ENGINEERED MULTIPARTITE TRANSCRIPTIONAL EFFECTORS SOURCED FROM HUMAN PROTEIN DOMAINS

The present disclosure is directed to designed fusion proteins derived from MTFs with strong potency to modulate transcription and designated these recombinant fusion proteins MSN and NMS. These powerful transactivators potently activate transcription from endogenous loci when recruited through CRISPR-dCas9, Zinc Finger, or TALE system proteins. This technology permits upregulation of gene expression in targeted manner devoid of viral transcription activation domains and is amenable to high-throughput screening. These synthetic transcription activators interact with all programable DNA binding proteins tested and have exhibited applicability in vitro for efficient lineage conversion.

SEQUENCE LISTING

This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on Jan. 30, 2023, is named RICEP0092WO.xml and is 6,945 bytes in size.

BACKGROUND

1. Field of the Disclosure

The present disclosure relates generally to the fields of molecular biology and gene expression. More particular, the disclosure relates to multipartite transcriptional effectors and uses thereof.

Nuclease deactivated CRISPR-Cas (dCas) systems can be used to modulate transcription in cells and organisms1-8. For CRISPR-based activation (CRISPRa) approaches, transcriptional activators can be recruited to genomic regulatory elements using direct fusions to dCas proteins9-13, antibody-mediated recruitment14, or using engineered gRNA architectures15, 16. High levels of CRISPRa-driven transactivation have been achieved by shuffling17, reengineering18, or combining9, 19, 20 transactivation domains (TADs) and/or chromatin modifiers. However, many of the transactivation components used in these CRISPRa systems have coding sizes that are restrictive for applications such as viral vector-based delivery. Moreover, most of the transactivation modules that display high potencies harbor components derived from viral pathogens and are poorly tolerated in clinically important cell types, which could hamper biomedical or in vivo use. Finally, there is an untapped repertoire of thousands of human transcription factors (TFs) and chromatin that has yet to be systematically tested and optimized as programmable modifiers21-24 transactivation components. This diverse repertoire of human protein building blocks could be used to reduce the size of transactivation components, obviate the use of viral TFs, and possibly permit cell and/or pathway specific transactivation.

Mechanosensitive transcription factors (MTFs) modulate transcription in response to mechanical cues and/or external ligands25, 26. When stimulated, MTFs are shuttled into the nucleus where they can rapidly transactivate target genes by engaging key nuclear factors including RNA polymerase II (RNAP) and/or histone modifiers27-30. The dynamic shuttling of MTFs can depend upon both the nature and the intensity of stimulation. Mammalian cells encode several classes of MTFs, including serum regulated MTFs (e.g., YAP, TAZ, SRF, MRTF-A and B, and MYOCD)26, 31, cytokine regulated/JAK-STAT family MTFs (e.g., STAT proteins)32, and oxidative stress/antioxidant regulated MTFs (e.g., NRF2)33; each of which can potently activate transcription when appropriately stimulated.

SUMMARY

Thus, in accordance with the present disclosure, recombinant transcription activators comprising transcription activation domains from MRTF-A, STAT1 and eNRF2 are described. The recombinant transcription activators may further comprise a genomic regulatory element targeting domain and/or RNA-binding protein. The RNA-binding protein can be any protein that specifically binds RNA, such as one containing an MCP or PCP domain. Other examples include RNA-binding proteins/domains from PP7, Pumilio or RNA-binding Cas species distinct from the genomic regulatory element. The genomic regulatory element targeting domain may be a Cas protein, such as Cas6, AsdCas12a, SpdCas9, CjdCas9, or SadCas9. The genomic regulatory element targeting domain may also be a TALE DNA binding domain or a zinc finger DNA binding domain. The transcription activation domains may be ordered MRTF-A, STAT1 and eNRF2 in an N- to C-terminal order or may be ordered eNRF2, MRTF-A and STAT1 in an N- to C-terminal order. The transcription activation domains may be directly linked to said genomic regulatory element targeting domain or linked to said genomic regulatory element targeting domain through a linking moiety, such as where the linking moiety is GS or XTEN. The recombinant transcription activator may be about 250-500 or about 290 amino acid residues in length.

Also provided is a recombinant nucleic acid segment encoding a transcription activator comprising transcription activation domains MRTF-A, STAT1 and eNRF2. The nucleic acid may further comprise a nucleic acid segment encoding a genomic regulatory element targeting domain and/or RNA-binding protein. The RNA-binding protein can be any protein that specifically binds RNA, such as one containing an MCP or PCP domain. Other examples include RNA-binding proteins/domains from PP7, Pumilio or RNA-binding Cas species distinct from the genomic regulatory element. The genomic regulatory element targeting domain may be a Cas protein, such as Cas6, AsdCas12a, SpdCas9, CjdCas9, or SadCas9. The genomic regulatory element targeting domain may be a TALE DNA binding domain or a zinc finger DNA binding domain. The transcription activation domains may be ordered MRTF-A, STAT1 and eNRF2 in an N- to C-terminal order or may be ordered eNRF2, MRTF-A and STAT1 in an N- to C-terminal order. The transcription activation domains may be directly linked to said genomic regulatory element targeting domain or linked to said genomic regulatory element targeting domain through a linking moiety, such as where the linking moiety is GS or XTEN. The recombinant transcription activator may be about 750-1500 or about 870 bases in length. The promoter active may be eukaryotic cell is EFS or CMV.

In another embodiment, there is provided an artificial recombinant transcription factor comprising or consisting of at least 2 or at least 3 repeated 9aa TADs generated from MRTF-B and MYOCD or transcription factors. The recombinant transcription factor may be about or less than 300 amino acids in size. The MRTF-B and MYOCD features may be linked by linking moiety, such as the linking moieties GS and/or XTEN.

In still another embodiment, there is provided a method of editing gene expression in a eukaryotic cell comprising transferring into said cell the nucleic acid segment as defined above. The gene regulatory element targeting domain may be a Cas protein, and the method may comprise providing to said cell a guide RNA. The eukaryotic cell may be an isolated cell in culture, derived from a living organism, a human cell, non-human mammalian cell or a fibroblast. The editing may result in one or more of (a) increased gene expression of one or multiple genes, (b) induction of cellular differentiation, (c) induction of cellular de-differentiation. The editing may result in induction of pluripotency/stem cells from a differentiated cell. The editing may result in expression of a native/endogenous gene in a cell deficient in expression of said native gene/endogenous gene. The editing may result in expression of a non-native/exogenous gene such that said cell is protected from or at reduced risk of development of a disease state, disease condition or disorder. The editing system may be delivered via a viral mechanism, such as adeno-associated virus, lentivirus, retrovirus, herpesvirus, baculovirus, or adenovirus or delivered via a non-viral mechanism, such as electroporation, nucleofection, mechanical stress, or liposomal transfer.

