Patent Application
20250197462
Provided herein are compositions, systems, and methods for the generation, identification, and characterization of effector domains for activating and silencing gene expression. In particular, synthetic transcription factors comprising one or more of the effector domains and methods of using thereof are provided.
The contents of the electronic sequence listing titled STDU2-39797-601.xml (Size: 1,411,208 bytes; and Date of Creation: Aug. 17, 2022) is herein incorporated by reference in its entirety.
Previous efforts to engineer synthetic transcription factors have pulled activation and repressor domains from a small toolbox of previously discovered effector domains. New methods are needed to expand this toolbox.
Provided herein are compositions, systems, and method for the generation, identification, and characterization of effector domains for activating and silencing gene expression. In particular, high throughput systems are provided to discover and characterize effector domains. In some embodiments, provided herein is a high throughput approach to discover and characterize effector domains that greatly expands the toolbox. These domains satisfy a critical need to engineer enhanced synthetic transcription factors for applications in gene and cell therapy, synthetic biology, and functional genomics.
In some embodiments, the methods for identification of effector domains comprise: a) preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain linked to an inducible DNA binding domain; b) transforming reporter cells with the domain library, wherein a reporter cell comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a strong promoter, wherein the two-part reporter gene is capable of being silenced by a putative transcriptional repressor domain following treatment with an agent configured to induce the inducible DNA binding domain; c) treating the reporter cells with the agent for a length of time necessary for protein and mRNA degradation in the cell; d) separating reporter cells based on presence or absence of the surface marker, the fluorescent protein, or a combination thereof; e) sequencing the protein domains from the separated reporter cells; f) calculating for each protein domain sequence a ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof; and g) identifying protein domains as transcriptional repressor.
In some embodiments, the methods for identification of effector domains comprise: a) preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain linked to an inducible DNA binding domain; b) transforming reporter cells with the domain library, wherein the reporter cells comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a weak promoter, wherein the two-part reporter gene is capable of being activated by a putative transcriptional activator domain following treatment with an agent configured to induce the inducible DNA binding domain; c) treating the reporter cells with the agent for a length of time necessary for protein and mRNA production in the cell; d) separating reporter cells based on presence or absence of the surface marker, the fluorescent protein, or a combination thereof; e) sequencing the protein domains from the separated reporter cells; f) calculating for each protein domain sequence a ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof; and g) identifying protein domains as transcriptional activator.
In some embodiments, the methods further comprise stopping treatment of the reporter cells with the agent and repeating steps d-g one or more times. In some embodiments, steps d-g are repeated at least 48 hours after stopping treatment of the reported cells with the agent.
In some embodiments, each protein domain is less than or equal to 80 amino acids. In some embodiments, the protein domain is from a nuclear-localized protein. In some embodiments, the protein domain comprises amino acid sequences of the wild-type protein domains from nuclear-localized proteins. In some embodiments, the protein domain comprises mutated amino acid sequences of protein domains from nuclear-localized proteins.
In some embodiments, the inducible DNA binding domain comprises a tag.
In some embodiments, the methods further comprise measuring expression level of protein domains. In some embodiments, the expression level is determined by measuring a relative presence or absence of the tag on the DNA binding domain.
In some embodiments, the reporter cells are treated with the agent for at least 3 days. In some embodiments, the reporter cells are treated with the agent for at least 5 days. In some embodiments, the reporter cells are treated with the agent for at least 24 hours. In some embodiments, the reporter cells are treated with the agent for at least 48 hours.
In some embodiments, the protein domain is identified as a transcription repressor when log 2 of the ratio is at least two standard deviations from (e.g., higher than) the mean of a poorly expressed negative control.
In some embodiments, the protein domain is identified as a transcription activator when log 2 of the ratio is at least two standard deviations from (e.g., lower than) the mean of weakly expressing negative control.
Also provided herein are synthetic transcription factor comprising one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination thereof fused to a heterologous DNA binding domain. In some embodiments, at least one of the one or more transcriptional activator domains or at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-896. In some embodiments, at least one of the one or more transcriptional activator domains or at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1346-1401.
In some embodiments, the one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination thereof are selected from those found in any of Tables 1 to 7.
In some embodiments, the synthetic transcription factor comprises two or more transcriptional activator domains or two or more transcriptional repressors domains fused to a heterologous DNA binding domain. In select embodiments, the synthetic transcription factor comprises three transcriptional activator domains or transcriptional repressors domains fused to a heterologous DNA binding domain.
In some embodiments, at least one of the one or more transcriptional activator domain comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 563-664. In some embodiments, at least one of the one or more transcriptional activator domain is selected from those found in Table 2.
In some embodiments, the one or more transcriptional activator domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 563, 564, 565, 566, 575, 576, 579, and 580. In some embodiments, the one or more transcriptional activator domains comprise an amino acid sequence having: SEQ ID NO: 563 or SEQ ID NO: 564; SEQ ID NO: 565 or SEQ ID NO: 566; SEQ ID NO: 575 or SEQ ID NO: 576; SEQ ID NO: 579 or SEQ ID NO: 580; or a combination thereof. In some embodiments, the one or more transcriptional activator domains comprises an amino acid sequence having two or more of: SEQ ID NOs: 563, 565, 575, and 579. In some embodiments, the one or more transcriptional activator domains comprises an amino acid sequence having SEQ ID NOs: 563, 565, and 579. In some embodiments, the one or more transcriptional activator domains further comprise an amino acid sequence of SEQ ID NO: 575.
In some embodiments, the synthetic transcription factor comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1331-1344. In some embodiments, the synthetic transcription factor comprises an amino acid sequence of any of SEQ ID NOs: 1331-1344.
In some embodiments, the at least one of the one or more transcriptional repressor domain comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-562 and 665-896. In some embodiments, the at least one of the one or more transcriptional repressor domain comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1346-1401. In some embodiments, the at least one of the one or more transcriptional repressor domain is selected from those found in any of Tables 1, 3, 4 or 6.
In some embodiments, the one or more transcriptional repressor domains comprise an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 32, 36, 363, or a combination thereof. In some embodiments, the synthetic transcription factor comprises an amino acid sequence of SEQ ID NO: 1345.
In some embodiments, the one or more transcriptional activator domain or the one or more transcriptional repressor domain is identified by the methods disclosed herein.
In some embodiments, the heterologous DNA binding domain comprises a programmable DNA binding domain. In some embodiments, the DNA binding domain is derived from a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein. In some embodiments, the DNA binding domain is derived from Transcription activator-like effectors (TALEs) domains. In some embodiments, the DNA binding domain is an inducible DNA binding system.
Also provided herein are nucleic acids encoding a synthetic transcription factor or an effector domain, as disclosed herein. In some embodiments, the nucleic acid in under control of an inducible promoter. In some embodiments, the nucleic acid in under control of a tissue specific promoter. In some embodiments, the nucleic acid encodes at least one additional transcription factor or effector domain.
Further provided herein is a composition or system comprising a synthetic transcription factor, a nucleic acid, a vector, or a cell as disclosed herein. In some embodiments, the composition comprises two or more synthetic transcription factors, nucleic acids, vectors, or cells. In some embodiments, the composition further comprises a guide RNA or a nucleic acid encoding a guide RNA.
Additionally, provided are methods of modulating the expression of at least one target gene in a cell. In some embodiments, the at least one target gene in a cell is an endogenous gene, an exogenous gene, or a combination thereof. The methods comprise introducing into the cell at least one synthetic transcription factor, nucleic acid, vector, or composition or system, as described herein. The gene expression of the at least one target gene is modulated when gene expression levels of the at least one target gene are increased or decreased compared to normal gene expression levels for the at least one target gene. In some embodiments, the synthetic transcription factor comprises a Cas protein DNA binding domain and the method further comprises contacting the cell with at least one guide RNA.
In some embodiments, the cell is in vitro (e.g., ex vivo) or in a subject.
In some embodiments, the gene expression of at least two genes are modulated.
Systems and methods to generate a catalog of compact transcriptional effector domains is provided. Further, in some embodiments, this catalog of domains is fused onto DNA binding domains to engineer synthetic transcription factors. These find use to perform targeted and tunable regulation of gene expression in eukaryotic (or other) cells. This technology leverages a high-throughput platform to screen and characterize tens of thousands of synthetic transcription factors in cells. These synthetic transcription factors are fusions between a DNA binding domain and a transcriptional effector domain. The system has been used to generate hundreds of short effector domains (e.g., 80 amino acids) and a high-throughput process for shortening them further to the minimally sufficient sequences (e.g., 10 amino acids), which is an advantage for delivery (e.g., packaging in viral vectors). The targeting of these fusions generates local regulation of mRNA transcription, either negatively or positively depending on the effector domain. Some of these synthetic transcription factors mediate long-term epigenetic regulation that persists after the factor itself has been released from the target.
Previously, a limited number of transcriptional effector domains were available for the engineering of synthetic transcription factors. To address this limitation, provided herein is a high-throughput approach to screening and quantifying the function of transcriptional effectors domains. This approach enabled the discovery of hundreds of effector domains that can upregulate or downregulate transcription in a targeted manner when fused onto a DNA binding domain. This process also finds use to identify mutants of effector domains with enhanced activity. These effector domains find use to engineer synthetic transcription factors for applications in gene and cell therapy, synthetic biology, and functional genomics.
Exemplary applications include, but are not limited to:
Targeted repression/activation of endogenous genes with fusions of programmable DNA binding domains (e.g., dCas9, dCas12a, zinc finger, TALE) to transcriptional effector domains.
Targeted repression/activation of exogenous genes with fusions of programmable DNA binding domains (e.g., dCas9, dCas12a, zinc finger, TALE) to transcriptional effector domains.
Gene and cell therapy (e.g., to silence a pathogenic transcript in a patient) or in research.
Synthetic transcription factors find use to perturb the expression of multiple genes simultaneously (e.g., to perform high-throughput genetic interaction mapping with CRISPRi/a screens using multiple guide RNAs).
Use in synthetic transcription factors in genetic circuits, e.g., inducible gene expression or more complex circuits. These circuits find use in gene therapy (e.g., AAV delivery of antibodies) and cell therapy (e.g., ex vivo engineering of CAR-T cells) to achieve therapeutic gene expression outputs in response to environmental and small molecule inputs.
The new transcriptional effector domains provided herein have several advantages for applications that rely on synthetic transcription factors. Short domains were identified (e.g., 80 amino acids or less) and a high-throughput process was generated for shortening them further to the minimally sufficient sequence, which is an advantage for delivery (e.g., packaging in viral vectors). In some cases, potent effector domains were identified that were as short as 10 amino acids. In some embodiments, the domains are extracted from human proteins, which provides the advantage of reducing immunogenicity in comparison to viral effector domains. Most of the domains generated have not been reported as transcriptional effectors previously. In addition, a high-throughput process is provided for testing mutations in these domains in order to identify enhanced variants. The high-throughput approach is more readily aided by the development of an artificial cell surface marker that provides more efficient, inexpensive, and rapid screening of these libraries using magnetic separation. This is an advantage over the more conventional approach of sorting libraries based on fluorescent reporter gene expression.
