all timepoints. Note that in each example there was a more rapid onset of the repression mediated by dCas9-SALL1-SUDS3 than that mediated by dCas9-KRAB, and that the repression mediated by dCas9-SALL1-SUDS3 persisted at close to maximal levels for longer than the repression mediated by dCas9-KRAB.
• Example 5 Pooling sgRNAs [314] WTC-11 hiPSCs stably expressing dCas9-SALL1-SUDS3, and U2OS cells stably expressing dCas9-SALL1-SUDS3 were nucleofected or transfected with individual or a pool of three synthetic sgRNAs targeting PPIB (3 ⁇ M), SEL1L (3 ⁇ M), RAB11A (3 ⁇ M) - 3 ⁇ M of each sgRNA electroporated, BRCA1 (25 nM), PSDM7 (25 nM), SEL1L (25 nM), and ST3GAL4 (25 nM) delivered via lipid transfection. Cells were harvested 72 hours later.
• RNA was isolated and relative gene expression was measured using RT-qPCR. Relative gene expression was calculated with the ⁇ Cq method using GAPDH as the housekeeping gene and normalized to a non-targeted control.
• FIG. 8A shows, in the WTC-11 hiPSCs, the pooling was comparable to or better than the use of each individual sgRNA.
• figure 8B shows, in the US2OS hEF1 ⁇ dCas9-SALL1-SUDS3, the pooling was comparable to or better than the use of each individual sgRNA.
• Example 6 Multiplexing of gRNAs for simultaneous repression of multiple genes
• hiPSCs stably expressing dCas9-SALL1-SUDS3 were nucleofected with individual sgRNAs and pools of up to 6 sgRNAs targeting unique genes. Cells were harvested 72 hours later . The total RNA was isolated and the relative gene expression was measured using RT-qPCR. The relative gene expression was calculated with the ⁇ Cq method using GAPDH as the housekeeping gene and normalized to a non-targeted control [318] As figure 9 shows, when up to six genes were targeted for simultaneous repression in human iPS cells, the levels of target gene repression was comparable to when only one of the genes was targeted.
• Example 7 Fusion to N-terminal amino acid and to C-terminal amino acid of Cas protein
• the structures of three Cas fusion proteins are represented at the bottom of figure 10: dCas9-KRAB; dCas9-SALL1-SUDS3, and SUDS3-SALL1-dCas9.
• the Cas fusion proteins were expressed under the control of the human EF1 ⁇ promoter.
• U2OS Ubi[G76V]-EGFP cell lines were generated that stably expressed various bipartite dCas9 fusion proteins based, along with a cell line stably expressing dCas9-KRAB.
• Example 8 Plasmid:Plasmid Co-Transfection in A375 & U2OS cells
• Plasmid repressors: (1) hEF1 ⁇ -dCas9 KRAB; or (2) dCas9-SALL1-SUDS3 were co-transfected with guides (total 100 ng) using 0.6 ⁇ L/well of DharmaFECT® kb.
• Figure 11 shows the results when measuring gene expression by RT-qPCR at three days post-plasmid co-transfection of repressor and gene targets in A375 cells.
• Figure 12 shows the results when measuring gene expression by RT-qPCR at three days post-plasmid co-transfection of repressor and gene targets in U2OS cells. Both figures consistently show greater repression in systems that contained the plasmid for dCas9-SALL1-SUDS3.
• Example 9 Additional Repressors [327] U2OS Ubi[G76V]-EGFP cell lines were generated that stably expressed various bipartite dCas9 fusion proteins based, along with a cell line stably expressing dCas9-KRAB. Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be critical to proteasome function, as well with non-targeting controls.
• Example 10 Type V Cas protein-SALL1-SUDS3 fusion constructs
• a deactivated MAD7 (an engineered Cas12a protein)-SALL1-SUDS3 fusion construct was cloned (dMAD7-SALL1-SUDS3), and U2OS cells were generated that stably expressed it under control of the minimal CMV (mCMV) promoter.