It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The engineered upregulation of gene expression depends on seamless design, rational architecture and precise targeting of engineered transcription factors or epigenetic modifiers to the gene regulatory element of interest. Despite much progress in developing multipartite transcription factors and recruitment strategies to form multimeric complex at the target DNA sequence in human cells, there remain few options in the synthetic transcription factor (TF) toolbox.

Here, the inventors quantify the endogenous transactivation potency of dozens of different TADs derived from human MTFs in different combinations and across various dCas-based recruitment architectures. The inventors use these data to design new multipartite transactivation modules, called MSN, NMS, and eN3×9 and the inventors further apply the MSN and NMS effectors to build the CRISPR-dCas9 recruited enhanced activation module (DREAM) platform. The inventors demonstrate that CRISPR-DREAM potently stimulates transcription in primary human cells and cancer cell lines, as well as in murine and CHO cells.

The inventors also show that CRISPR-DREAM activates different classes of RNAs spanning diverse regulatory elements within the human genome. Further, the inventors find that the MSN/NMS effectors are portable to smaller engineered dCas9 variants, natural orthologues of dCas9, dCas12a, Type I CRISPR/Cas systems, and TALE and ZF proteins. Moreover, the inventors demonstrate that a dCas12a-NMS fusion enables superior multiplexing transactivation capabilities compared to existing systems.

The inventors also show that dCas9-NMS efficiently reprograms human fibroblasts to induced pluripotency and the inventors leverage the compact size of these new effectors to build potent dual and all-in-one CRISPRa AAVs. Finally, the inventors demonstrate that MSN, NMS, and eN3×9 are better tolerated than viral-based TADs in primary human MSCs and T cells. Overall, the engineered transactivation modules that the inventors have developed here are small, highly potent, devoid of viral sequences, versatile across programmable DNA binding systems, and enable robust multiplexed transactivation in human cells-important features that can be leveraged to test new biological hypotheses and engineer complex cellular functions. These and other aspects of the disclosure are described in detail below.

Mechanosensitive transcription factors (MTFs) are highly regulated robust and efficient transcriptional modulators, which respond to mechanical cues or external ligands. Upon activation they can be shuttled to the cell nucleus, rapidly induce transcription and then subsequently can be exported from nucleus. These dynamics are controlled by the nature and the intensity of stimulation. MTFs coordinate this rapid transcription by engaging many nuclear factors including RNA polymerase, histone writers, readers, and/or erasers. Here, the inventors evaluated and selected particular TADs from a variety of factors including serum regulated (YAP-TAZ-TEAD and SRF-MRTF/MYOCD) transcription factors, cytokine regulated JAK-STAT family transcription factors and oxidative stress/antioxidant regulated NRF2. A discussion of these factors and the design of new recombinant transcription regulators is provided below.

A. Transcription Factors and Activation Domains

A transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The function of TFs is to regulate gene expression to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism. Groups of TFs function in a coordinated fashion to direct cell division, cell growth, and cell death throughout life; cell migration and organization during embryonic development; and intermittently in response to signals from outside the cell, such as a hormone. Transcription factors are members of the proteome as well as regulome.

TFs work alone or with other proteins in a complex, by promoting (as an activator), or blocking (as a repressor) the recruitment of RNA polymerase (the enzyme that performs the transcription of genetic information from DNA to RNA) to specific genes.

A defining feature of TFs is that they contain at least one DNA-binding domain (DBD), which attaches to a specific sequence of DNA adjacent to the genes that they regulate. TFs are grouped into classes based on their DBDs. Other proteins such as coactivators, chromatin remodelers, histone acetyltransferases, histone deacetylases, kinases, and methylases are also essential to gene regulation, but lack DNA-binding domains, and therefore are not TFs.

Transcription factors are essential for the regulation of gene expression and are, as a consequence, found in all living organisms. The number of transcription factors found within an organism increases with genome size, and larger genomes tend to have more transcription factors per gene.

There are approximately 2800 proteins in the human genome that contain DNA-binding domains, and 1600 of these are presumed to function as transcription factors, though other studies indicate it to be a smaller number. Therefore, approximately 10% of genes in the genome code for transcription factors, which makes this family the single largest family of human proteins. Furthermore, genes are often flanked by several binding sites for distinct transcription factors, and efficient expression of each of these genes requires the cooperative action of several different transcription factors (see, for example, hepatocyte nuclear factors). Hence, the combinatorial use of a subset of the approximately 2000 human transcription factors easily account for the unique regulation of each gene in the human genome during development.

Transcription factors bind to either enhancer or promoter regions of DNA adjacent to the genes that they regulate. Depending on the transcription factor, the transcription of the adjacent gene is either up- or down-regulated. Transcription factors use a variety of mechanisms for the regulation of gene expression. These mechanisms include:

Transactivation domains or trans-activating domains (TADs) are transcription factor scaffold domains which contain binding sites for other proteins such as transcription coregulators. These binding sites are frequently referred to as activation functions (AFs). TADs are named after their amino acid composition. These amino acids are either essential for the activity or simply the most abundant in the TAD. Transactivation by the Gal4 transcription factor is mediated by acidic amino acids, whereas hydrophobic residues in Gcn4 play a similar role. Hence, the TADs in Gal4 and Gcn4 are referred to as acidic or hydrophobic, respectively.

MRTF-A (myocardin related transcription factor A), also known as MKL/megakaryoblastic leukemia 1 is a protein that in humans is encoded by the MKL1 gene. The protein encoded by this gene is regulated by the actin cytoskeleton and is shuttled between the cytoplasm and the nucleus in response to actin dynamics. In the nucleus, it coactivates the transcription factor serum response factor, a key regulator of smooth muscle cell differentiation, in an interaction mediated by its Basic domain. It is closely related to MKL2 and myocardin, with which it shares five key conserved structural domains. This gene is involved in a specific translocation event that creates a fusion of this gene and the RNA-binding motif protein-15 gene. This translocation has been associated with acute megakaryocytic leukemia. It also functions in the process of normal megakaryocyte maturation.

Reference sequences for MRTF-A mRNA and protein can be found at NM_001282660 and NP_001269589, respectively.

Signal transducer and activator of transcription 1 (STAT1) is a transcription factor which in humans is encoded by the STAT1 gene. It is a member of the STAT protein family. All STAT molecules are phosphorylated by receptor associated kinases, that causes activation, dimerization by forming homo- or heterodimers and finally translocate to nucleus to work as transcription factors. Specifically, STAT1 can be activated by several ligands such as Interferon alpha (IFNα), Interferon gamma (IFNγ), Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), Interleukin 6 (IL-6), or IL-27.