The collection of domains identified is large and diverse, and the platform readily enables new combinations of domains to be tested as fusions in high-throughput to create synthetic transcription factors with new properties (e.g., compositions of two repressor domains to achieve a combination of fast silencing and permanent silencing).
Hundreds of previously uncharacterized or unknown effector domains that can silence or active transcription and can be fused onto DNA binding domains. For example, a high-throughput approach for screening single domains and pairs of domains using lentiviral screens in human cells is provided. The high-throughput approach is more readily enabled by the development of an artificial cell surface marker that provides more efficient, inexpensive, and rapid screening of these libraries using a magnetic separation.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
The term “antibody,” as used herein, refers to a protein that is endogenously used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Typically, an antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (κ) or lambda (λ), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the β sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). The framework regions are connected by three CDRs. As discussed above, the three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding. The CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions. While the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular toxicity via interactions with effector molecules and cells.
The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding fragment” of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Any antigen-binding fragment of the antibody described herein is within the scope of the invention. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab′ fragment, which results from breaking the disulfide bridge of an F(ab′)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
As used herein, a “nucleic acid” or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41 (14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97:5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122:8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide” and “protein,” are used interchangeably herein.
As used herein, the term “percent sequence identity” refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Hence, in case a nucleic acid according to the technology is longer than a reference sequence, additional nucleotides in the nucleic acid, that do not align with the reference sequence, are not taken into account for determining sequence identity. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3×, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215 (3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106 (10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21 (7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25 (17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).
A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.
The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified,” “mutant,” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
Disclosed herein are methods for identifying transcriptional effector (e.g., activator and repressor) domains. In some embodiments, the methods comprise: preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain from nuclear-localized proteins linked to an inducible DNA binding domain; transforming reporter cells with the domain library, wherein the reporter cells comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a promoter, wherein the two-part reporter gene is capable of being modulated by a putative transcriptional effector domain following treatment with an agent configured to induce the inducible DNA binding domain; treating the reporter cells with the agent for a length of time necessary for protein and mRNA levels to be altered in the cell (e.g., increased due to production or decreased due to degradation); sequencing the protein domains from the separated reporter cells; calculating for each protein domain sequence a ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof, and identifying protein domains as transcriptional repressors or activators.
The methods comprise preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain from nuclear-localized proteins linked to an inducible DNA binding domain. The protein domain may be less than or equal to 80 amino acids. In some embodiments, the protein domain may be about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 15 amino acids, about 10 amino acids, or about 5 amino acids.
The protein domain may be derived from any known protein. In some embodiments, the protein domain is from a nuclear-localized protein. A nuclear-localized protein includes those proteins which are or can localize to the nucleus fully or partially during the life-cycle of the protein. In some embodiments, the protein domain comprises amino acid sequences of the wild-type protein domain from nuclear-localized proteins. In some embodiments, the protein domain comprises mutated amino acid sequences of protein domains from nuclear-localized proteins.
The inducible DNA binding domain may use any system for induction of DNA binding, including, but not limited to, tetracycline Tet/DOX inducible systems, light inducible systems, Abscisic acid (ABA) inducible systems, cumate systems, 40HT/estrogen inducible systems, ecdysone-based inducible systems, and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.
In some embodiments, the inducible DNA binding domain comprises a tag. The tag may include any tag known in the art, including tags removable by chemical or enzymatic means. Suitable tags for use in the present method include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), a polyhistidine (PolyHis) tag, an ALFA-tag, a V5-tag, a Myc-tag, a hemagglutinin (HA)-tag, a Spot-tag, a T7-tag, an NE-tag, a Calmodulin-tag, a polyglutamate tag, a polyarginine tag, a FLAG tag, and the like.
The methods comprise transforming reporter cells with the domain library, wherein the reporter cell comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a promoter, wherein the two-part reporter gene is capable of being modulated by a putative transcriptional effector domain following treatment with an agent configured to induce the inducible DNA binding domain.
The promoter may confer a high rate of transcription (a strong promoter) or confer a low rate of transcription (weak promoter). Many promoter libraries have been established experimentally and choice of promoter and promoter strength is dependent on cell type. In some embodiments, when identifying transcriptional activator domains, a weak promoter may be used. In some embodiments, when identifying transcriptional repressor domains, a strong promoter may be used.
Cell surface markers include proteins and carbohydrates which are attached to the cellular membrane. Cell surface markers are generally known in the art for a variety of cell types and can be expressed in a reporter cell of choice based on known molecular biology methods. The surface marker may be a synthetic surface marker comprising marker polypeptide attached to a transmembrane domain. For example, the marker polypeptide may include an antibody or a fragment thereof (e.g., Fc region) attached to a transmembrane domain. In some embodiments, the marker polypeptide is human IgG1 Fc region and the synthetic surface marker comprises human IgG1 Fc region attached to a transmembrane domain.
Fluorescent proteins are well known in the art and include proteins adapted to fluoresce in various cellular compartments and as a result of varying wavelengths of incoming light. Examples of fluorescent proteins include phycobiliproteins, cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced orange fluorescent protein (OFP), enhanced green fluorescent protein (eGFP), modified green fluorescent protein (emGFP), enhanced yellow fluorescent protein (eYFP) and/or monomeric red fluorescent protein (mRFP) and derivatives and variants thereof.
The methods comprise separating reporter cells based on presence or absence of the surface marker, the fluorescent protein, or a combination thereof. A number of cell separation techniques are known in the art are suitable for use with the methods disclosed herein, including, for example, immunomagnetic cell separation, fluorescent-activated cell sorting (FACS), and microfluidic cell sorting. In some embodiments, cell separation comprises immunomagnetic cell separation.
In some embodiments, the method further comprises stopping treatment of the reporter cells with the agent and repeating the separating, sequencing, calculating, and identifying steps one or more times. In some embodiments, the steps are repeated at least 48 hours after stopping treatment of the reported cells with the agent.
In some embodiments, the method further comprises measuring expression level of protein domains. The expression level of the protein domains can be determined using any methods known in the art, including immunoblotting and immunoassays for the protein itself or any tags or labels thereof. In some embodiments, the expression level is determined by measuring a relative presence or absence of the tag on the DNA binding domain.
In some embodiments, the methods identify a transcriptional repressor domain. In some embodiments, the methods comprise, a) preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain linked to an inducible DNA binding domain; b) transforming reporter cells with the domain library, wherein a reporter cell comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a strong promoter, wherein the two-part reporter gene is capable of being silenced by a putative transcriptional repressor domain following treatment with an agent configured to induce the inducible DNA binding domain; c) treating the reporter cells with the agent for a length of time necessary for protein and mRNA degradation in the cell; d) separating reporter cells based on presence or absence of the surface marker, the fluorescent protein, or a combination thereof; e) sequencing the protein domains from the separated reporter cells; f) calculating for each protein domain sequence a ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof; and g) identifying protein domains as transcriptional repressor.
In some embodiments, the reporter cells are treated with the agent for at least 3 days. For, example the reporter cells may be treated with the agent for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 14 days, or more. In some embodiments, the reporter cells at treated with the agent for 3-12 days, 3-10 days, 3-7 days, or 3-5 days.
The protein domain is identified as a transcriptional repressor when log 2 of the ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof is at least two standard deviations from (e.g., greater than) the mean of a negative control (See
In some embodiments, the methods identify a transcriptional activator domain. In some embodiments, the methods comprise, a) preparing a domain library comprising a plurality of nucleic acid sequences each configured to express a fusion protein comprising a protein domain linked to an inducible DNA binding domain; b) transforming reporter cells with the domain library, wherein the reporter cells comprises a two-part reporter gene comprising a surface marker and a fluorescent protein under the control of a weak promoter, wherein the two-part reporter gene is capable of being activated by a putative transcriptional activator domain following treatment with an agent configured to induce the inducible DNA binding domain; c) treating the reporter cells with the agent for a length of time necessary for protein and mRNA production in the cell; d) separating reporter cells based on presence or absence of the surface marker, the fluorescent protein, or a combination thereof; e) sequencing the protein domains from the separated reporter cells; f) calculating for each protein domain sequence a ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof; and g) identifying protein domains as transcriptional repressor.
In some embodiments, the reporter cells are treated with the agent for at least 24 hours. For, example the reporter cells may be treated with the agent for at least 24 hours (1 day), at least 36 hours, at least 48 hours (2 days), at least 60 hours, at least 72 hours (3 days), at least 94 hours, at least 106 hours (4 days) or more. In some embodiments, the reporter cells are treated for between 24 and 72 hours or between 36 and 60 hours.
The protein domain is identified as a transcriptional activator when log 2 of the ratio of sequencing counts from reporter cells not having the surface marker, the fluorescent protein, or a combination thereof to sequencing counts from reporter cells having the surface marker, the fluorescent protein, or a combination thereof is at least two standard deviations from (e.g., less than) the mean of a negative control. (See
The present disclosure also provides synthetic transcription factors comprising one or more transcriptional effector domains fused to a heterologous DNA binding domain. As used herein, the term “transcription factor” refers to a protein or polypeptide that interacts with, directly or indirectly, specific DNA sequences associated with a genomic locus or gene of interest to block or recruit RNA polymerase activity to the promoter site for a gene or set of genes.
In some embodiments the synthetic transcription factor comprises one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination thereof fused to a heterologous DNA binding domain. In some embodiments, the at least one of the one or more transcriptional activator domains or at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, 99%) identity to any of SEQ ID NOs: 1-896. In some embodiments, at least one of the one or more transcriptional activator domains or at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1346-1401.
In some embodiments, the one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination thereof are selected from those found in any of Tables 1 to 7.
In some embodiments, the one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 35, 36, 37, 38, 41, 42, 67, 68, 81, 82, 83, 84, 85, 86, 91, 92, 99, 100, 107, 108, 109, 110, 119, 120, 123, 124, 135, 136, 141, 142, 153, 154, 167, 168, 169, 170, 177, 178, 183, 184, 193, 194, 203, 204, 205, 206, 211, 212, 229, 230, 237, 238, 241, 242, 259, 260, 271, 272, 281, 282, 283, 284, 285, 286, 293, 294, 325, 326, 327, 328, 329, 330, 333, 334, 347, 348, 349, 350, 355, 356, 363, 364, 365, 366, 379, 380, 387, 388, 391, 392, 395, 396, 401, 402, 409, 410, 413, 414, 415, 416, 421, 422, 425, 426, 437, 438, 441, 442, 457, 458, 467, 468, 489, 490, 499, 500, 509, 510, 513, 514, 523, 524, 525, 526, 527, 528, 539, 540, 541, 542, 547, 548, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 577, 578, 579, $80, 583, 584, 585, 586, 593, 594, 597, 598, 599, 600, 601, 602, 605, 606, 613, 614, 615, 616, 623, 624, 627, 628, 633, 634, 639, 640, 643, 644, 647, and 648. In some embodiments, the one or more transcriptional activator domains, one or more transcriptional repressor domains, or a combination comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 35, 36, 37, 38, 41, 42, 67, 68, 81, 82, 83, 84, 85, 86, 91, 92, 99, 100, 107, 108, 109, 110, 119, 120, 123, 124, 135, 136, 141, 142, 153, 154, 167, 168,169, 170, 177, 178, 183, 184, 193, 194, 203, 204, 205, 206, 211, 212, 229, 230, 237, 238, 241, 242, 259, 260, 271, 272, 281, 282, 283, 284, 285, 286, 293, 294, 325, 326, 327, 328, 329, 330, 333, 334, 347, 348, 349, 350, 355, 356, 363, 364, 365, 366, 379, 380, 387, 388, 391, 392, 395, 396, 401, 402, 409, 410, 413, 414, 415, 416, 421, 422, 425, 426, 437, 438, 441, 442, 457, 458, 467, 468, 489, 490, 499, 500, 509, 510, 513, 514, 523, 524, 525, 526, 527, 528, 539, 540, 541, 542, 547, 548, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 577, 578, 579, 580, 583, 584, 585, 586, 593, 594, 597, 598, 599, 600, 601, 602, 605, 606, 613, 614, 615, 616, 623, 624, 627, 628, 633, 634, 639, 640, 643, 644, 647, and 648.
