RNA-Guided Targeting of Genetic and Epigenomic Regulatory Proteins to Specific Genomic Loci

Abstract

Methods and constructs for RNA-guided targeting of transcriptional activators to specific genomic loci.

Claims

1. A fusion protein comprising catalytically inactive CRISPR associated 9 (Cas9) protein linked to a heterologous functional domain.

2. The fusion protein of claim 1, wherein the heterologous functional domain is a transcriptional activation domain.

3. The fusion protein of claim 2, wherein the transcriptional activation domain is from VP64 or NF-B p65.

4. The fusion protein of claim 1, wherein the catalytically inactive Cas9 protein is from S. pyogenes.

5. The fusion protein of claim 1, wherein the catalytically inactive Cas9 protein comprises mutations at D10A and H840A.

6. The fusion protein of claim 1, wherein the heterologous functional domain is linked to the N terminus or C terminus of the catalytically inactive Cas9 protein, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.

7. The fusion protein of claim 1, further comprising one or both of a nuclear localization sequence and one or more epitope tags on the N-terminus, C-terminus, or in between the catalytically inactive CRISPR associated 9 (Cas9) protein and the heterologous functional domain, optionally with one or more intervening linkers.

8. The fusion protein of claim 7, wherein the one or more epitope tags is selected from the group consisting of c-myc, 6His, and FLAG tags.

9. A nucleic acid encoding the fusion protein of claim 1.

10. A nucleic acid encoding the fusion protein of claim 2.

11. A nucleic acid encoding the fusion protein of claim 3.

12. A nucleic acid encoding the fusion protein of claim 4.

13. An expression vector comprising the nucleic acid of claim 9.

14. An expression vector comprising the nucleic acid of claim 10.

15. An expression vector comprising the nucleic acid of claim 11.

16. An expression vector comprising the nucleic acid of claim 12.

Description

DESCRIPTION OF DRAWINGS

[0017] FIG. 1A is a schematic illustration showing a single guide RNA (sgRNA) recruiting Cas9 nuclease to a specific DNA sequence.

[0018] FIG. 1B is a schematic illustration showing a longer version of the sgRNA used to introduce targeted alterations.

[0019] FIG. 1C is a schematic illustration showing a Cas9 protein containing D10A and H840A mutations to render the nuclease portion of the protein catalytically inactive fused to a transcriptional activation domain.

[0020] FIG. 2 is a bar graph showing levels of VEGFA protein expression in cells transfected with gRNA and Cas9-VP64. Fold activation was calculated relative to off-target gRNA control. Error bars represent standard error of the mean of three independent replicates. 118=18 guide RNAs targeted to various sites in the human VEGF-A gene; Cas9-Vp64=Fusion of catalytically inactive Cas9 (bearing D10A/H840A mutations) fused to the VP64 Activation domain; eGFP gRNA=a guide RNA targeted to an off-target site located in an EGFP Reporter gene

[0021] FIG. 3A is a bar graph showing VEGFA protein expression in cells transfected with multiple gRNAs and Cas9-VP64, demonstrating synergistic activation of VEGFA. Fold activation was calculated relative to off-target gRNA control. Error bars represent standard error of the mean of three independent replicates.

[0022] FIG. 3B is a bar graph showing VEGFA protein expression in cells transfected with multiple gRNAs and Cas9-VP64. The number underneath each bar indicate the amount in nanograms (ng) of Cas-activator (C) plasmid or guide RNA (g) plasmid transfected.

[0023] FIG. 4 is an exemplary sequence of a Guide RNA expression vector.

[0024] FIG. 5 is an exemplary sequence of CMV-T7-Cas9 D10A/H840A-3FLAG-VP64.

[0025] FIG. 6 is an exemplary sequence of CMV-T7-Cas9 recoded D10A/H840A-3FLAG-VP64.

[0026] FIG. 7 is an exemplary sequence of a Cas9-activator. An optional 3FLAG sequence is underlined; the nuclear localization signal PKKKRKVS (SEQ ID NO:1) is in lower case; two linkers are in bold; and the VP64 transcriptional activator sequence, DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML (SEQ ID NO:2), is boxed.