Type I interferons (IFN-α, IFN-β) bind to receptors, cause signaling via kinases, phosphorylate and activate the Jak kinases TYK2 and JAK1 and STAT1 and STAT2. STAT molecules form dimers and bind to ISGF3G/IRF-9, which is Interferon stimulated gene factor 3 complex with Interferon regulatory Factor 9. This allows STAT1 to enter the nucleus. STAT1 has a key role in many gene expressions that cause survival of the cell, viability or pathogen response. There are two possible transcripts (due to alternative splicing) that encode 2 isoforms of STAT1. STAT1α, the full-length version of the protein, is the main active isoform, responsible for most of the known functions of STAT1. STAT1β, which lacks a portion of the C-terminus of the protein, is less-studied, but has variously been reported to negatively regulate activation of STAT1 or to mediate IFN-γ-dependent anti-tumor and anti-infection activities.

STAT1 is involved in upregulating genes due to a signal by either type I, type II, or type III interferons. In response to IFN-γ stimulation, STAT1 forms homodimers or heterodimers with STAT3 that bind to the GAS (Interferon-Gamma-Activated Sequence) promoter element; in response to either IFN-α or IFN-β stimulation, STAT1 forms a heterodimer with STAT2 that can bind the ISRE (Interferon-Stimulated Response Element) promoter element. In either case, binding of the promoter element leads to an increased expression of ISG (Interferon-Stimulated Genes).

Reference sequences for STAT1 mRNA and protein can be found at NM_007315 and NP_009330, respectively.

Nuclear factor erythroid 2-related factor 2 (NRF2), also known as nuclear factor erythroid-derived 2-like 2, is a transcription factor that in humans is encoded by the NFE2L2 gene. NRF2 is a basic leucine zipper (bZIP) protein that may regulate the expression of antioxidant proteins that protect against oxidative damage triggered by injury and inflammation, according to preliminary research. In vitro, NRF2 binds to antioxidant response elements (AREs) in the nucleus leading to transcription of ARE genes. NRF2 increases heme oxygenase 1 leading to an increase in phase II enzymes in vitro. NRF2 also inhibits the NLRP3 inflammasome.

NRF2 appears to participate in a complex regulatory network and performs a pleiotropic role in the regulation of metabolism, inflammation, autophagy, proteostasis, mitochondrial physiology, and immune responses. Several drugs that stimulate the NFE2L2 pathway are being studied for treatment of diseases that are caused by oxidative stress. A mechanism for hormetic dose responses is proposed in which Nrf2 may serve as an hormetic mediator that mediates a vast spectrum of chemopreventive processes.

NRF2 is a basic leucine zipper (bZip) transcription factor with a Cap “n” Collar (CNC) structure. NRF2 possesses six highly conserved domains called NRF2-ECH homology (Neh) domains. The Neh1 domain is a CNC-bZIP domain that allows Nrf2 to heterodimerize with small Maf proteins (MAFF, MAFG, MAFK). The Neh2 domain allows for binding of NRF2 to its cytosolic repressor Keap1. The Neh3 domain may play a role in NRF2 protein stability and may act as a transactivation domain, interacting with component of the transcriptional apparatus. The Neh4 and Neh5 domains also act as transactivation domains but bind to a different protein called cAMP Response Element Binding Protein (CREB), which possesses intrinsic histone acetyltransferase activity. The Neh6 domain may contain a degron that is involved in a redox-insensitive process of degradation of NRF2. This occurs even in stressed cells, which normally extend the half-life of NRF2 protein relative to unstressed conditions by suppressing other degradation pathways.

Reference sequences for NRF2 mRNA and protein can be found at NM_006164 and NP_001138884, respectively.

E. MSN and NMS

As discussed above, the inventors explored the transcription activation properties of a variety TAD domains from human transcription factors. After selecting the most potent, they examined all possible anchoring positions (direct fusion in N-terminal and C-terminal, MS2-MCP and SunTag) with a dCas9-sgRNA complex. They broadly divided these transcription factors into general categories based on their regulation of transcription and tested their ability to activate transcription in all anchoring architectures. Surprisingly, none of the TADs of STAT family members alone were able to activate transcription from the inventors' testbed. Among the three TAD domains of NRF2, two fused TAD domains of NRF2 namely Neh4 and Neh5 (designated eNRF2) were the most promising. In addition, MS2-MCP mediated recruitment showed significantly higher degree of upregulation among all anchoring architectures both for MRTF-A, MRTF-B and eNRF2. To assess the broad effectiveness of these TADs (MRTF-A, MRTF-B and eNRF2), the inventors tested their efficacy on endogenous protein coding genes in pooled gRNA settings (HBG1), single gRNA settings (SBNO2), LncRNA (GRASLND) and eRNA (NET1) and as expected these selected TADs can upregulate all tested target genes from 5 to 2000-fold (FIGS. 8a-f and FIGS. 10a-e).

To further increase transcriptional activity, the inventors made tripartite fusions by fusing eNRF2, MRTF-A and STAT1 and constructed two highly potent engineered transcription factors designated MSN (MRTF-A-STAT1-eNRF2) and NMS (eNRF2-MRTF-A-STAT1). These molecules were tested and compared for gene activation potential with all state of art CRISPR based activators (MCP-p65-HSF1, MCP-VP64, MCP-VPR, MCP-p300), MCP-MRTF-a-STAT1-eNRF2 (MCP-MSN) showed higher activation than all as tested on OCT4 locus (FIGS. 13a-c).

The inventors then investigated the potency of tripartite fusions (both MSN and NMS) by transferring them to another robust, versatile, easily programmable and multiplexable orthogonal system, namely, dCas12a. These were compared against available gold standard activator dCas12a-[Activ] and the result clearly demonstrate that dCas12a-NMS is able to induce transcription comparable or better than dCas12a-[Activ] and in tested ASCL1, ILIR2 loci (2 crRNA for each gene). Finally, it has been shown that Cas12a can process up to 20 crRNA and can activate 10 different genes, so the inventors took similar strategy and cloned 20 crRNA in an array targeting 16 different endogenous genes, targeting either promoter, enhancer, eRNA and LncRNA and the data showed the activation of 16 genes using dCas12a-NMS (FIGS. 4f-i and Supplementary FIGS. 31a-g).