In some embodiments, the one or more transcriptional activator domain, the one or more transcriptional repressor domain, or combination thereof is identified by the methods disclosed herein.
In some embodiments, the synthetic transcription factor comprises two or more transcription effector domains (e.g., transcriptional activator domains, transcriptional repressor domains, or a combination thereof) fused to a heterologous DNA binding domain. In some embodiments, the synthetic transcription factor comprises two or more transcriptional activator domains or two or more transcriptional repressors domains fused to a heterologous DNA binding domain. The two or more effector domains can be fused to the DNA binding domain in any orientation, and may be separated from each other with an amino acid linker. In select embodiments, the synthetic transcription factor comprises two or more transcription effector domains (e.g., transcriptional activator domains, transcriptional repressor domains, or a combination thereof) fused to a heterologous DNA binding domain.
In some embodiments, when the synthetic transcription factor comprises more than one transcription effector domains, the synthetic transcription factor may comprise at least one transcriptional activator domain or at least one transcriptional repressor domain as disclosed herein with at least one additional effector domain known in the art. See for example, Tycko J. et al., Cell. 2020 Dec. 23; 183 (7): 2020-2035, incorporated herein by reference in its entirety. In some embodiments, the one or more transcriptional activator domain, the one or more transcriptional repressor domain is identified by the methods described herein.
In some embodiments, when the synthetic transcription factor comprises more than one transcription effector domains, at least one of the one or more transcriptional activator domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 563-664. In some embodiments, at least one of the one or more transcriptional activator domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 563-596. In some embodiments, at least one of the one or more transcriptional activator domain is selected from those found in Table 2.
In some embodiments, the one or more transcriptional activator domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 563, 564, 565, 566, 575, 576, 579, and 580.
In some embodiments, the one or more transcriptional activator domains comprise: SEQ ID NO: 563 or SEQ ID NO: 564; SEQ ID NO: 565 or SEQ ID NO: 566; SEQ ID NO: 575 or SEQ ID NO: 576; SEQ ID NO: 579 or SEQ ID NO: 580; or a combination thereof. In some embodiments, the one or more transcriptional activator domains comprises two or more of SEQ ID NOs: 563, 565, 575, and 579. For example, the one or more transcriptional activator domains may include SEQ ID NOs: 563 and 565; SEQ ID NOs: 563 and 575; SEQ ID NOs: 563 and 579; SEQ ID NOs: 565 and 575; SEQ ID NOs: 565 and 579; or SEQ ID NOs: 575 and 579.
The one or more transcriptional activator domains may comprise three of more of SEQ ID NOs: 563, 565, 575, and 579. In some embodiments, the one or more transcriptional activator domains comprise SEQ ID NOs: 563, 565, and 579. In some embodiments, the one or more transcriptional activator domains comprise SEQ ID NOs: 563, 565, 575, and 579.
In some embodiments, the synthetic transcription factor comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1331-1344. In some embodiments, the synthetic transcription factor comprises an amino acid sequence of any of SEQ ID NOs: 1331-1344.
In some embodiments, when the synthetic transcription factor comprises more than one transcription effector domains, at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1-562 and 665-896. In some embodiments, when the synthetic transcription factor comprises more than one transcription effector domains, at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 1346-1401. In some embodiments, at least one of the one or more transcriptional repressor domains comprises an amino acid sequence having at least 70% identity to any of SEQ ID NO: 666. In some embodiments, at least one of the one or more transcriptional repressor domains is selected from those found in any of Tables 1, 3, 4 or 6.
In some embodiments, the one or more transcriptional repressor domains comprise an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 32, 36, 363, or a combination thereof. In some embodiments, the one or more transcriptional repressor domains comprises SEQ ID NO: 32. In some embodiments, the one or more transcriptional repressor domains comprises SEQ ID NO: 36. In some embodiments, the one or more transcriptional repressor domains comprises SEQ ID NO: 363. In some embodiments, the synthetic transcription factor comprises at least one transcriptional repressor domains comprising an amino acid sequence having at least 70% identity to any of SEQ ID NOs: 32, 36, or 363, and at least one additional transcriptional repressor domain. The at least one additional transcriptional repressor domain may comprise any additional repressor domain, disclosed herein or known in the art. In some embodiments, the at least one additional transcriptional repressor domain comprises SEQ ID NO: 1347. In some embodiments, the synthetic transcription factor comprises an amino acid sequence of SEQ ID NO: 1345.
The DNA binding domain is any polypeptide which is capable of binding double- or single-stranded DNA, generally or with sequence specificity. DNA binding domains include those polypeptides having helix-turn-helix motifs, zinc fingers, leucine zippers, HMG-box (high mobility group box) domains, winged helix region, winged helix-turn-helix region, helix-loop-helix region, immunoglobulin fold, B3 domain, Wor3 domain, TAL effector DNA-binding domain and the like. The heterologous DNA binding domains may be a natural binding domain. In some embodiments, the heterologous DNA binding domain comprises a programmable DNA binding domain, e.g., a DNA binding domain engineered, for example by altering one or more amino acid of a natural DNA binding domain to bind to a predetermined nucleotide sequence.
In some embodiments, the DNA binding domain is capable of binding directly to the target DNA sequences.
The DNA-binding domain may be derived from domains found in naturally occurring Transcription activator-like effectors (TALEs), such as AvrBs3, Hax2, Hax3 or Hax4 (Bonas et al. 1989. Mol Gen Genet 218 (1): 127-36; Kay et al. 2005 Mol Plant Microbe Interact 18 (8): 838-48). TALEs have a modular DNA-binding domain consisting of repetitive sequences of residues; each repeat region consists of 34 amino acids. A pair of residues at the 12th and 13th position of each repeat region determines the nucleotide specificity and combining of the regions allows synthesis of sequence-specific TALE DNA-binding domains. In some embodiments, the TALE DNA binding domains may be engineered using known methods to provide a DNA binding domain with chosen specificity for any target sequence. The DNA binding domain may comprise multiple (e.g., 2, 3, 4, 5, 6, 10, 20, or more) Tal effector DNA-binding motifs. In particular, any number of nucleotide-specific Tal effector motifs can be combined to form a sequence-specific DNA-binding domain to be employed in the present transcription factor.
In some embodiments, the DNA binding domain associates with the target DNA in concert with an exogenous factor.
In some embodiments, the DNA binding domain is derived from a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein (e.g., catalytically dead Cas9) and associates with the target DNA through a guide RNA. The gRNA itself comprises a sequence complementary to one strand of the DNA target sequence and a scaffold sequence which binds and recruits Cas9 to the target DNA sequence. The transcription factors described herein may be useful for CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa).
The guide RNA (gRNA) may be a crRNA, crRNA/tracrRNA (or single guide RNA, sgRNA). The gRNA may be a non-naturally occurring gRNA. The terms “gRNA,” “guide RNA” and “guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the binding specificity of the Cas protein. A gRNA hybridizes to (complementary to, partially or completely) the DNA target sequence.
The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be any length necessary for selective hybridization. gRNAs or sgRNA(s) can be between about 5 and about 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer).
To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLOS ONE, 10 (3): (2015)); Zhu et al. (PLOS ONE, 9 (9) (2014)); Xiao et al. (Bioinformatics. January 21 (2014)); Heigwer et al. (Nat Methods, 11 (2): 122-123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases.
The present disclosure also provides synthetic transcription factors comprising one or more transcriptional effector domains fused to an exogenous factor which associates with a second exogenous factor comprising a DNA binding domain. Such inducible systems include, but not limited to, tetracycline Tet/DOX inducible systems, light inducible systems, Abscisic acid (ABA) inducible systems, cumate systems, 40HT/estrogen inducible systems, ecdysone-based inducible systems, and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.
The present disclosure also provides nucleic acids encoding a synthetic transcription factor or a transcriptional effector (e.g., activator or repressor) domain, as disclosed herein. For example, the effector domains may be encoded by nucleic acids disclosed in Tables 1-3. In some embodiments, the effector domains may be encoded by nucleic acids having at least 70% identity to any of SEQ ID NOs: 897-1329. In some embodiments, the nucleic acid encodes one or more synthetic transcription factor or one or more effector domain.
Nucleic acids of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable, or inducible, cell type specific, tissue-specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter/regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1-alpha (EF1-α) promoter with or without the EF1-α intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell.
Moreover, inducible expression can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible promoter/regulatory sequence. Promoters that are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter/regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.
The present disclosure also provides for vectors containing the nucleic acids and cells containing the nucleic acids or vectors, thereof. The vectors may be used to propagate the nucleic acid in an appropriate cell and/or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
To construct cells that express the present transcription factors, expression vectors for stable or transient expression of the present system may be constructed via conventional methods and introduced into cells. For example, nucleic acids encoding the components the disclose transcription factors, or other nucleic acids or proteins, may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors/plasmids/viral vectors should be suitable for integration and replication in eukaryotic cells.
In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference.
The vectors of the present disclosure may direct the expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining.
Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene for selection of stable or transient transfectants in host cells; transcription termination and RNA processing signals; 5′- and 3′-untranslated regions; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, neomycin, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae.
When introduced into a cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA.
Thus, the disclosure further provides for cells comprising a synthetic transcription factor, a nucleic acid, or a vector, as disclosed herein.
Conventional viral and non-viral based gene transfer methods can be used to introduce the nucleic acids into cells, tissues, or a subject. Such methods can be used to administer the nucleic acids to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle.
Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. A variety of viral constructs may be used to deliver the present nucleic acids to the cells, tissues and/or a subject. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1): 33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference.
The nucleic acids or transcription factors may be delivered by any suitable means. In certain embodiments, the nucleic acids or proteins thereof are delivered in vivo. In other embodiments, the nucleic acids or proteins thereof are delivered to isolated/cultured cells in vitro or ex vivo to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition.
Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of host cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and/or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.
Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110 (6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, the construct containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, the construct or the nucleic acid encoding the components of the present system is a DNA molecule. In some embodiments, the nucleic acid encoding the components of the present system is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the components of the present system is an RNA molecule, which may be electroporated to cells.
Additionally, delivery vehicles such as nanoparticle- and lipid-based delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1:27) and Ibraheem et al. (Int J Pharm. 2014 Jan. 1; 459 (1-2): 70-83), incorporated herein by reference.
As such, the disclosure provides an isolated cell comprising the vector(s) or nucleic acid(s) disclosed herein. Preferred cells are those that can be easily and reliably grown, have reasonably fast growth rates, have well characterized expression systems, and can be transformed or transfected easily and efficiently. Examples of suitable prokaryotic cells include, but are not limited to, cells from the genera Bacillus (such as Bacillus subtilis and Bacillus brevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces, Salmonella, and Envinia. Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells, and mammalian cells. Examples of suitable yeast cells include those from the genera Kluyveromyces, Pichia, Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Exemplary insect cells include Sf-9 and HIS (Invitrogen, Carlsbad, Calif.) and are described in, for example, Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67:4566-4579 (1993), incorporated herein by reference. Desirably, the cell is a mammalian cell, and in some embodiments, the cell is a human cell. A number of suitable mammalian and human host cells are known in the art, and many are available from the American Type Culture Collection (ATCC, Manassas, Va.). Examples of suitable mammalian cells include, but are not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), as well as the CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host cells include primate, rodent, and human cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, HEK, A549, HepG2, mouse L-929 cells, and BHK or HaK hamster cell lines.
Methods for selecting suitable mammalian cells and methods for transformation, culture, amplification, screening, and purification of cells are known in the art.
The present invention is also directed to compositions or systems comprising a synthetic transcription factor, a nucleic acid, a vector, or a cell, as described herein. In some embodiments, the compositions or system comprises two or more synthetic transcription factors, nucleic acids, vectors, or cells.
In some embodiments, the composition or system further comprises a gRNA. The gRNA may be encoded on the same nucleic acid as a synthetic transcription factor or a different nucleic acid. In some embodiments, the vector encoding a synthetic transcription factor may further encode a gRNA, under the same or different promoter. In some embodiments, the gRNA is encoded on its own vector, separated from that of the transcription factor.
The present disclosure also provides methods of modulating the expression of at least one target gene in a cell, the method comprising introducing into the cell at least one synthetic transcription factor, nucleic acid, vector, or composition or system as described herein. In some embodiments, the gene expression of at least two genes is modulated.
In some embodiments, the gene is an endogenous gene. In some embodiments, the gene is an exogenous gene. In some embodiments, the gene is on an exogenous vector. In some embodiments, the exogenous gene was introduced into the cell as part of a gene therapy regime. For example, a controllable and activatable vector expressing secreted hepatocyte growth factor has broad therapeutic potential due to its capacity to induce regeneration of health tissues when transduced into the tissue or interest or neighboring tissues (e.g., liver to regenerate damaged liver or kidney, heart for prevention of/and regeneration after heart attack, brain for neurogenesis in Alzheimer's and Parkinson's diseases).
Modulation of expression comprises increasing or decreasing gene expression compared to normal gene expression for the target gene. When the gene expression of at least two genes is modulation, both genes may have increased gene expression, both gene may have decreased gene expression, or one gene may have increased gene expression and the other may have decreased gene expression.
The cell may be a prokaryotic or eukaryotic cell. In preferred embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is ex vivo.
In some embodiments, the cell is in an organism or host, such that introducing the disclosed systems, compositions, vectors into the cell comprises administration to a subject. The method may comprise providing or administering to the subject, in vivo, or by transplantation of ex vivo treated cells, at least one synthetic transcription factor, nucleic acid, vector, or composition or system as described herein.
A “subject” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human.
As used herein, the terms “providing,” “administering,” “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a subject by a method or route which results in at least partial localization of the system to a desired site. The systems can be administered by any appropriate route which results in delivery to a desired location in the subject.
Also within the scope of the present disclosure are kits including at least one or all of at least one nucleic acid encoding an effector domain, or a DNA binding domain, or a combination thereof, at least one synthetic transcription factor, or nucleic acid encoding thereof, vectors encoding at least one effector domain or at least one synthetic transcription factor, a composition or system as described herein, a cell comprising an effector domain, a DNA binding domain, a synthetic transcription factor, or a nucleic acid encoding any of thereof, a reporter cell as described herein and a two-part reporter gene as described herein or a nucleic acid encoding thereof.
The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of use of the components for the methods of identifying repressor domains or methods of modulating gene expression.
The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.
The kit may further comprise a device for holding or administering the present system or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and/or a syringe.
The present disclosure also provides for kits for performing the methods or producing the components in vitro. The kit may include the components of the present system. Optional components of the kit include one or more of the following: (1) buffer constituents, (2) control plasmid, (3) sequencing primers.
Human gene expression is regulated by thousands of proteins that activate or repress transcription. We lack a complete and quantitative description of these proteins' effector domains, the domains sufficient to mediate changes in gene expression. To systematically measure transcriptional effector domains in human cells, provided herein is a high-throughput assay in which libraries of protein domains are fused to a DNA-binding domain and recruited to a reporter gene. The cells are then separated by reporter expression level and the library of protein domains is sequenced. The reporter is a synthetic surface marker that facilitates simple separation of tens of millions of cells into high- and low-expression populations, using magnetic beads.
Gene silencing and epigenetic memory was quantified after recruitment of all nuclear protein domains of ≤80 amino acids. Using the measurements for the complete families of >300 KRAB domains and >200 Homeodomains, relationships were discovered between transcription factor's repressor domain strength and their evolutionary history and developmental role. Further, a deep mutational scan of the ZNF10 KRAB effector function and identified substitutions with enhanced stability and repression compared to the KRAB domain used in CRISPRi. To search for effector domains beyond previously annotated regions, the sequence of 238 repressor complex proteins was tiled and novel repressor domains as short as 10 amino acids were discovered in unannotated regions of large chromatin regulators, including the non-canonical polycomb 1.6 recruitment protein MGA. Greater than 20 repressors were individually characterized and all of them were found to silence a reporter gene in an all-or-nothing fashion at the single-cell level, but with distinct dynamics of silencing and epigenetic memory.
In addition, new activator domains in nuclear proteins were discovered, including a highly divergent acidic KRAB domain variant.
Together, these results demonstrate a strategy for systematic measurement of transcriptional effector domain activity in human cells, and expand the number of compact transcriptional effector domains that can be applied in synthetic transcription and epigenetic perturbation technologies.
Problems addressed by the present technology:
The systems and methods provided herein can measure regulatory domains of activators and repressors capability to change the output from a reporter promoter. Historically, this requires low throughput work so relatively few effector domains have been measured. The systems and methods provided herein off an alternative high-throughput assay.
The systems and methods find use, for example, for: a. understanding gene regulation, predicting function of non-coding regulatory elements that these proteins bind to; and b. identifying effector domains for epigenome perturbation tools.
Previously, a limited number of transcriptional effector domains were available for the engineering of synthetic transcription factors. To address this limitation, provided herein is a high-throughput approach to screening and quantifying the function of transcriptional effectors domains. This approach enabled the discovery of hundreds of effector domains that can upregulate or downregulate transcription in a targeted manner when fused onto a DNA binding domain. This process also identifies mutants of effector domains with enhanced activity. These effector domains can be used to engineer synthetic transcription factors for applications in gene and cell therapy, synthetic biology, and functional genomics.
The new transcriptional effector domains provided herein have several advantages for applications that rely on synthetic transcription factors. We identify short domains (≤80 amino acids) and a high-throughput process for shortening them further to the minimally sufficient sequence, which is an advantage for delivery (e.g., packaging in viral vectors). In some cases, we identify potent effector domains that are as short as 10 amino acids. The domains are extracted from human proteins, which provides the advantage of reducing immunogenicity in comparison to viral effector domains. Most of these domains have not been reported as transcriptional effectors previously.
By performing high-throughput recruitment with the Pfam domain library against both a strong pEF promoter and a weak minCMV promoter, both repressor and activator domains were able to be measured. One possible reason that many more repressors were found is that they are more often autonomous stably-folding sequences which meet the Pfam definition of a domain while TADs are more often disordered or low-complexity regions that are not annotated as domains. Another possible reason could be that co-activators are more limiting in the nucleus than co-repressors (Gillespie Mol Cell 2020), which implies lower expression of activator domains could result in greater activation strength, but this effect would not be expected to completely mask signal in the screen. New library designs that tile transcription factors or focus on regions with TAD-like signatures (e.g., acidity) will uncover additional activator domains.
In addition, a high-throughput process for testing mutations in these domains in order to identify enhanced variants is disclosed herein. The high-throughput approach is more readily enabled by development of an artificial cell surface marker that provides more efficient, inexpensive, and rapid screening of these libraries using magnetic separation. This is an advantage over the more conventional approach of sorting libraries based on fluorescent reporter gene expression.