DETAILED DESCRIPTION

[0027] Described herein are fusion proteins of transcriptional activation domains fused to a catalytically inactivated version of the Cas9 protein for the purpose of enabling RNA-guided targeting of these functional domains to specific genomic locations in cells and living organisms.

[0028] The CRISPR/Cas system has evolved in bacteria as a defense mechanism to protect against invading plasmids and viruses. Short protospacers, derived from foreign nucleic acid, are incorporated into CRISPR loci and subsequently transcribed and processed into short CRISPR RNAs (crRNAs). These RNAs then use their sequence complementarity to the invading nucleic acid to guide Cas9-mediated cleavage, and consequent destruction of the foreign nucleic acid. Last year, Doudna and colleagues demonstrated that a single guide RNA (sgRNA) can mediate recruitment of Cas9 nuclease to specific DNA sequences in vitro (FIG. 1C; Jinek et al., Science 2012).

[0029] More recently, a longer version of the sgRNA has been used to introduce targeted alterations in human cells and zebrafish (FIG. 1B; Mali et al. Science 2013, Hwang and Fu et al., Nat Biotechnol. 2013 March; 31(3):227-9).

[0030] As described herein, in addition to guiding Cas9-mediated nuclease activity, it is possible to use CRISPR-derived RNAs to target heterologous functional domains fused to Cas9 to specific sites in the genome (FIG. 1C). As described herein, it is possible to use single guide RNAs (sgRNAs) to target Cas9-transcriptional activators (hereafter referred to as Cas9-activators) to the promoters of specific genes and thereby increase expression of the target gene. Cas9-activators can be localized to sites in the genome, with target specificity defined by sequence complementarity of the guide RNA.

[0031] In some embodiments, the present system utilizes the Cas9 protein from S. pyogenes, either as encoded in bacteria or codon-optimized for expression in mammalian cells, containing D10A and H840A mutations to render the nuclease portion of the protein catalytically inactive (FIG. 1C). The Cas9-activators are created by fusing a transcriptional activation domain, e.g., from either VP64 or NF-B p65, to the N-terminus or C-terminus of the catalytically inactive Cas9 protein.

[0032] The sequence of the catalytically inactive Cas9 used herein is as follows; the mutations are in bold and underlined.

TABLE-US-00001 (SEQIDNO:3) 102030405060 MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE 708090100110120 ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG 130140150160170180 NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSD 190200210220230240 VDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN 250260270280290300 LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI 310320330340350360 LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA 370380390400410420 GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELH 430440450460470480 AILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE 490500510520530540 VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL 550560570580590600 SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI 610620630640650660 IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG 670680690700710720 RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL 730740750760770780 HEHIANLAGSRAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER 790800810820830840 MKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDA 850860870880890900 IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL 910920930940950960 TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS 970980990100010101020 KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRK 103010401050106010701080 MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF 109011001110112011301140 ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVA 115011601170118011901200 YSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK 121012201230124012501260 YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE 127012801290130013101320 QHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA 1330134013501360 PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0033] The transcriptional activation domains can be fused on the N or C terminus of the Cas9. In addition, although the present description exemplifies transcriptional activation domains, other heterologous functional domains (e.g., transcriptional repressors, enzymes that modify the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins), or enzymes that modify histone subunit (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), or histone demethylases)) as are known in the art can also be used. A number of sequences for such domains are known in the art, e.g., a domain that catalyzes hydroxylation of methylated cytosines in DNA. Exemplary proteins include the Ten-Eleven-Translocation (TET)1-3 family, enzymes that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA.

[0034] Sequences for human TET1-3 are known in the art and are shown in the following table:

TABLE-US-00002 GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP_085128.2 NM_030625.2 TET2* NP_001120680.1 (var 1) NM_001127208.2 NP_060098.3 (var 2) NM_017628.4 TET3 NP_659430.1 NM_144993.1 *Variant (1) represents the longer transcript and encodes the longer isotorm (a). Variant (2) differs in the 5 UTR and in the 3 UTR and coding sequence compared to variant 1. The resulting isoform (b) is shorter and has a distinct C-terminus compared to isoform a.