• mCMV minimal CMV
• a deactivated CasPhi8 (a Cas12J protein)-SALL1-SUDS3 fusion construct was cloned (dCAsPhi8-SALL1-SUDS3), and U2OS cells were generated that stably expressed it under control of the mCMV promoter. These cells, along with U2OS cells stably expressing dMAD7 or dCasPhi8, were lipid transfected with synthetic guides designed for the respective Cas proteins, in each case delivered at 25 nM. Transcriptional repression was assessed 48 hours post-transfection.
• Figure 14A shows CRISPRi induced transcriptional repression in U2OS cells stably expressing either dMAD7 or dMAD7-SALLl-SUDS3 for two individual synthetic guide RNAs against each of BRCA1 and PPIB, as well as for a pool of synthetic guide RNA, and an NTC.
• the figure shows significantly greater repression effected by dMAD7-SALLl-SUDS3 as compared to dMAD7.
• Figure 14B shows CRISPRi induced transcriptional repression in U2OS cells stably expressing either dCasPhi8 or dCasPhi8-SALLl-SUDS3 for three iterations of individual synthetic guide RNAs targeting the same site in BRCA1, and basal BRCA1 expression in untreated U2OS cells.
• the figure shows significantly greater repression effected by dCasPhi8-SALLl-SUDS3 as compared to dCasPhi8.
• U2OS cells stably expressing SUDS3-SALLl-WtCas9 under the control of the hEFla promoter were transfected with 25 nM pools of guide RNAs designed for both CRISPRi and CRISPR editing.
• Guides designed for CRISPRi contained a truncated 14-mer targeting region.
• Guides designed for CRISPR editing contained the full 20- mer targeting region.
• Cells were harvested 72 hours later post-transfection.
• the total RNA was isolated and the relative gene expression was measured using RT-qPCR. The relative gene expression was calculated with the AACq method using GAPDH as the housekeeping gene and normalized to a non-targeted control. Genomic DNA was isolated, target regions were amplified using PCR and Sanger sequenced, and indel formation was analyzed using TIDE.
• Figure 15B shows MRE11A can be repressed while LBR is simultaneously edited.
• Figure 15C shows MRE11A can be repressed while PPIB is simultaneously edited.
• Figure 15D shows SEL1L can be repressed while LBR is simultaneously edited.
• Figure 15E shows SEL1L can be repressed while PPIB is simultaneously edited.
• Example 12 Comparison of single repressor domains as dCas9-fusion Protein [338]
• U2OS Ubi[G76V]-EGFP cell lines were generated that stably expressed various single repressor dCas9 fusion proteins (BCL6, CbpA, H-NS, MBD3, NIPP1, SALL1, and SUDS3), along with a cell line stably expressing dCas9-KRAB, all under the control of the human EF1 ⁇ promoter.
• Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be critical to proteasome function, as well as non- targeting controls.
• Example 13 Comparison of dCas9-SUDS3 repressor to dCa9-KRAB and dCas9-KRAB-MeCP2 systems
• U2OS Ubi[G76V]-EGFP cell lines were generated that stably expressed either dCas9-KRAB, dCas9-KRAB MeCP2, or dCas9-SUDS3 under the control of the human EF1 ⁇ promoter.
• Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be critical to proteasome function, as well as non-targeting controls.
• Example 14 Proteasome Functional Reporter assay and Transcriptional Repression
• U2OS Ubi[G76V]-EGFP cell lines were generated that stably expressed either dCas9-KRAB or dCas9-SALL1-SUDS3 under the control of the human EF1 ⁇ promoter.
• Cells were transfected with 25 nM synthetic sgRNAs targeting genes known to be critical to proteasome function, as well as non-targeting controls. The fluorescence of each transfection condition was determined at 72 hours posttransfection, with an EnVision® plate reader and values were normalized to those of the untreated cell line.
• the U2OS cell line stably expressed a mutant Ubiquitin fused to enhanced green fluorescent protein (Ubi[G76V]-EGFP).
• Ubi[G76V]-EGFP enhanced green fluorescent protein
• the expressed ubiquitin EGFP is constitutively degraded, leaving only background fluorescence, whereas cells with inhibited proteasome function display an accumulation of EGFP. Repression of target genes therefore results in increased fluorescence.