Recently, the prototypic and well-studied Type I CRISPR system (E. coli K12) was engineered to robustly modulate transcription from endogenous loci. To leverage the efficacy of MSN and NMS domains and Type I CRISPR system, the inventors transferred MSN and NMS domains to Cas6 and compared its efficiency against already benchmarked Cas6-p300 system. These data demonstrate that Cas6-MSN acts superior to Cas6-p300 in the targeted TTN and HBG1 loci. Further, like dCas12a, Type I cascade system can process its own crRNA array and shown to activate 2 genes in arrayed crRNA settings, here, the inventors further extend the crRNA array up to 6, targeting 4 different genes and found that MSN is superior to p300 in multiplex activation platform (FIGS. 4c-e).

II. Genomic Regulatory Element Targeting Domains, RNA Binding Domains and Linkers

As discussed above, the utility of the TADs described above has been demonstrated using a variety of targeting domains. While the precise nature and function of the targeting domains is secondary, and virtually any such domain could function, the following discussion highlights highly relevant examples. In addition, an optional element further includes RNA binding elements such as MCPs, PCPs and Pumilio proteins. These elements would expand the toolbox of recruitment strategies of these domains, enabling the targeting of multiple effectors in combination with the MSN and NMS.

Cas (CRISPR associated protein) molecules play a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids and is heavily utilized in genetic engineering applications. Its main function is to cut DNA and thereby alter a cell's genome.

Cas9 is a perhaps the most studied of all the Cas molecules. It is a dual RNA-guided DNA endonuclease enzyme associated with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) adaptive immune system in Streptococcus pyogenes. S. pyogenes utilizes CRISPR to memorize and Cas9 to later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for sites complementary to the 20 bP spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes.

Apart from its original function in bacterial immunity, the Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and Transcription activator-like effector nuclease (TALEN) proteins, Cas9 is becoming a prominent tool in the field of genome editing.

Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA. Because the target specificity of Cas9 stems from the guide RNA: DNA complementarity and not modifications to the protein itself (like TALENs and zinc fingers), engineering Cas9 to target new DNA is straightforward. Versions of Cas9 that bind but do not cleave cognate DNA can be used to locate transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression. Native Cas9 requires a guide RNA composed of two disparate RNAs that associate—the CRISPR RNA (crRNA), and the trans-activating crRNA (tracrRNA). Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA (chiRNA).

B. TALE DNA Binding Domain

TAL (transcription activator-like) effectors (often referred to as TALEs, but not to be confused with the three amino acid loop extension homeobox class of proteins) are proteins secreted by some β- and γ-proteobacteria. Most of these are Xanthomonads. Plant pathogenic Xanthomonas bacteria are especially known for TALEs, produced via their type III secretion system. These proteins can bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection. They recognize plant DNA sequences through a central repeat domain consisting of a variable number of ˜34 amino acid repeats. There appears to be a one-to-one correspondence between the identity of two critical amino acids in each repeat and each DNA base in the target sequence. These proteins are interesting to researchers both for their role in disease of important crop species and the relative ease of retargeting them to bind new DNA sequences. Similar proteins can be found in the pathogenic bacterium Ralstonia solanacearum and Burkholderia rhizoxinica, as well as yet unidentified marine microorganisms. The term TALE-likes is used to refer to the putative protein family encompassing the TALEs and these related proteins.

The most distinctive characteristic of TAL effectors is a central repeat domain containing between 1.5 and 33.5 repeats that are usually 34 residues in length (the C-terminal repeat is generally shorter and referred to as a “half repeat”). A typical repeat sequence is LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), but the residues at the 12th and 13th positions are hypervariable (these two amino acids are also known as the repeat variable di-residue or RVD). There is a simple relationship between the identity of these two residues in sequential repeats and sequential DNA bases in the TAL effector's target site. The crystal structure of a TAL effector bound to DNA indicates that each repeat comprises two alpha helices and a short RVD-containing loop where the second residue of the RVD makes sequence-specific DNA contacts while the first residue of the RVD stabilizes the RVD-containing loop. Target sites of TAL effectors also tend to include a thymine flanking the 5′ base targeted by the first repeat; this appears to be due to a contact between this T and a conserved tryptophan in the region N-terminal of the central repeat domain. However, this “zero” position does not always contain a thymine, as some scaffolds are more permissive.

TAL effectors can induce susceptibility genes that are members of the NODULIN3 (N3) gene family. These genes are essential for the development of the disease. In rice two genes, Os-8N3 and Os-11N3, are induced by TAL effectors. Os-8N3 is induced by PthXo1 and Os-11N3 is induced by PthXo3 and AvrXa7. Two hypotheses exist about possible functions for N3 proteins-first, that they are involved in copper transport, resulting in detoxification of the environment for bacteria (the reduction in copper level facilitates bacterial growth), and second, that they are involved in glucose transport, facilitating glucose flow (this mechanism provides nutrients to bacteria and stimulates pathogen growth and virulence).

This simple correspondence between amino acids in TAL effectors and DNA bases in their target sites makes them useful for protein engineering applications. Numerous groups have designed artificial TAL effectors capable of recognizing new DNA sequences in a variety of experimental systems. Such engineered TAL effectors have been used to create artificial transcription factors that can be used to target and activate or repress endogenous genes in tomato, Arabidopsis thaliana, and human cells.

Genetic constructs to encode TAL effector-based proteins can be made using either conventional gene synthesis or modular assembly. A plasmid kit for assembling custom TALEN and other TAL effector constructs is available through the public, not-for-profit repository Addgene. Webpages providing access to public software, protocols, and other resources for TAL effector-DNA targeting applications include the TAL Effector-Nucleotide Targeter and taleffectors.com.

Engineered TAL effectors can also be fused to the cleavage domain of FokI to create TAL effector nucleases (TALEN) or to meganucleases (nucleases with longer recognition sites) to create “megaTALs.” Such fusions share some properties with zinc finger nucleases and may be useful for genetic engineering and gene therapy applications. TALEN-based approaches are used in the emerging fields of gene editing and genome engineering. TALE-induced non-homologous end joining modification has been used to produce novel disease resistance in rice.

C. Zinc Finger DNA Binding Domains

A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions (Zn2+) to stabilize the fold. It was originally coined to describe the finger-like appearance of a hypothesized structure from the African clawed frog (Xenopus laevis) transcription factor IIIA. However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. Xenopus laevis TFIIIA was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by the Krüppel factor in Drosophila. It often appears as a metal-binding domain in multi-domain proteins.

Proteins that contain zinc fingers (zinc finger proteins) are classified into several different structural families. Unlike many other clearly defined supersecondary structures such as Greek keys or β hairpins, there are a number of types of zinc fingers, each with a unique three-dimensional architecture. A particular zinc finger protein's class is determined by this three-dimensional structure, but it can also be recognized based on the primary structure of the protein or the identity of the ligands coordinating the zinc ion. In spite of the large variety of these proteins, however, the vast majority typically function as interaction modules that bind DNA, RNA, proteins, or other small, useful molecules, and variations in structure serve primarily to alter the binding specificity of a particular protein.