In order to turn the classical recruitment reporter assay into a high-throughput assay of transcriptional domains, two problems were solved: (1) modification of the reporter to make it compatible with rapid screening of libraries of tens of thousands of domains, and (2) development of a strategy to generate a library of candidate effector domains. To improve on the previously published fluorescent reporter (Bintu et al., 2016), a synthetic surface marker was engineered to enable facile magnetic separation of large numbers of cells and the reporter was integrated in a suspension cell line amenable to cell culture in large volume spinner flasks. Specifically, K562 reporter cells with 9×TetO binding sites upstream of a strong constitutive pEF1a promoter that drives expression of a two-part reporter consisting of a synthetic surface marker (the human IgG1 Fc region linked to an Igk leader and PDGFRβ transmembrane domain) and a fluorescent citrine protein (
Sequences were pulled from the UniProt database for Pfam-annotated domains in human proteins that can localize to the nucleus (including non-exclusively nuclear-localized proteins). In total, 14,657 domains were retrieved. Of these, 72% were less than or equal to 80 amino acids (AA) long (
Before assaying for transcriptional activity, it was determined which protein domains were well-expressed in K562 cells using a high-throughput approach (
The Pfam domain library was screened for transcriptional repressors. The pooled library of cells was treated with doxycycline for 5 days, which gave sufficient time after transcriptional silencing for the reporter mRNA and protein to degrade and dilute out due to cell division, resulting in a clear bimodal mixture of ‘ON’ and ‘OFF’ cells (
One of the strongest hits was the YAF2_RYBP, a domain present in the RING1- and YY1-binding protein (RYBP) and its paralog YY1-associated Factor 2 (YAF2), which are both components of the polycomb repressive complex 1 (PRC1) (Chittock et al., 2017; García et al., 1999). The domain from the RYBP protein as annotated by Pfam (which is just 32 amino acids, thus shorter than the version synthesized in the 80 AA domain library) was individually tested and rapid silencing of the reporter gene was confirmed (
To quantify repression kinetics, the citrine level distributions were gated to calculate a percentage of silenced cells with normalization of the uniform low level of background silencing in the untreated cells, and then the data was fit to a model with an exponential silencing rate during doxycycline treatment and an exponential decay (or reactivation) after doxycycline removal that plateaus at a constant irreversibly silent percentage of cells (
Identification of Domains of Unknown Function that Repress Transcription
Over 22% of the Pfam domain families are labeled as Domains of Unknown Function (DUFs), while others are not named using this label but are nevertheless DUFs (El-Gebali et al., 2019). These domains have recognizable sequence conservation but lack experimental characterization. As such, the high-throughput domain screen described herein offered the opportunity to associate initial functions with DUFs. First, DUF3669 domains were identified as repressor hits and individually validated by flow cytometry (
All three of the IRF-2BP1_2 N-terminal zinc finger domains (Childs and Goodbourn, 2003), an uncharacterized domain found in the interferon regulatory factor 2 (IRF2) co-repressors IRF2BP1, IRF2BP2, and IRF2BPL, were repressor hits. The Cyt-b5 domain in the DNA repair factor HERC2 E3 ligase (Mifsud and Bateman, 2002) was another functionally uncharacterized domain that was validated as a strong repressor hit (
A Random Sequence with Strong Repressor Activity
Random sequences have not previously been tested for repressor activity. Surprisingly, one of the random 80 AA sequences, which were designed as negative controls, was a strong repressor hit with an average log 2(OFF:ON)=4.0, despite having a weak expression level below the threshold. Individual validation by flow cytometry confirmed that this sequence fully silenced the population of reporter cells after 5 days of recruitment with moderate epigenetic memory up to two weeks after doxycycline removal (
The data provided an opportunity to analyze the function of all effector domains in the largest family of transcription factors: the KRAB domains. The KRAB gene family includes some of the strongest known repressor domains (such as the KRAB in ZNF10). Previous studies of a subset of repressive KRAB domains revealed that they can repress transcription by interacting with the co-repressor KAP1, which in turn interacts with chromatin regulators such as SETDB1 and HP1 (Cheng et al., 2014). However, it remains unclear how many of the KRAB domains are repressors, and whether the recruitment of KAP1 is necessary or sufficient for repression across all KRABs.
The library included 335 human KRAB domains, and 92.1% were found as repressor hits after filtering for domains that were well-expressed. 9 repressor hit and 2 non-hit KRAB domains were individually validated by flow cytometry and these categorizations were confirmed in every case (
Interestingly, repressive KRAB domains were mostly found in proteins with the simplest domain architecture consisting of just a KRAB domain and a zinc-finger array, while the non-repressive KRAB domains were mostly found in genes that also include a DUF3669 or SCAN domain (
The compound domain architecture that included a SCAN or DUF3669 is more common in evolutionary old KRAB genes (Imbeault et al., 2017). Here, a clear relationship was observed between the evolutionary age of the KRAB genes and the KRAB repressor strength, with KRAB domains from genes pre-dating the marsupial-human common ancestor having no repressor activity, and KRAB domains from genes that evolved later consistently functioning as strong repressors (
Deep Mutational Scan of the CRISPRi ZNF10 KRAB Effector Identifies Mutations that Modulate Gene Silencing
The KRAB domain from ZNF10 has been extensively used in synthetic biology applications for gene repression and is fused to dCas9 in the programmable epigenetic and transcriptional control tool known as CRISPR interference (Gilbert et al., 2014). To better understand its sequence-function relationships, a deep mutational scan (DMS) of this KRAB was performed domain using HT-recruit. A library with all possible single substitutions and all consecutive double and triple substitutions was designed (
The ZNF10 KRAB effector has 3 components: the A-box which is necessary for binding KAP1 (Peng et al., 2009), the B-box which is thought to potentiate KAP1 binding (Peng et al., 2007), and an N-terminal extension that is natively found on a separate exon upstream of the KRAB domain (
These substitutions were mapped onto an aligned mouse KRAB A-box structure (PDB: 1v65, 55% identity, 69% similarity in A-box [V13-Y54],
In contrast to the A-box, B-box mutations showed relatively little effect at the end of recruitment (day 5), with only one statistically significant position (P59) showing consistent but weak effects. Meanwhile P59 and 4 other positions (K58, I62, L65, E66) showed a significant effect on memory after doxycycline removal as measured at day 9 (
Lastly, the KRAB N-terminus contained residues where many substitutions consistently enhanced silencing relative to wild-type (
This silencing enhancement may have been a result of enhanced KRAB protein expression level. To investigate the relationship between protein expression level and KRAB silencing strength, the high-throughput FLAG-tag expression level measurements for the set of KAP1-binding KRAB domains was inspected and a significant correlation was found between KRAB expression level and silencing at day 13 (r2=0.49,
Homeodomain Repressor Strength is Colinear with Hox Gene Organization
The second largest domain family that included repressor hits in the screen was the homeodomain family. Homeodomains are composed of 3 helices and are sequence-specific DNA binding domains that make base contacts through Helix 3 (Lynch et al., 2006). In some cases, they are also known to act as repressors (Holland et al., 2007; Schnabel and Abate-Shen, 1996). The library included the homeodomains from 216 human genes, and 26% were repressor hits. The repressors were found in 4 out of the 11 subclasses of homeodomains: PRD, NKL, HOXL, and LIM (
Next, the HOXL subclass results were inspected more closely. This subclass contained the Hox genes, a subset of 39 homeodomain transcription factors that are master regulators of cell fate and specify regions of the body plan along the anterior-posterior axis during embryogenesis. These genes are found in four Hox paralog clusters (A to D) arranged co-linearly from 3′ to 5′ corresponding to the temporal order and spatial patterning of their expression along the anterior-posterior axis (Gilbert, 1971). Interestingly, the repressor strength of their homeodomains was also collinear with their arrangement in the Hox clusters, such that the more 5′ gene homeodomains were stronger repressors (Spearman's p=0.82,
Multiple sequence alignment of the Hox homeodomains revealed an RKKR (SEQ ID NO: 1330) motif present in the N-terminal arm of the 11 strongest repressor domains (
Outside the Hox homeodomains, 99.5% of the repressor hits in the Pfam nuclear protein domain library did not contain the RKKR (SEQ ID NO: 1330) motif, while many non-hits did. Also, there was no correlation between net domain charge and repression strength at day 5 when considering the full library of domains (R2=0.04). Together, these results suggested the RKKR (SEQ ID NO: 1330) motif and charge contributed to Hox homeodomain repression in the recruitment assay, but they were not sufficient for repression when found in the context of other domains.
It was established that a reporter K562 line with a weak minimal CMV (min CMV) promoter that could be activated upon recruitment of fusions between rTetR and activation domains (
In total, 48 hits from 26 domain families were found. Beyond the three known activator domain families above, the remaining families with an activator hit were not previously annotated on Pfam as activator domains (
Several hits were not sourced from sequence-specific transcription factors where classical activator domains are expected but were instead nonclassical activators from co-activator and transcriptional machinery proteins including Med9, TFIIEβ, and NCOA3. In particular, the Med9 domain, whose ortholog directly binds other mediator complex components in yeast (Takahashi et al., 2009), was a strong activator with an average log 2(OFF:ON)=−5.5, despite its weak expression level. Nonclassical activators have previously been reported to work individually in yeast (Gaudreau et al., 1999) but only weakly when individually recruited in mammalian cells (Nevado et al., 1999). One exception is TATA-binding protein (Dorris and Struhl, 2000). By screening more nonclassical sequences, more exceptions to this notion were found.
For all tested domains, doxycycline-dependent activation of the reporter gene was confirmed using both the extended 80 AA sequence from the library and the trimmed Pfam-annotated domain (
Surprisingly, the strongest activator in the library was the KRAB domain from ZNF473 (
It was individually validated that the KRAB from ZNF473 as a strong activator and KRAB_2 from ZFP28 as a moderate strength activator (
Pfam annotations provided one useful means of filtering the nuclear proteome to generate a relatively compact library, but Pfam is likely currently missing many of the human effector domains. In order to discover effector domains in unannotated regions of proteins, a tiling library was designed by curating a list of 238 proteins from silencer complexes and tiling their sequences with 80 amino acids separated by a 10 amino acid tiling window (
Novel unannotated repressor domains were also discovered. For example, BAZ2A (also known as TIP5) is a nuclear remodeling complex (NoRC) component that mediates transcriptional silencing of some rDNA (Guetg et al., 2010), but does not have any annotated effector domains. The BAZ2A tiling data showed a peak of repressor function in a glutamine-rich region and it was individually validated as a moderate strength repressor (
A MGA, which is thought to repress transcription by binding the genome at E-box motifs and recruiting the non-canonical polycomb 1.6 complex (Blackledge et al., 2014; Jolma et al., 2013; Stielow et al., 2018), tiling experiment revealed two domains with repressor function, located adjacent to the two known DNA binding domains, called here Repressor 1 and Repressor 2 (
Next, it was attempted to identify the minimal necessary sequence for repressor function in each independent domain by examining the overlap in all tiles covering a protein region that shows repressor function and determining which contiguous sequence of amino acids is present in all the repressive tiles (
Transcriptional effector functions were measured systematically and quantitatively across biological contexts by performing high-throughput recruitment (HT-recruit) screens using two different DNA-binding domains (rTetR and dCas9) to recruit effectors to various gene targets and cell-types (