[0035] In some embodiments, all or part of the full-length sequence of the catalytic domain can be included, e.g., a catalytic module comprising the cysteine-rich extension and the 2OGFeDO domain encoded by 7 highly conserved exons, e.g., the Tet1 catalytic domain comprising amino acids 1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprising amino acids 966-1678. See, e.g., FIG. 1 of Iyer et al., Cell Cycle. 2009 Jun. 1; 8(11):1698-710. Epub 2009 Jun. 27, for an alignment illustrating the key catalytic residues in all three Tet proteins, and the supplementary materials thereof (available at ftp site ftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html) for full length sequences (see, e.g., seq 2c); in some embodiments, the sequence includes amino acids 1418-2136 of Tet1 or the corresponding region in Tet2/3.

[0036] Other catalytic modules can be from the proteins identified in Iyer et al., 2009.

[0037] Methods of Use

[0038] The described Cas9-activator system is a useful and versatile tool for modifying the expression of endogenous genes. Current methods for achieving this require the generation of novel engineered DNA-binding proteins (such as engineered zinc finger or transcription activator-like effector DNA binding domains) for each site to be targeted. Because these methods demand expression of a large protein specifically engineered to bind each target site, they are limited in their capacity for multiplexing. Cas9-activators, however, require expression of only a single Cas9-activator protein, which can be targeted to multiple sites in the genome by expression of multiple short gRNAs. This system could therefore easily be used to simultaneously induce expression of a large number of genes. This capability will have broad utility, e.g., for basic biological research, where it can be used to study gene function and to manipulate the expression of multiple genes in a single pathway, and in synthetic biology, where it will enable researchers to create circuits in cell that are responsive to multiple input signals. The relative ease with which this technology can be implemented and adapted to multiplexing will make it a broadly useful technology with many wide-ranging applications.

[0039] The methods described herein include contacting cells with a nucleic acid encoding the Cas9-activators described herein, and nucleic acids encoding one or more guide RNAs directed to a selected gene, to thereby modulate expression of that gene. Guide RNAs, and methods of designing and expressing guide RNAs, are known in the art. See, e.g., Jinek et al., Science 2012; 337:816-821; Mali et al., Science. 2013 Feb. 15; 339(6121):823-6; Cong et al., Science. 2013 Feb. 15; 339(6121):819-23; and Hwang and Fu et al., Nat Biotechnol. 2013 March; 31(3):227-9). In some embodiments, the guideRNAs are directed to a region that is 100-800, e.g., about 500 bp upstream of the transcription start site. In some embodiments, vectors (e.g., plasmids) encoding more than one gRNA are used, e.g., plasmids encoding, 2, 3, 4, 5, or more gRNAs directed to different sites in the same region of the target gene.

[0040] Polypeptide Expression Systems

[0041] In order to use the fusion proteins described, it may be desirable to express the engineered proteins from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the fusion protein can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the fusion protein or for production of the fusion protein. The nucleic acid encoding the fusion protein can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

[0042] To obtain expression, the fusion protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0043] The promoter used to direct expression of the fusion protein nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the fusion protein is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the fusion protein. In addition, a preferred promoter for administration of the fusion protein can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

[0044] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the fusion protein, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

[0045] The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the fusion protein, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ. A preferred tag-fusion protein is the maltose binding protein (MBP). Such tag-fusion proteins can be used for purification of the engineered TALE repeat protein. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG

[0046] Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0047] Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the fusion protein encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

[0048] The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.

[0049] Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

[0050] Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.

[0051] In some embodiments, the fusion protein includes a nuclear localization domain which provides for the protein to be translocated to the nucleus. Several nuclear localization sequences (NLS) are known, and any suitable NLS can be used. For example, many NLSs have a plurality of basic amino acids, referred to as a bipartite basic repeats (reviewed in Garcia-Bustos et al, 1991, Biochim. Biophys. Acta, 1071:83-101). An NLS containing bipartite basic repeats can be placed in any portion of chimeric protein and results in the chimeric protein being localized inside the nucleus. In preferred embodiments a nuclear localization domain is incorporated into the final fusion protein, as the ultimate functions of the fusion proteins described herein will typically require the proteins to be localized in the nucleus. However, it may not be necessary to add a separate nuclear localization domain in cases where the DBD domain itself, or another functional domain within the final chimeric protein, has intrinsic nuclear translocation function.