Since their original discovery and the elucidation of their structure, these interaction modules have proven ubiquitous in the biological world and may be found in 3% of the genes of the human genome. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities. Engineering zinc fingers to have an affinity for a specific sequence is an area of active research, and zinc finger nucleases and zinc finger transcription factors are two of the most important applications of this to be realized to date.

Zinc finger (Znf) domains are relatively small protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not, instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise the finger-like folds. They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein, and/or lipid substrates. Their binding properties depend on the amino acid sequence of the finger domains and on the linker between fingers, as well as on the higher-order structures and the number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. Znf motifs occur in several unrelated protein superfamilies, varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g., some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organization, epithelial development, cell adhesion, protein folding, chromatin remodeling, and zinc sensing, to name but a few. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target.

Initially, the term zinc finger was used solely to describe DNA-binding motif found in Xenopus laevis; however, it is now used to refer to any number of structures related by their coordination of a zinc ion. In general, zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. Originally, the number and order of these residues was used to classify different types of zinc fingers (e.g., Cys2His2, Cys4, and Cys6). More recently, a more systematic method has been used to classify zinc finger proteins instead. This method classifies zinc finger proteins into “fold groups” based on the overall shape of the protein backbone in the folded domain. The most common “fold groups” of zinc fingers are the Cys2His2-like (the “classic zinc finger”), treble clef, and zinc ribbon.

Various protein engineering techniques can be used to alter the DNA-binding specificity of zinc fingers and tandem repeats of such engineered zinc fingers can be used to target desired genomic DNA sequences. Fusing a second protein domain such as a transcriptional activator or repressor to an array of engineered zinc fingers that bind near the promoter of a given gene can be used to alter the transcription of that gene. Fusions between engineered zinc finger arrays and protein domains that cleave or otherwise modify DNA can also be used to target those activities to desired genomic loci. The most common applications for engineered zinc finger arrays include zinc finger transcription factors and zinc finger nucleases, but other applications have also been described. Typical engineered zinc finger arrays have between 3 and 6 individual zinc finger motifs and bind target sites ranging from 9 basepairs to 18 basepairs in length. Arrays with 6 zinc finger motifs are particularly attractive because they bind a target site that is long enough to have a good chance of being unique in a mammalian genome.

Linkers are short peptide segments that permit the “fusion” of two often larger peptide or polypeptide regions such that the functionalities of the larger regions are not impaired or physically constrained by direct linkage at their termini. Linkers are often characterized by polar uncharged or charged residues, flexibility (although some applications benefit from rigid linkers) and secondary structures of particular nature.

III. Recombinant Vector Systems

Systems using MSN and NMS can not only be delivered as proteins per se (after appropriate recombinant production in bacterial or eurkaryotic hosts) but by expression from genetic construct as well. Plasmids or linear DNA encoding the NMS/MSN construct and the necessary gene regulatory elements can be delivered by virus, nanoparticles, or other methods. Similarly, RNA encoding these constructs and necessary regulatory elements or RNA modifications can be delivered via similar vehicles.

Expression requires that appropriate signals be provided in the vectors and include various regulatory elements in addition to the such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.

Use of the term “expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i.e., is under the control of a promoter. The phrase “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An “expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

In certain embodiments, viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.

Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

IV. Target Genes and Cells of Interest

The target cells in which the presently disclosed molecules can be used are virtually limitless. Of particular interest are diseased cells that do not express or have low expression of a particular gene. Others are cells where induction of gene expression will differentiate the cell into a cell needed by a host, such as for wound healing or recovery from a traumatic insult such as a stroke or myocardial infarction. The presently disclosed molecules are also of particular use in generating iPSCs by inducing gene expression patterns capable of de-differentiating cells such as fibroblasts. Further, other cell types include cells of the immune system or those with immunomodulatory potential, eye, central nervous system (CNS)-related, and/or muscle cells.

V. Treatment of Disease

The present disclosure has the potential to treat genetic disorders, in particular disorders of haploinsufficiency. Haploinsufficiency describes a model of dominant gene action in diploid organisms, in which a single copy of the wild-type allele at a locus in heterozygous combination with a variant allele is insufficient to produce the wild-type phenotype. Haploinsufficiency may arise from a de novo or inherited loss-of-function mutation in the variant allele, such that it produces little or no gene product (often a protein). Although the other, standard allele still produces the standard amount of product, the total product is insufficient to produce the standard phenotype. This heterozygous genotype may result in a non- or sub-standard, deleterious, and (or) disease phenotype. Haploinsufficiency is the standard explanation for dominant deleterious alleles.

In the alternative case of haplosufficiency, the loss-of-function allele behaves as above, but the single standard allele in the heterozygous genotype produces sufficient gene product to produce the same, standard phenotype as seen in the homozygote. Haplosufficiency accounts for the typical dominance of the “standard” allele over variant alleles, where the phenotypic identity of genotypes heterozygous and homozygous for the allele defines it as dominant, versus a variant phenotype produced by only by the genotype homozygous for the alternative allele, which defines it as recessive. The systems could also be used to induce a co-delivered gene not normally found in the target cells, for example, a cancer killing protein.

The following table provides examples of genes for which haploinsufficiency can lead to disease:

Genes Associated With Haploinsufficiency Diseases

Gene
Gene
mo-

Symbol
ID
some
Disorder/Syndrome

prostate cancer

and epilepsy with 1p36 deletion

syndrome

tumor multiplicity

deficiency syndrome

similar to Peters' anomaly

(CNS) malformations and urinary

tract defects

CCN1
3491
1
delayed formation of the ventricular

septum in the embryo and persistent

pigmentosa-19, and macular

stature and colobomata

enlargement of the affected jaw)