In order to extend the approach to recruiting libraries of effectors to endogenous targets, the library was fused onto dCas9 and targeted endogenous surface marker genes. Targeting surface markers allowed use or fluorescent antibodies to immunostain cells, thus providing a way to monitor single-cell gene expression variability during individual recruitment assays by flow cytometry and to magnetically separate ON and OFF cells with ProG beads during HT-recruit (
Using these components, new HT-recruit and FLAG-based expression screens were performed and combined with the previous screens across 10 rTetR-targeted synthetic reporter contexts in two cell types and 5 dCas9-targeted screens, in total charting the effector context-specificity landscape for over 5000 human nuclear protein domains across 15 contexts (
The cell type-specificity of repressors was investigated by comparing the HT-recruit using rTetR to target Pfam domains to the pEF1a reporter in HEK293T and K562 cells. Repression was stronger in K562, both in the screen (
Applying a similar approach, the cell-type specificity of activators was investigated by comparing HT-recruit using rTetR to target the minCMV reporter in HEK293T and K562 cells. A number of activator hits in the K562 screen were also activator hits in the HEK293T screen, including strong activators like FOXO-TAD, LMSTEN, NCOA3, and the ZNF473 KRAB domain (
One of the K562-specific activators identified in the screen is the N-terminus QLQ domain, a Gln-Leu-Gln motif, from SMARCA2. SMARCA2 is a component of the ATP-dependent BAF chromatin remodeling complex involved in regulating transcription by altering the chromatin structure around genes. When fused to dCas9 and targeted to the CD2 endogenous gene, QLQ activated CD2 in K562 but not in HEK293T cells (
In addition, WW domains from multiple TFs (KIBRA, APBB1, WWP2) and PHD domains (from PYGO1 and CXXC1) were activators in K562 and not in HEK293T cells (
Since more effector domains work in K562 versus HEK293T, K562 cells were predominantly used to investigate how the activity of activators and repressors change at different promoters. Repressor promoter-specificity was investigated using rTetR to target the library members to pEF, PGK, and UbC promoters in K562 cells. Repression at PGK and UbC was highly correlated (
In order to expand measurements to endogenous genes, the Pfam library of domains was fused to dCas9 instead of rTetR. The dCas9 fusions were tested by recruiting them at the TetO sites upstream of the pEF reporter. In general, fewer repressor hits were garnered, but the top KRAB domains were still able to repress (
Core promoters were found to respond similarly to activators with rTetR recruitment in K562. Similar activator activity was validated across core promoters (
It is generally thought that effector domains are classified as activators or repressors; however, like the transcription factors that consists of these domains, these effectors may have a dual activator-repressor function that is context-dependent on gene target or promoter. HT-recruit results were compared from repressible and activatable targets. Many helix-loop-helix (HLH) domains activate the CD2 endogenous gene upon dCas9 recruitment with the sg717 guide in K562 cells (
Surprisingly, many of the same HLH effectors can repress the constitutive pEF1a promoter when recruited with rTetR (
There are other cases of effectors that activate in one context and repress in another as identified through the screen (
Synergistic Repression with RYBP-MGA
Non-KRAB repressors have been used for gene silencing and epigenomics, but these have generally not demonstrated a similar efficiency as KRAB for CRISPRi applications. In the HT-recruit screens, non-KRAB repressors were less robust across contexts (
Others have reported enhancement of silencing by combining multiple repressors with ZNF10 KRAB and even reported KRAB-like efficiencies with MBD2B repressor fusions, combinations of these non-KRAB repressors were rested. First, when MGA [381-390] and MGA[2431-2460] were combined together, super-additive repression (23% versus 0% and 10% individually) was observed at the HEK293T reporter (
In a CRISPRi benchmarking screen targeting promoters that were previously shown to be important with a ZNF10 KRAB screen, RYBP-MGA showed much greater efficiency than dCas9 alone but somewhat lower efficiency than ZNF10 KRAB across a number of sgRNAs (
To find the most useful activators for synthetic applications, additional dCas9 HT-recruit screens were performed at the activatable endogenous genes CD20 and CD28, and then activators from the HT-recruit measurements were ranked performed across different contexts (
These trimmed activators were fused and, as controls, two commonly used activators (VP64 and VPR) were also fused, individually to dCas9 in a lentiviral vector with BFP as a delivery marker. As in the HT-recruit screens, ZNF473 KRAB activated CD2 with sg717 while the other domains were poor activators (
The most striking feature of NFZ was the improvement in lentiviral delivery with dCas9-NFZ versus dCas9-VPR, as marked by a higher fraction of BFP+ cells (
The activity of dCas9-NFZ was confirmed across three endogenous surface marker genes (CD2, CD20, CD28) in K562 cells. NFZ was better delivered than VPR, with unconcentrated or concentrated virus (
In contrast to what was seen with lentivirus, by plasmid transfection into HEK293T cells, NFZ was similarly delivered as VPR (
By changing the N, F, Z domain orientation, N+Z+F was identified as a slightly stronger activator combination than other configurations, with lentiviral delivery efficiencies between VPR and NFZ (
To further test the limits of combining activators, another short activator domain, SMARCA2 QLQ (
In addition, the Q, N, and F (but not Z) domains can synergistically activate CD2 when fused as homotypic bipartite combinations (
Inducible expression with the compact tripartite activator This combination was ported to the rTetR DBD and in the presence of dox, rTetR-NZF activated minCMV or partially reactivated the background-silenced PGK reporter in HEK293T cells (
A new compact inducible AAV system by using the following components: (1) the compact NFZ activator, (2) minimal promoters and terminators, and (3) fewer repeats of FKBP (with the assumption fewer FKBP repeats are needed if the activators are stronger). In sum, these modifications expanded the transgene cargo capacity from 1.1 to 2.3 kb (
As an initial test, the newly designed inducible AAV construct that contains a Citrine transgene was transiently transfected. Rapamycin-dependent Citrine inducibility and low levels of leakyness were observed with NFZ, whereas in the previously used P65+HSF1 there was high level of leakiness and with reduced transgene cargo capacity (
CRISPR Interference with the ZNF705F KRAB Repressor Domain
Performing an additional screen at the GATA1 locus allowed comparison of repressors at an enhancer region (
Before using growth as a selection strategy for a CRISPR HT-recruit screen, the growth effects of expressing the dCas9-effector fusions, were inspected in a negative control screen with a safe-target sgRNA called N4294. This safe-targeting sgRNA does not cause growth effects when co-expressed with dCas9 or dCas9-KRAB (from ZNF10). In this control screen, some domains were associated with cell toxicity or growth defects, with the strongest being the cyclin-dependent kinase inhibitor (CDI) domains from CDKN1A/B (
To identify strong KRAB repressors that function well across many different contexts, the KRAB paralogs were ranked across all the HT-recruit screens and in addition to ZNF705F KRAB, the KRAB domain from ZNF471 was identified as another top repressor (
Previously, using a deep mutational scan of the ZNF10 KRAB, a mutant, WSR7EEE, in the N-terminal KRAB domain region was identified as providing increased expression in cells and silencing strength when fused to rTetR. The same was true when this mutant was fused to dCas9 (
When targeted to the CD43 TSS with dCas9, both of the newly identified KRAB paralogs outperformed the ZNF10 KRAB[WSR7EEE] (
To assess the KRAB repressors in a different DBD context where there is more room to improve efficiency beyond that of ZNF10 KRAB, dCas12a, a compact CRISPR DBD which is generally weaker than dCas9 and which was not included in the HT-recruit screens was used. With dCas12a, it was similarly observed that ZNF705F KRAB recruitment consistently resulted in ˜1.5× more complete silencing of a population of cells than ZNF10 KRAB (
Cell lines and cell culture All experiments were carried out in K562 cells (ATCC CCL-243). Cells were cultured in a controlled humidified incubator at 37° C. and 5% CO2, in RPMI 1640 (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), streptomycin (10,000 μg/mL), and L-glutamine (2 mM). HEK293FT and HEK293T-LentiX cells were grown in DMEM (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), and streptomycin (10,000 μg/mL) and used to produce lentivirus. Reporter cell lines were generated by TALEN-mediated homology-directed repair to integrate a donor construct into the AAVS1 locus as follows: 1.2×106 K562 cells were electroporated in Amaxa solution (Lonza Nucleofector 2b, setting TO-16) with 1000 ng of reporter donor plasmid, and 500 ng of each TALEN-L (Addgene #35431) and TALEN-R (Addgene #35432) plasmid (targeting upstream and downstream the intended DNA cleavage site, respectively). After 7 days, the cells were treated with 1000 ng/mL puromycin antibiotic for 5 days to select for a population where the donor was stably integrated in the intended locus, which provides a promoter to express the PuroR resistance gene. Fluorescent reporter expression was measured by microscopy and by flow cytometry (BD Accuri).
Nuclear protein Pfam domain library design The UniProt database (UniProt Consortium, 2015) was queried for human genes that can localize to the nucleus. Subcellular location information on UniProt was determined from publications or ‘by similarity’ in cases where there was only a publication on a similar gene (e.g., ortholog) and was manually reviewed. Pfam-annotated domains were then retrieved using the ProDy searchPfam function (Bakan et al., 2011). domains that were 80 amino acids or shorter were filtered for and the C2H2 Zinc finger DNA-binding domains, which are highly abundant, repetitive, were excluded and not expected to function as transcriptional effectors. The sequence of the annotated domain was retrieved and it was extended equally on either side to reach 80 amino acids total. Duplicate sequences were removed, then codon optimization was performed for human codon usage, removing BsmBI sites and constraining GC content to between 20% and 75% in every 50 nucleotide window (performed with DNA chisel (Zulkower and Rosser, 2020)). 499 random controls of 80 amino acids lacking stop codons were computationally generated as controls. 362 elements tiling the DMD protein in 80 amino acid tiles with a 10 amino acid sliding window were also included as controls because DMD was not thought to be a transcriptional regulator. In total, the library consists of 5,955 elements.
Silencer tiling library design 216 proteins involved in transcriptional silencing were curated from a database of transcriptional regulators (Lambert et al., 2018). 32 proteins likely to be involved in transcriptional silencing were manually added and then an unbiased protein tiling library was generated. To do this, the canonical transcript for each gene was retrieved from the Ensembl BioMart (Kinsella et al., 2011) using the Python API. If no canonical transcript was found, the longest transcript with a CDS was retrieved. The coding sequences were divided into 80 amino acid tiles with a 10 amino acid sliding window between tiles. For each gene, a final tile was included, spanning from 80 amino acids upstream of the last residue to that last residue, such that the C-terminal region would be included in the library. Duplicate protein sequences were removed, and codon optimization was performed for human codon usage, removing BsmBI sites and constraining GC content to between 20% and 75% in every 50 nucleotide window (performed with DNA chisel (Zulkower and Rosser, 2020)). 361 DMD tiling negative controls were included, as in the previous library design, resulting in 15,737 library elements in total.
KRAB deep mutational scan library design A deep mutational scan of ZNF10 KRAB domain sequence, as used in CRISPRi (Gilbert et al., 2014), was designed with all possible single substitutions and all consecutive double and triple substitutions of the same amino acid (e.g., substitution with AAA). These amino acid sequences were reverse translated into DNA sequences using a probabilistic codon optimization algorithm, such that each DNA sequence contains some variation beyond the substituted residues, which improves the ability to unambiguously align sequencing reads to unique library members. In addition, all Pfam-annotated KRAB domains from human KRAB genes found on InterPro were included, similarly as in the previous nuclear Pfam domain library. Tiling sequences, as designed in the previous tiling library, were also included for five KRAB Zinc Finger genes. 300 random control sequences and 200 tiles from the DMD gene were included as negative controls. During codon optimization, BsmBI sites were removed and GC content was constrained to be between 30% and 70% in every 80 nucleotide window (performed with DNA chisel (Zulkower and Rosser, 2020)). The total library size was 5,731 elements.