• Total RNA was also isolated and expression of the target genes was assessed via RT-qPCR. Relative expression was calculated with the AACq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
• Figure 18A shows dCas9-SALLl-SUDS3 effected significantly more phenotypic knockdown than dCas9-KRAB. (A higher mean GFP expression correlates to greater repression.)
• Figure 18B shows that the more pronounced phenotype observed with dCas9-SALLl-SUDS3 correlated with increased transcriptional repression of the targeted proteasome genes.
• Figure 19A shows the transcriptional repression of PPIB and SEL1L in U2OS cells stably expressing either dCas9-SALLl-SUDS3 and a guide RNA from a single lentiviral vector or from two separate vectors.
• Figure 19B shows the transcriptional repression of PPIB and SEL1L in HCT 116 cells stably expressing either dCas9- SALL1-SUDS3 and a guide RNA from a single lentiviral vector or from two separate vectors.
• Lentiviral vectors were used to generate U2OS and HCT 116 cells that stably expressed dCas9-SALLl-SUDS3 under the control of the human EFla promoter (hEFla) or mouse CMV promoter (mCMV) respectively. These cells were subsequently transduced with lentiviral particles containing vectors that expressed individual guide RNAs from the human U6 promoter and targeted PPIB, SEL1L, or contained a non-targeting control sequence.
• hEFla human EFla promoter
• mCMV mouse CMV promoter
• Parental U2OS and HCT 116 cells were transduced with lentiviral particles containing a single vector that expressed dCas9- SALL1-SUDS3 under the control of the hEFla (U2OS) or mCMV (HCT 116) promoters, and an individual guide RNA from the human U6 promoter.
• These single vector systems also targeted PPIB or SEL1L, or contained a non-targeting control sequence. Twenty-four hours post-transduction, media containing 2.5 ⁇ g/mL puromycin was added to enrich for transduced cells. Cells were cultured in this media for 7 days and passaged every 3 to 4 days.
• Figure 19A shows that either a single lentiviral or dual lentiviral vector system can be used to express dCas9-SALL1-SUDS3 and a guide RNA to robustly repress a target gene in U2OS cells.
• Figure 19B shows that either a single lentiviral or dual lentiviral system can be used to express dCas9-SALL1-SUDS3 and a guide RNA to robustly repress a target gene in HCT 116 cells.
• Example 16 Plasmid sgRNAs vs. Synthetic sgRNAs
• U2OS and A375 cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1 ⁇ promoter were transfected with individual, matched 25 nM synthetic sgRNAs or 100 ng of plasmid sgRNA targeting BRCA1, PSMD7, SEL1L, and ST3GAL4.
• FIG. 20A demonstrates that dCas9-SALL1-SUDS3 mediates substantially greater target gene expression when delivered with synthetic sgRNAs than when delivered with plasmid sgRNAs in U2OS cells.
• Figure 20B demonstrates that dCas9-SALL1-SUDS3 mediates substantially more target gene expression when delivered with synthetic sgRNAs than when delivered with plasmid sgRNAs in A375 cells.
• Example 17 Synthetic sgRNA vs crRNA:tracrRNA [351]
• U2OS cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1 ⁇ promoter were transfected with pooled 25 nM synthetic sgRNAs or synthetic crRNA:tracrRNA complexes. Cells were harvested 72 hours post-transfection, total RNA was isolated, and the relative gene expression of each target genes was measured using RT-qPCR. Relative gene expression was calculated with the ⁇ Cq method using GAPDH as the housekeeping gene and normalized to a non-targeted control.
• Figure 21 demonstrates that while repression is markedly more pronounced with pooled synthetic sgRNAs, both synthetic sgRNA and synthetic crRNA:tracrRNA complexes can be delivered with dCas9-SALL1-SUDS3 to cause target gene repression.
• Example 18 5' Truncated Spacer [353] U2OS cells stably expressing dCas9-SALL1-SUDS3 under the control of the hEF1 ⁇ promoter were transfected with 25 nM pools of guide RNAs containing either truncated 14-mer targeting regions or full length 20-mer targeting regions. Cells were harvested 72 hours post-transfection.