TP53BP2
7159
1
no suppression of tumor growth

MYCN
4613
2
reduced brain size and intestinal

atresias in Feingold syndrome

GCKR
2646
2
one form of maturity onset diabetes

of the young

FSHR
2492
2
degenerative changes in the central

nervous system

of tumor growth

IV, and with aortic and arterial

cleft palate

levels and shortened telomeres

with an Albright hereditary

and skin carcinogenesis

RASSF1A
11186
3
pathogenesis of a variety of cancers,

no suppression of tumor growth

TKT
7086
3
reduced adipose tissue and

female fertility

associated with ovarian dysfunction

congenita (ADDC), a rare inherited

bone marrow failure syndrome

and epilepsy

neurologic abnormalities

including the most common genetic

form of dwarfism, achondroplasia

of arteriovenous malformations

defects of dental enamel formation)

kidney disease

characterized by malformations

of the anterior segment of the eye,

failure of the periumbilical skin to

involute, and dental hypoplasia

development

developmental disorders

susceptibility associated with

genomic instability

and acute myeloid leukemias

TCOF1
6949
5
depletion of neural crest cell

syndrome

heart disease

and ectodermal organ formation

the anterior eye chamber

disruption of tissue structure,

integrity and changes in

EEF1E1
9521
6
no suppression of tumor growth

TNX
7148
6
Elastic fiber abnormalities in

hypermobility type

function and neonatal lethality

deficiency

anorexia and activation of

paraventricular nucleus neurons

chondrodysplasia and Japanese

and Pallister-Hall syndromes

diabetes of the young, type 2

(MODY2) and persistent

hyperinsulinemic hypoglycemia of

ELN
2006
7
cardiovascular disease and

connective tissue abnormalities

neurodevelopmental disorder

RFC2
5982
7
growth deficiency as well as

developmental disturbances in

Williams syndrome

granulomatous disease

characterised by abnormally

joint subluxation or hypodontia

stature, microcephaly and deafness

hippocampus synaptic function

FOXP2
93986
7
Speech and language impairment

ST7
7982
7
no suppression of tumor growth

syndrome

dysplasia, and autosomal dominant

and sporadic cases of congenital

cataracts and ocular anterior

segment anomalies

syndromes

DMRT1
1761
9
failure of testicular development

and feminization in male

karyotypic males and impaired

ovary function in karyotypic females

melanoma and nervous system

tumors

GCNT1
2650
9
T lymphoma cells resistant to

cell death

tumors formation

sex reversal, and adrenal failure

the bladder

COL5A1
1289
9
Structural abnormalities of the

cornea and lid

malformation and aortic valve

syndrome

and tumorigenesis

deafness and renal dysplasia)

syndrome

BUB3
9184
10
short life span that is associated

with the early onset of

disease and tumorigenesis

EXT2
2132
11
type II form of multiple exostoses

mirror image polydactyly,

dysmorphism and genital hypoplasia

neurodegenerative diseases

with multiple abnormalities

including growth retardation, facial

palate, and minor digital anomalies

ATM
472
11
High incidence of cancer

of various tumors

lymphoblastic leukemia

tumor growth

MYF6
4618
12
myopathy and severe course of

Becker muscular dystrophy

and postnatal growth

system morphology and function in

M levels

pancreatic and other cancers

abnormalities

phenotype and secondary

hyperthyrotropinemia, and diseases

due to transcription factor defects

hypoplasia, and ear anomalies

and dopa-responsive dystonia

pituitary anomalies

lymphomagenesis and thymocyte

development

syndrome

major defects in arterial and

vascular development

craniosynostosis syndrome

development and prevent spina

insufficiency

disorder Bloom syndrome

postnatal growth

retardation and developmental

SOX8
30812
16
the mental retardation found in

kidney disease

and feeding difficulties

CTCF
10664
16
loss of imprinting of insulin-like

growth factor-II in Wilms tumor

WWOX
51741
16
initiation of tumor development

FOXF1
2294
16
defects in formation and branching

of primary lung buds

YWHAE
7531
17
pathogenesis of small cell lung

cancer

migration and differentiation in

the adult dentate gyrus

liability to pressure palsies

abnormalities of melatonin in

including overgrowth

IL-7-induced mortality and

disease development

IL-7-induced mortality and

disease development

suppressor function

neurodegenerative disorders such as

cortico-basal degeneration and

progressive supranuclear palsy

deficiency in muscle fibers and

results in the clinical phenotype

multiple neoplasia syndrome

autosomal dominant retinitis

VENTRICULAR DYSPLASIA

syndromic mental disorder

growth

neoplastic transformation

hyperactivity and toxicity

and minicore myopathy with

external ophthalmoplegia

adenocarcinoma

dystrophy

type III and the autosomal

reduced penetrance

diabetes mellitus type I

and untimely degradation of securin

GNAS
2778
20
reduced activation of a downstream

target in epithelial tissues

syndrome

developmental delay

cardiac and thymic malformations)