Domain library cloning Oligonucleotides with lengths up to 300 nucleotides were synthesized as pooled libraries (Twist Biosciences) and then PCR amplified. 6×50 μl reactions were set up in a clean PCR hood to avoid amplifying contaminating DNA. For each reaction, 5 ng of template, 0.1 μl of each 100 μM primer, 1 μl of Herculase II polymerase (Agilent), 1 μl of DMSO, 1 μl of 10 nM dNTPs, and 10 μl of 5× Herculase buffer was used. The thermocycling protocol was 3 minutes at 98° C., then cycles of 98° C. for 20 seconds, 61° C. for 20 seconds, 72° C. for 30 seconds, and then a final step of 72° C. for 3 minutes. The default cycle number was 29×, and this was optimized for each library to find the lowest cycle that resulted in a clean visible product for gel extraction (in practice, 25 cycles was the minimum). After PCR, the resulting dsDNA libraries were gel extracted by loading ≥4 lanes of a 2% TBE gel, excising the band at the expected length (around 300 bp), and using a QIAgen gel extraction kit. The libraries were cloned into a lentiviral recruitment vector pJT050 with 4×10 μl GoldenGate reactions (75 ng of pre-digested and gel-extracted backbone plasmid, 5 ng of library, 0.13 μl of T4 DNA ligase (NEB, 20000 U/μl), 0.75 μl of Esp3I-HF (NEB), and 1 μl of 10× T4 DNA ligase buffer) with 30 cycles of digestion at 37° C. and ligation at 16° C. for 5 minutes each, followed by a final 5 minute digestion at 37° C. and then 20 minutes of heat inactivation at 70° C. The reactions were then pooled and purified with MinElute columns (QIAgen), eluting in 6 μl of ddH2O. 2 μl per tube was transformed into two tubes of 50 μl of electrocompetent cells (Lucigen DUO) following the manufacturer's instructions. After recovery, the cells were plated on 3-7 large 10″×10″ LB plates with carbenicillin. After overnight growth at 37° C., the bacterial colonies were scraped into a collection bottle and plasmid pools were extracted with a HiSpeed Plasmid Maxiprep kit (QIAgen). 2-3 small plates were prepared in parallel with diluted transformed cells in order to count colonies and confirm the transformation efficiency was sufficient to maintain at least 30× library coverage. To determine the quality of the libraries, the domains were amplified from the plasmid pool and from the original oligo pool by PCR with primers with extensions that include Illumina adapters and sequenced. The PCR and sequencing protocol were the same as described below for sequencing from genomic DNA, except these PCRs use 10 ng of input DNA and 17 cycles. These sequencing datasets were analyzed as described below to determine the uniformity of coverage and synthesis quality of the libraries. In addition, 20-30 colonies from the transformations were Sanger sequenced (Quintara) to estimate the cloning efficiency and the proportion of empty backbone plasmids in the pools.
High-throughput recruitment to measure repressor activity Large scale lentivirus production and spinfection of K562 cells were performed. To generate sufficient lentivirus to infect the libraries into K562 cells, HEK293T cells were plated on four 15-cm tissue culture plates. On each plate, 9×105 HEK293T cells were plated in 30 mL of DMEM, grown overnight, and then transfected with 8 μg of an equimolar mixture of the three third-generation packaging plasmids and 8 μg of rTetR-domain library vectors using 50 μl of polyethylenimine (PEI, Polysciences #23966). After 48 hours and 72 hours of incubation, lentivirus was harvested. The pooled lentivirus was filtered through a 0.45-μm PVDF filter (Millipore) to remove any cellular debris. For the nuclear Pfam domain repressor screen, 4.5×107 K562 reporter cells were infected with the lentiviral library by spinfection for 2 hours, with two separate biological replicates of the infection. Infected cells grew for 3 days and then the cells were selected with blasticidin (10 μg/mL, Sigma). Infection and selection efficiency were monitored each day using flow cytometry to measure mCherry (BD Accuri C6). Cells were maintained in spinner flasks in log growth conditions each day by diluting cell concentrations back to a 5×105 cells/mL, with at least 1.5×108 cells total remaining per replicate such that the lowest maintenance coverage was >25,000× cells per library element (a very high coverage level that compensates for losses from incomplete blasticidin selection, library preparation, and library synthesis errors). On day 6 post-infection, recruitment was induced by treating the cells with 1000 ng/ml doxycycline (Fisher Scientific) for 5 days, then cells were spun down out of doxycycline and blasticidin and maintained in untreated RPMI media for 8 more days, up to Day 13 counting from the addition of doxycycline. 2.5×108 cells were taken for measurements at each timepoint (days 5, 9, and 13). The protocol was similar for the KRAB DMS, but doxycycline was added on day & post-infection, >12,500× coverage, and 2×108-2.2×108 cells were taken for each timepoint. The protocol was similar for the tiling screen, but 9.6×107 cells were infected, doxycycline was added on day 8 post-infection, at least 2×108 cells were maintained at each passage for >12,500× coverage, and 2×108-2.7×108 cells were taken for each timepoint.
High-throughput recruitment to measure transcriptional activation activity For the nuclear Pfam domain activator screen, lentivirus for the nuclear Pfam library in the rTetR(SE-G72P)-3×FLAG vector was generated as for the repressor screen, and 3.8×107 K562-pDY32 minCMV reporter cells were infected with the lentiviral library by spinfection for 2 hours, with two separate biological replicates of the infection. Infected cells grew for 2 days and then the cells were selected with blasticidin (10 μg/mL, Sigma). Infection and selection efficiency were monitored each day using flow cytometry to measure mCherry (BD Accuri C6). Cells were maintained in spinner flasks in log growth conditions each day by diluting cell concentrations back to a 5×105 cells/mL, with at least 1×108 total cells remaining per replicate such that the lowest maintenance coverage was >18,000× cells per library element. On day 7 post-infection, recruitment was induced by treating the cells with 1000 ng/ml doxycycline (Fisher Scientific) for 2 days, then cells were spun down out of doxycycline and blasticidin and maintained in untreated RPMI media for 4 more days. 2×108 cells were taken for measurements at the day 2 time point. There was no evidence of activation memory at day 4 post-doxycycline removal, as determined by the absence of citrine positive cells by flow cytometry, so no additional time points were collected.
Magnetic separation of reporter cells At each timepoint, cells were spun down at 300×g for 5 minutes and media was aspirated. Cells were then resuspended in the same volume of PBS (Gibco) and the spin down and aspiration was repeated, to wash the cells and remove any IgG from serum. Dynabeads™ M-280 Protein G (ThermoFisher 10003D) were resuspended by vortexing for 30 seconds. 50 mL of blocking buffer was prepared per 2×108 cells by adding 1 gram of biotin-free BSA (Sigma Aldrich) and 200 μl of 0.5 M pH 8.0 EDTA (ThemoFisher 15575020) into DPBS (Gibco), vacuum filtering with a 0.22-μm filter (Millipore), and then kept on ice. 60 μl of beads was prepared for every 1×107 cells, by adding 1 mL of buffer per 200 μl of beads, vortexing for 5 seconds, placing on a magnetic tube rack (Eppendorf), waiting one minute, removing supernatant, and finally removing the beads from the magnet and resuspending in 100-600 μl of blocking buffer per initial 60 μl of beads. For the KRAB DMS only, 30 μl of beads was prepared for every 1×107 cells, in the same way. Beads were added to cells at no more than 1×107 cells per 100 μl of resuspended beads, and then incubated at room temperature while rocking for 30 minutes. For a sample with 2×108 cells, 1.2 mL of beads were used, resuspended in 12 mL of blocking buffer, in a 15 mL Falcon tube and a large magnetic rack. For a sample with <5×107 cells, non-stick Ambion 1.5 mL tubes and a small magnetic rack were used. After incubation, the bead and cell mixture were placed on the magnetic rack for >2 minutes. The unbound supernatant was transferred to a new tube, placed on the magnet again for >2 minutes to remove any remaining beads, and then the supernatant was transferred and saved as the unbound fraction. Then, the beads were resuspended in the same volume of blocking buffer, magnetically separated again, the supernatant was discarded, and the tube with the beads was kept as the bound fraction. The bound fraction was resuspended in blocking buffer or PBS to dilute the cells (the unbound fraction is already dilute). Flow cytometry (BD Accuri) was performed using a small portion of each fraction to estimate the number of cells in each fraction (to ensure library coverage was maintained) and to confirm separation based on citrine reporter levels (the bound fraction should be >90% citrine positive, while the unbound fraction is more variable depending on the initial distribution of reporter levels). Finally, the samples were spun down and the pellets were frozen at −20° C. until genomic DNA extraction.
High-throughput measurement of domain fusion protein expression level The expression level measurements were made in K562-pDY32 cells (with citrine OFF) infected with the 3×FLAG-tagged nuclear Pfam domain library. 1×108 cells per biological replicate were used after 5 days of blasticidin selection (10 μg/mL, Sigma), which was 7 days post-infection. 1×106 control K562-JT039 cells (citrine ON, no lentiviral infection) were spiked into each replicate. Fix Buffer I (BD Biosciences, BDB557870) was preheated to 37° C. for 15 minutes and Permeabilization Buffer III (BD Biosciences, BDB558050) and PBS (Gibco) with 10% FBS (Hyclone) were chilled on ice. The library of cells expressing domains was collected and cell density was counted by flow cytometry (BD Accuri). To fix, cells were resuspended in a volume of Fix Buffer I (BD Biosciences, BDB557870) corresponding to pellet volume, with 20 μl per 1 million cells, at 37° C. for 10-15 minutes. Cells were washed with 1 mL of cold PBS containing 10% FBS, spun down at 500×g for 5 minutes and then supernatant was aspirated. Cells were permeabilized for 30 minutes on ice using cold BD Permeabilization Buffer III (BD Biosciences, BDB558050), with 20 μl per 1 million cells, which was added slowly and mixed by vortexing. Cells were then washed twice in 1 ml PBS+10% FBS, as before, and then supernatant was aspirated. Antibody staining was performed for 1 hour at room temperature, protected from light, using 5 μl/1×106 cells of α-FLAG-Alexa647 (RNDsystems, IC8529R). The cells were washed and resuspended at a concentration of 3×107 cells/ml in PBS+10% FBS. Cells were sorted into two bins based on the level of APC-A fluorescence (Sony SH800S) after gating for mCherry positive viable cells. A small number of unstained control cells was also analyzed on the sorter to confirm staining was above background. The spike-in citrine positive cells were used to assess the background level of staining in cells known to lack the 3×FLAG tag, and the gate for sorting was drawn above that level. After sorting, the cellular coverage ranged from 336-1,295 cells per library element across samples. The sorted cells were spun down at 500×g for 5 minutes and then resuspended in PBS. Genomic DNA extraction was performed following the manufacturer's instructions (QIAgen Blood Maxi kit was used for samples with >1×107 cells, and QIAamp DNA Mini kit with one column per up to 5×106 cells was used for samples with ≤1×107 cells) with one modification: the Proteinase K+AL buffer incubation was performed overnight at 56° C.