• RNA was isolated and the relative gene expression of the target genes was measured using RT-qPCR. Relative gene expression was calculated with the ⁇ Cq method using GAPDH as the housekeeping gene and normalized to a non-targeted control.
• Figure 22 shows that the targeting region of a guide RNA can be shortened at the 5’ end by at least 6-mer and still effect transcriptional repression when delivered with dCas9-SALL1-SUDS3.
• Example 19 LNA modified sgRNAs
• U2OS Ubi[G76V]-EGFP cells stably expressing dCas9-SALL1-SUDS3 under the control of the human EF1 ⁇ promoter were transfected with 25 nM synthetic sgRNAs targeting two genes known to be critical to proteasome function, as well as non-targeting controls.
• the guides contained various combinations of 2′-O-methyl and phosphorothioate linkages and locked nucleic acids at the ends of the sgRNA molecule, and in the 20-mer targeting region, position 1 to position 20 from the 5’ end.
• FIG. 23B shows the impact of the incorporation of locked nucleic acid positions into the sgRNA targeting region on dCas9-SALL1-SUDS3 mediated functional knockdown. Locked nucleic acids can be incorporated at some positions of the sgRNA targeting region to improve target gene repression.
• Example 20 RNA -repressor complex recruitment
• tracrRNA designs containing different MS2 ligand binding moiety sequences and positions were tested against each gene target and compared to complexes containing a tracrRNA without an MS2 ligand binding moiety, labeled crRNA:tracrRNA w/out MS2.
• Cells were harvested 72 hours post-transfection, total RNA was isolated, and the relative gene expression of each target genes was measured using RT-qPCR. Relative gene expression was calculated with the ⁇ Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
• Figure 24A demonstrates that MCP-SALL1-SUDS3 can be recruited to dCas9 through the C-5 MS2 sequence positioned at the either sgRNA stem loop 2 or at the 3’ terminus of the tracrRNA molecule.
• the recruitment of MCP-SALL1-SUDS3 can enhance the repressive effect of dCas9 binding, represented here as crRNA:tracrRNA w/out MS2.
• Figure 24B shows that MCP-SALL1-SUDS3 can be recruited to dCas9 through both the C-5 MS2 sequence and the F-5 MS2 sequence containing a 2dAP chemical mod to significantly improve the repressive effect of dCas9 binding.
• Example 21 Knockdown in T-cells
• Primary human CD4+ T cells were nucleofected with dCas9-SALLl-SUDS3 mRNA and pooled synthetic sgRNA via a Lonza 96-well Shuttle system. 24 and 72 hours post-nucleofection, functional knockdown of CXCR3 was assessed as a percent of cells expressing the target gene by FACS analysis. Cells were stained for CD4 as a positive expression control using an Alexa Fluor 488 conjugated antibody and compared to CXCR3 using APC conjugated primary antibodies. Total RNA was isolated at each timepoint and mRNA expression of CXCR3 was assessed via RT- qPCR. The relative expression of CXCR3 was calculated with the AACq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
• Figure 25A is a graph that shows the transcriptional repression and protein level knockdown of CXCR3 in primary human CD4+ T cells nucleofected with dCas9-SALLl-SUDS3 and either a synthetic non-targeting control or a pool of 3 guides targeting the gene of interest 1 and 3 days post-nucleofection.
• Figures 25B shows that the onset of knockdown with dCas9-SALLl-SUDS3 was rapid and persisted for several days in this clinically relevant primary cell type, comparing protein expression in the non-transfected control system on day 1, protein expression in the non-transfected control system on day 3, protein expression in the CXCR3 pool system on day 1 and protein expression in the CXCR3 pool system on day 3.
• Target region is bolded, chemical modifications are italicized.
• Target region is bolded, chemical modifications are italicized.
• Target region is bolded, chemical modifications are italicized.
• Target region is bolded, chemical modifications are italicized.
• Target region is bolded, chemical modifications are italicized.
• Table 8 Lentiviral guide RNAs (Sp Cas9) delivered via particles or as plasmids

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