schizophrenia

(schizoaffective disorders are

common features in patients

with DiGeorge/velocardiofacial

SOX10
6663
22
the etiology of

syndrome, characterized by severe

laxity, dolichocephaly, and minor

SHOX
6473
X
congenital form of growth failure,

the aetiology of “idiopathic” short

stature and the growth deficits and

skeletal anomalies in Leri Weill,

Langer and Turner syndrome

NLGN4X
57502
X
autism and Asperger syndrome

CSF2RA
1438
X
growth deficiency

and in the regulation of retinal

andiogenesis in response to hypoxia

carcinoma and Rodriguez

breast cancer

AR
367
X
Kennedy's disease and

ARX
170302
X
cognitive disability and epilepsy

chronic myelomonocytic leukemia

spinal muscular atrophy, and

occipital horn syndrome

disabilities

ATRX
546
X
cognitive disabilities as well as

syndrome

intellectual disability, and

developmental delay

BCL11A
53335
2
Autism and intellectual development

BCOR
54880
X
sarcoma of the kidney

BRWD3
254065
X
cognitive disabilities and

CASK
8573
X
FG syndrome 4, intellectual

disability and microcephaly

gastric and ovarian cancer

also known as X-linked West

CLCN5
1184
X
Dent disease and renal tubular

disorders complicated by

CNKSR2
22866
X
Intellectual disability

including Gilles de la Tourette

intellectual disability

syndrome and with Marshall

syndrome

Disease and idiopathic osteoporosis

and acute myeloid leukemia

CUL4B
8450
X
Intellectual disability

and lissencephaly syndrome

syndrome

and cardiomyopathy

dysplasia-11, and cancer

with microcephaly

and epithelial cancer

F8
2157
X
hemophilia A

F9
2158
X
hemophilia B or Christmas disease

FAM58A
92002
X
STAR syndrome

syndrome

syndrome and a syndromatic form

of X-linked cognitive disability

infection syndrome and Emberger

syndrome

progressive cataracts, and

GK
2710
X
glycerol kinase deficiency

GLA
2717
X
Fabry disease

postaxial polydactyly types A1 and B

GRIA3
2892
X
Intellectual disability

autism, attention deficit hyperactivity

disorder, epilepsy and schizophrenia

intellectual disability

IDS
3423
X
Hunter syndrome

and immunodeficiencies

IL1RAPL1
11141
X
intellectual disability

KIAA2022
340533
X
cognitive disability and epilepsy

acute myeloid leukemias

L1CAM
3897
X
Masa syndrome and L1 syndrome

and melorheostosis

MAGT1
84061
X
intellectual disability

impairment, and seizures

and cerebral malformation

cancer

endometrial cancer

with malignant fibrous

NDP
4693
X
Norrie disease

multiple synostoses syndrome

NR0B1
190
X
congenital adrenal hypoplasia and

and schizophrenia

NSDHL
50814
X
CHILD syndrome

night blindness

Lowe and also Dent disease

I and Simpson-Golabi-Behmel

syndrome type 2

PAK3
5063
X
intellectual disability

PCDH19
57526
X
epileptic encephalopathy and

autism

PHF6
84295
X
cognitive disability and epilepsy

PHF8
23133
X
Mental retardation and cleft palate

and spastic paraplegia type 2

and Arts syndrome

and macrocephaly

Parkes Weber syndrome

and aneurysms

polyposis syndrome, and

hereditary hemorrhagic

telangiectasia syndrome

syndrome

syndrome

SMS
6611
X
intellectual disability

cancer

neuronal degeneration such as

Rett syndrome

Aneurysm Syndrome

TSPAN7
7102
X
cognitive disability and

neuropsychiatric diseases

Cardiovascular malformations

Example 1—Materials and Methods

Plasmid Transfection and Nucleofection. HEK293T cell transfections were performed in 24-well plates using 375 ng of dCas9 expression plasmid and 125 ng of equimolar pooled or individual gRNAs/crRNAs. 1.25×105 HEK293T cells were plated the day before transfection and then transfected using Lipofectamine 3000 (Invitrogen, USA) as per manufacturer's instruction. For two component systems (dCas9+MCP or dCas9+scFv systems) 187.5 ng of each plasmid was used. For multiplex gene activation experiments using DREAM platforms, 25 ng of each gRNA encoding plasmid targeting each respective gene was used. Transfections in HeLa, A549, SK-BR-3, U2OS, HCT-116, HFF, NIH3T3, and CHO-K1 were performed in 12-well plates using Lipofectamine 3000 and 375 ng dCas9 plasmid, 375 ng of MCP-effector fusion proteins, and 250 ng DNA of MS2-modified gRNA encoding plasmid. For transfections using dCas12a fusion proteins where single genes were targeted, 375 ng of dCas12a-effector fusion plasmids and 125 ng of crRNA plasmids were transfected using lipofectamine 3000 per manufacturer's instruction. For multiplex gene activation experiments using dCas12a, 375 ng of dCas12a-effector fusion encoding plasmid and 250 ng of multiplex crRNA expression plasmids were used. For experiments using E. coli and P. aeruginosa Type I CRISPR systems, the inventors followed the same stoichiometries used in previous studies. For transfection of ICAM1-ZF effectors, 500 ng of each ICAM1 targeting ZF fusion was transfected. Transfections using IL1RN-TALE fusion proteins were performed using 500 ng of either single TALE or a pool of 4 TALEs using 125 ng of each TALE fusion. All ZF and TALE transfections were performed in HEK293T cells in 24-well format using Lipofectamine 3000 as per manufacturers instruction. For K562 cells, 1×106 cells were nucleofected using the Lonza SF Cell Line 4D-Nucleofector Kit (Lonza V4XC-2012) and a Lonza 4D Nucleofector (Lonza, AAF1002X) using the FF-120 program. 2000 ng of total plasmids were nucleofected in each condition using 1×106 K562 cells and 667 ng each of; dCas9 plasmid, MCP fusion plasmid, and pooled MS2-sgRNA expression plasmid was nucleofected per condition. Immediately after nucleofection, K562 cells were transferred to prewarmed media containing 6-well plates. hTERT-MSCs were electroporated with using the Neon transfection system (Thermo Fisher Scientific) using the 100 μL kit. 5×105 hTERT-MSCs were resuspended in 100 μL resuspension buffer R and 10 μg total DNA (3.75 μg dCas9, 3.75 μg MCP-fusion effector plasmid, and 2.5 μg MS2-modified gRNA encoding plasmid). Electroporation was performed using the settings recommended by the manufacturers for mesenchymal stem cells: Voltage: 990V, Pulse width: 40 ms, Pulse number: 1. For fibroblast reprogramming experiments, the inventors used the Neon transfection system using the amounts of endotoxin free DNA described previously17 and below. Dual AAV (500 ng of each) and All-in-one (AIO) AAV (1 μg) construct transfections were performed in Neuro-2a cells in 12-well format using Lipofectamine 3000 as per manufacturers instruction.

PBMC Isolation, Culture, and Nucleofection. De-identified white blood cell concentrates (buffy coats) were obtained from the Gulf Coast Regional Blood Center in Houston, Texas. PBMCs were isolated from buffy coats using Ficoll gradient separation and cryopreserved in liquid nitrogen until later use. 1×106 PBMCs per well were stimulated for 48h in a CD3/CD28 (Tonbo Biosciences, 700037U100 and 70289U100, respectively)-coated 24-well plate containing RPMI media supplemented with 10% FBS (Sigma-Aldrich), 1% Pen/Strep (Gibco), 10 ng/ml IL-15 (Tonbo Biosciences, 218157U002), and 10 ng/mL IL-7 (Tonbo Biosciences, 218079U002). Stimulated PBMCs were electroporated using the Neon transfection system (Thermo Fisher Scientific) 100 μL kit per manufacturer protocol. Briefly, PBMCs were centrifuged at 300 g for 5 min and resuspended in Neon Resuspension Buffer T to a final density of 1×107 cells/mL. 100 μL of the resuspended cells (1×106 cells) were then mixed with 12 μg total plasmid DNA (4.5 μg of dCas9 fusion encoding plasmids, 4.5 μg of MCP fusion encoding plasmids, and 3 μg of four equimolar pooled MS2-modified gRNA encoding plasmids) and electroporated with the following program specifications using a 100 μL Neon Tip: pulse voltage 2,150v, pulse width 20 ms, pulse number 1. Endotoxin free plasmids were used in all experiments. After electroporation, PBMCs were incubated in prewarmed 6-well plates containing RPMI media supplemented with 10% FBS (Sigma-Aldrich), 1% Pen/Strep (Gibco), 10 ng/mL IL-15, and 10 ng/mL IL-7. PBMCs were maintained at 37° C., 5% CO2 for 48h before RNA isolation and QPCR.