Library preparation and sequencing Genomic DNA was extracted using a Blood & Tissue kit (QIAgen) following the manufacturer's instructions with up to 1.25×108 cells per column. DNA was eluted in EB and not AE to avoid subsequence PCR inhibition. The domain sequences were amplified by PCR with primers containing Illumina adapters as extensions. A test PCR was performed using 5 μg of genomic DNA in a 50 μl (half-size) reaction to verify if the PCR conditions would result in a visible band at the expected size for each sample. Then, 12-24×100 μl reactions were set up on ice (in a clean PCR hood to avoid amplifying contaminating DNA), with the number of reactions depending on the amount of genomic DNA available in each experiment. 10 μg of genomic DNA, 0.5 μl of each 100 UM primer, and 50 μl of NEBnext 2× Master Mix (NEB) was used in each reaction. The thermocycling protocol was to preheat the thermocycler to 98° C., then add samples for 3 minutes at 98° C., then 32× cycles of 98° C. for 10 seconds, 63° C. for 30 seconds, 72° C. for 30 seconds, and then a final step of 72° C. for 2 minutes. All subsequent steps were performed outside the PCR hood. The PCR reactions were pooled and ≥140 μl were run on at least three lanes of a 2% TBE gel alongside a 100-bp ladder for at least one hour, the library band around 395 bp was cut out, and DNA was purified using the QIAquick Gel Extraction kit (QIAgen) with a 30 μl elution into non-stick tubes (Ambion), A confirmatory gel was run to verify that small products were removed. These libraries were then quantified with a Qubit HS kit (Thermo Fisher), pooled with 15% PhiX control (Illumina), and sequenced on an Illumina NextSeq with a High output kit using a single end forward read (266 or 300 cycles) and 8 cycle index reads.
Domain sequencing analysis Sequencing reads were demultiplexed using bcl2fastq (Illumina). A Bowtie reference was generated using the designed library sequences with the script ‘makelndices.py’ and reads were aligned with 0 mismatch allowance using the script ‘makeCounts.py’. The enrichments for each domain between OFF and ON (or FLAGhigh and FLAGlow) samples were computed using the script ‘makeRhos.py’. Domains with <5 reads in both samples for a given replicate were dropped from that replicate (assigned 0 counts), whereas domains with <5 reads in one sample would have those reads adjusted to 5 in order to avoid the inflation of enrichment values from low depth. For all of the nuclear domain screens, domains with ≤5 counts in both replicates of a given condition were filtered out of downstream analysis. For the nuclear domain expression screen, well-expressed domains were those with a log 2(FLAGhigh:FLAGlow)≥1 standard deviation above the median of the random controls. For the nuclear Pfam domain repressor screen, hits were domains with log 2(OFF:ON)≥2 standard deviations above the mean of the poorly expressed domains. For the nuclear domain activator screen, hits were domains with log 2(OFF:ON)≤2 standard deviations below the mean of the poorly expressed domains. For the silencer tiling screen, tiles with ≤20 counts in both replicates of a given condition were filtered out and hits were tiles with log 2(OFF:ON)≥2 standard deviations above the mean of the random and DMD tiling controls. Gene ontology analysis enrichments were computed using the PantherDB web tool (www.pantherdb.org). The background sets were all proteins containing domains that were well-expressed and measured in the experiment after count filters were applied. P-values for statistical significance were calculated using Fisher's exact test, the False Discovery Rate (FDR) was computed, and only the most significant results, all with FDR<10%, were shown.
Western blot and co-immunoprecipitation Cells transduced with a lentiviral vector containing an rTetR-fusion-T2A-mCherry-BSD were selected with blasticidin (10 μg/mL) were selected until mCherry was >80%. Cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, Protease inhibitor cocktail). Protein amounts were quantified using the DC Protein Assay kit (Bio-Rad). Equal amounts were loaded onto a gel and transferred to a nitrocellulose or PVDF membrane. Membrane was probed using GATA1 antibody (1:1000, rabbit, Cell Signaling Technologies cat no. 3535S) and GAPDH antibody (1:2000, mouse, ThermoFisher cat no. AM4300) or FLAG M2 monoclonal antibody (1:1000, mouse, Sigma-Aldrich, catalog number F1804) and Histone 3 antibody (1:1000, mouse, Abcam cat no. AB1791) as primary antibodies. Donkey anti-rabbit IRDye 680 LT and goat anti-mouse IRDye 800CW (1:20,000 dilution, LI-COR Biosciences, cat nos. 926-68023 and 926-32210, respectively) or Goat anti-mouse IRDye 680 RD and goat anti-rabbit IRDye 800CW (1:20,000 dilution, LI-COR Biosciences, cat nos. 926-68070 and 926-32211, respectively) were used as secondary antibodies, respectively
Blots were imaged on a LiCor Odyssey CLx. Band intensities were quantified using ImageJ.
Individual repressor recruitment assays Individual effector domains were cloned as fusions with rTetR or rTetR(SE-G72P) with or without a 3×FLAG tag (see figure legends), upstream of a T2A-mCherry-BSD marker using GoldenGate cloning into backbones pJT050 or pJT126. K562-pJT039-pEF-citrine reporter cells were then transduced with this lentiviral vector and, 3 days later, selected with blasticidin (10 μg/mL) until >80% of the cells were mCherry positive (6-7 days). Cells were split into separate wells of a 24-well plate and either treated with doxycycline (Fisher Scientific) or left untreated. After 5 days of treatment, doxycycline was removed by spinning down the cells, replacing media with DPBS (Gibco) to dilute any remaining doxycycline, and then spinning down the cells again and transferring them to fresh media. Timepoints were measured every 2-3 days by flow cytometry analysis of >7,000 cells (either a BD Accuri C6 or Beckman Coulter CytoFLEX). Data was analyzed using Cytoflow and custom Python scripts. Events were gated for viability and for mCherry as a delivery marker. To compute a fraction of OFF cells during doxycycline treatment, a 2 component Gaussian mixture model was fitted to the untreated rTetR-only negative control cells which fits both the ON peak and the subpopulation of background-silenced OFF cells, and then set a threshold that was 2 standard deviations below the mean of the ON peak in order to label cells that have silenced as OFF. Using the time-matched untreated control, the background normalized percentage of cells was calculated CellsOFF,normalized=CellsOFF,+dox/(1−CellsOFF,untreated). Two independently transduced biological replicates were used. A gene silencing model, consisting of the increasing form of the exponential decay (e.g., exponential decay subtracted from 1) during the doxycycline treatment phase and an exponential decay during the doxycycline removal phase with additional parameters for lag times before silencing and reactivation initiate, was fit to the normalized data using SciPy.
Individual activator recruitment assays Domains were cloned as a fusion with rTetR(SE-G72P) upstream of a T2A-mCherry-BSD marker, using GoldenGate cloning in the backbone pJT126. K562 pDY32 minCMV citrine reporter cells were then transduced with each lentiviral vector and, 3 days later, selected with blasticidin (10 μg/mL) until >80% of the cells were mCherry positive (6-7 days). Cells were split into separate wells of a 24-well plate and either treated with doxycycline or left untreated. Timepoints were measured by flow cytometry analysis of >15,000 cells (Biorad ZE5). To compute a fraction of ON cells during doxycycline treatment, a Gaussian model was fitted to the untreated rTetR-only negative control cells which fits the OFF peak, and then set a threshold that was 2 standard deviations above the mean of the OFF peak in order to label cells that have activated as ON. Two independently transduced biological replicates were used.
Flow cytometry for FLAG-tagged protein levels Staining of FLAG-tagged fusion protein levels was performed. Specifically, K562 cells were transduced with lentivirus to express the fusion proteins, selected with blasticidin, and then were fixed with Fix Buffer I (BD Biosciences) for 15 minutes at 37° C. Cells were washed with cold PBS with 10% FBS once and then permeabilized on ice for 30 min using Perm Buffer III (BD Biosciences). Cells were washed twice and then stained with anti-FLAG (XX) for 1 hour at 4° C. After a final round of washing, flow cytometry was performed using a CytoFLEX (Beckman Coulter) flow cytometer. The data was analyzed with CytoFlow by gating the cells on mCherry expression and then plot the FLAG-tagged protein level in mCherry+ and non-transduced cells. This approach controls for variability in staining efficiency as the two cell groups are mixed within the same sample.
Phylogenetic and alignment analyses KRAB and homeodomain sequences were retrieved from Pfam and extended, using surrounding native sequence, to reach 80 AA. Well-expressed domains were selected for alignment. Phylogenetic trees and sequence alignments were obtained using the alignment website Clustal Omega using default parameters (McWilliam et al., 2013; Sievers et al., 2011), and the 52 phylogenetic neighbor-joining tree without distance corrections was built with default parameters in Jalview (Waterhouse et al., 2009). Alignment visualization was performed in Jalview.
Analysis of amino acid residue conservation Protein sequences were submitted to the ConSurf webserver and analyzed using the ConSeq method. Briefly, ConSeq selects up to 150 homologs for a multiple string alignment, by sampling from the list of homologs with 35-95% sequence identity. Then, a phylogenetic tree is re-constructed and conservation is scored using Rate4Site. ConSurf provides normalized scores, so that the average score for all residues is zero, and the standard deviation is one. The conservation scores calculated by ConSurf are a relative measure of evolutionary conservation at each residue in the protein and the lowest score represents the most conserved position in the protein. The uniqueness of the ZNF10 KRAB N-terminal extension was determined by protein BLAST to all human proteins and searching for other zinc finger protein among the BLAST matches (Johnson et al., 2008).
ChIP-seq and ChIP-exo analysis External ChIP datasets were retrieved from multiple sources. ENCODE ChIP-seq data was processed with the uniform processing pipeline of ENCODE (ENCODE Project Consortium et al., 2020), and narrow peaks below IDR threshold 0.05 were retrieved. KRAB ZNF ChIP-exo data from tagged KRAB ZNF overexpression in HEK293 cells and KAP1 ChIP-exo data from H1 hESCs was obtained from GEO accession GSE78099 (Imbeault et al., 2017). Reads were trimmed to a uniform length of 36 basepairs and mapped to the hg38 version of the human genome using Bowtie (version 1.0.1; (Langmead et al., 2009)), allowing for up to 2 mismatches and only retaining unique alignments. Peak were called using MACS2 (version 2.1.0) (Feng et al., 2012) with the following settings: “-g hs-f BAM --keep-dup all --shift -75 --extsize 150 --nomodel”. Browser tracks were generated using Python scripts. For some KRAB ZNFs where ChIP-exo data was not available, ChIP-seq data from tagged KRAB ZNF overexpression in HEK293 cells was obtained from GEO accessions GSE76496 (Schmitges et al., 2016) and GSE52523 (Najafabadi et al., 2015). KRAB ZNF peaks were defined as solo binding sites if no other KRAB ZNF in the dataset had a peak less than 250 basepairs away. ENCODE H3K27ac ChIP-seq datasets for H1 cells were processed with the ENCODE pipeline (ENCODE Project Consortium et al., 2020), narrow peaks were called with MACS2, and peaks below IDR threshold 0.05 were retrieved.
External datasets ChIP-seq and ChIP-exo data for KRAB ZNF, KAP1, and H3K27ac (ENCODE Project Consortium et al., 2020; Imbeault et al., 2017; Najafabadi et al., 2015; Schmitges et al., 2016), KRAB ZNF gene evolutionary age (Imbeault et al., 2017), KRAB ZNF protein co-immunoprecipitation/mass spectrometry data (Helleboid et al., 2019), and CAT assays for KRAB repressor activity (Margolin et al., 1994; Witzgall et al., 1994) were retrieved from previously published studies.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims the benefit of U.S. Provisional Application No. 63/234,096, filed Aug. 17, 2021, the content of which is herein incorporated by reference in its entirety.
This invention was made with Government support under contract GM128947 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/075082 | 8/17/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63234096 | Aug 2021 | US |