[0052] The present invention includes the vectors and cells comprising the vectors.

Examples

[0053] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Engineering CRISPR/Cas Activator System

[0054] To express guide RNAs (gRNAs) in human cells, we engineered a vector that would express the full length chimeric gRNA (a fusion of crRNA and tracrRNA originally described by Jinek et al. (Science 2012)) driven by a U6 promoter. To create site-specific gRNAs, a pair of 26 nucleotide oligos are annealed and ligated into the BsmBI-digested vector backbone. See FIG. 4.

[0055] To engineer a Cas9-activator we introduced the D10A, H840A catalytic mutations (previously described in Jinek et al. Science 2012)) into either the wildtype or a codon-optimized Cas9 sequence (FIG. 5). These mutations render the Cas9 catalytically inactive so that it will no longer induce double-strand breaks. In one construct, a triple flag tag, nuclear localization signal and the VP64 activation domain were fused to the C-terminus of the inactive Cas9 (FIG. 6). Expression of this fusion protein is driven by the CMV promoter.

[0056] Cell Culture, Transfection and ELISA Assays were performed as follows.

[0057] Flp-In T-Rex 293 cells were maintained in Advanced DMEM supplemented with 10% FBS, 1% penstrep and 1% Glutamax (Invitrogen). Cells were transfected by Lipofectamine LTX (Invitrogen) according to manufacturer's instructions. Briefly, 160,000 293 cells were seeded in 24-well plates and transfected the following day with 250 ng gRNA plasmid, 250 ng Cas9-VP64 plasmid, 30 ng GFP, 0.5 ul Plus Reagent and 1.65 ul Lipofectamine LTX. Tissue culture media from transfected 293 cells was harvested 40 hours after transfection, and secreted VEGF-A protein assayed using R&D System's Human VEGF-A ELISA kit Human VEGF Immunoassay.

[0058] 17 gRNAs were engineered to target three different regions (500, 0 and +500 bp relative to the start site of transcription) in the human VEGFA promoter. Each gRNA was cotransfected with Cas9-VP64 into Hek293 cells and expression levels of VEGF-A protein was measured by ELISA. Of the 17 gRNAs, nine increased expression of VEGFA by three-fold or more as compared to an off-target gRNA control (FIG. 2). The greatest increase in VEGFA was observed in cells transfected with gRNA3, which induced protein expression by 18.7-fold. Interestingly, the three best gRNAs, and 6 of the 9 gRNAs capable of inducing expression by 3-fold or more, target the 500 region (500 bp upstream of the transcription start site).

[0059] Plasmids encoding one, or more, e.g., two or five, different guide RNAs targeted to the human VEGFA promoter were transfected together with a plasmid encoding the Cas9-activator and assessed for their abilities to activate transcription of the VEGFA promoter. Combinations of multiple gRNAs further increased the level of VEGFA activation (FIGS. 3A-B). Co-transfection of all 6 gRNAs targeted to the 500 region and all possible combinations of 5 of these 6 gRNAs resulted in a synergistic increase in VEGFA protein expression (FIG. 3A).

[0060] These experiments demonstrate that co-expression of a Cas9-activator protein (harboring the VP64 transcriptional activation domain) and a gRNA with 20 nt of sequence complementarity to sites in the human VEGF-A promoter in human HEK293 cells can result in upregulation of VEGF-A expression. Increases in VEGF-A protein were measured by ELISA assay and it was found that individual gRNAs can function together with a Cas9-activator fusion protein to increase VEGF-A protein levels by up to 18-fold (FIG. 2). Additionally, it was possible to achieve even greater increases in activation through transcriptional synergy by introducing multiple gRNAs targeting various sites in the same promoter together with Cas9-activator fusion proteins (FIGS. 3A-B).

Other Embodiments

[0061] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.