Human Primary T Cell and Primary Umbilical Cord MSC Culture and Lentiviral Transduction. PBMCs were isolated from de-identified white blood cell concentrates (buffy coats) using Ficoll gradient separation. T cells were isolated using negative selection via the EasySep™ Human T Cell Isolation Kit (StemCell, 17951). T cells were frozen in Bambanker Cell Freezing Media (Bulldog Bio Inc, BB01) and stored in liquid nitrogen until use. Umbilical cord derived MSCs (ATCC, PCS-500-010) were cultured in MSC basal media (ATCC, PCS-500-030) supplemented with Mesenchymal Stem Cell Growth Kit (PCS-500-040) containing rhFGF basic (5 ng/ml), rhFGF acidic (5 ng/mL), rhEGF (5 ng/mL), FBS (2%), and L-Alanyl-L-Glutamine (2.4 mM). MSC media was also supplemented with 1% Pen-strep (Gibco, 15140122). MSCs were maintained at 37° C., 5% CO2. Lentiviral transduction was performed in stimulated T cells as previously described34. Briefly, 1×106 T cells per well were stimulated for 24 h with Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation (Thermo Fisher Scientific, 11161D) according to manufacturer's instructions in a 24-well plate containing X-VIVO 15 media (Lonza, 04418Q) supplemented with 5% FBS (Sigma-Aldrich), 55 mM 2-Mercaptoethanol (Gibco, 21985023), 4 mM N-acetyl-L-cysteine (Thermo Fisher Scientific, 160280250), and 500 IU/ml of recombinant human IL-2 (Biolegend, 589104). Stimulated T cells were co-transduced via spinoculation at 931×g, 37° C. for 2 hours in a plate coated with Retronectin (Takara Bio, T100B) with an MOI of ˜5.0 for each lentivirus (dCas9 lentivirus at MOI ˜5.0 and gRNA-MCP-fusion effector lentivirus). After spinoculation, T cells were maintained at 37° C., 5% CO2 for 48h before downstream experiments. MSCs were co-transduced with an MOI of ˜10.0 (dCas9 lentivirus at MOI ˜10.0 and gRNA-MCP-fusion effector lentivirus at MOI ˜10.0) for each lentivirus via reverse transduction by seeding 1.25×105 cells into each well of a 12-well plate containing the virus in MSC media supplemented with 8 μg/mL polybrene. Media was changed after 16 hours. Further experimental analyses were performed 72 hours post-transduction.

Mouse Primary Neuron Culture and AAV8 transduction. Mouse C57 Cortex Neurons (Lonza, M-CX-300) were cultured in Primary Neuron Basal Medium (PNBM) supplemented with 2 mM L-glutamine, GA-1000 and 2% NSF. In brief, 4×105 cells were seeded in poly-D-lysine and laminin coated 24 well plates and cultured for 7 days for neuronal differentiation. On day 8, cells from each well were transduced with 1×1010 AAV8 viral particles (2.5×104/cell). 5 days post-transduction cells were harvested for RNA isolation and QPCR analysis.

Plasmid Cloning. Lenti-dCas9-VP64 (Addgene #61425), dCas9-VPR (Addgene #63798), dCas9-p300 (Addgene #83889), MCP-p65-HSF1 (Addgene #61423), scFv-VP64 (Addgene #60904), SpgRNA expression plasmid (Addgene #47108), MS2-modified gRNA expression plasmid (Addgene #61424), AsCas12a (Addgene #128136), E. Coli Type I Cascade system (Addgene #106270-106275) and Pae Type I Cascade System (Addgene #153942 and 153943), YAP-S5A (Addgene #33093) have been described previously. The eNRF2 TAD fusion was synthetically designed and ordered as a gBlock from IDT. To generate an isogenic C-terminal effector domain cloning backbone, the dCas9-p300 plasmid (Addgene #83889) was digested with BamHI and then a synthetic double-stranded ultramer (IDT) was incorporated using NEBuilder HiFi DNA Assembly (NEB, E2621) to generate a dCas9-NLS-linker-BamHI-NLS-FLAG expressing plasmid. This plasmid was further digested with AfeI and then a synthetic double-stranded ultramer (IDT) was incorporated using NEBuilder HiFi DNA Assembly to generate a FLAG-NLS-MCS-linker-dCas9 expressing Plasmid for N-terminal effector domain cloning. For fusion of effector domains to MCP, the MCP-p65-HSF1 plasmid (Addgene #61423) was digested with BamHI and NheI and respective effector domains were cloned using NEBuilder HiFi DNA Assembly. For SunTag components, the scFv-GCN4-linker-VP16-GB1-Rex NLS sequence was PCR amplified from pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS (Addgene #60904) and cloned into a lentiviral backbone containing an EF1-alpha promoter. Then VP64 domain was removed and an AfeI restriction site was generated and used for cloning TADs using NEBuilder HiFi DNA Assembly. The pHRdSV40-dCas9-10×GCN4_v4-P2A-BFP (Addgene #60903) vector was used for dCas9-based scFv fusion protein recruitment to target loci. All MTF TADs were isolated using PCR amplified from a pooled cDNA library from HEK293T, HeLa, U2OS and Jurkat-T cells. TADs were cloned into the MCP, dCas9 C-terminus, dCas9 N-terminus, and scFv backbones described above using NEBuilder HiFi DNA Assembly. Bipartite N-terminal fusions between MCP-MRTF-A or MCP-MRTF-B TADs and STAT 1-6 TADs were generated by digesting the appropriate MCP-fusion plasmid (MCP-MRTF-A or MCP-MRTF-B) with BamHI and then subcloning PCR-amplified STAT 1-6 TADs using NEBuilder HiFi DNA Assembly. Bipartite C-terminal fusions between MCP-MRTF-A or MCP-MRTF-B TADs and STAT 1-6 TADs were generated by digesting the appropriate MCP-fusion plasmid (MCP-MRTF-A or MCP-MRTF-B) with NheI and then subcloning PCR-amplified STAT 1-6 TADs using NEBuilder HiFi DNA Assembly. Similarly, eNRF2 was fused to the N- or C-terminus of the bipartite MRTF-A-STAT1 TAD in the MCP-fusion backbone using either BamHI (N-terminal; MCP-eNRF2-MRTF-A-STAT1 TAD) or NheI (C-terminal; MCP-MRTF-A-STAT1-eNRF2 TAD) digestion and NEBuilder HiFi DNA Assembly to generate the MCP-NMS or MCP-MSN tripartite TAD fusions, respectively. SadCas9 (with D10A and N580A mutations derived using PCR) was PCR amplified and then cloned into the SpdCas9 expression plasmid backbone created in this study digested with BamHI and XbaI. This SadCas9 expression plasmid was digested with BamHI and then PCR-amplified VP64 or VPR TADs were cloned in using NEBuilder HiFi DNA Assembly. CjCas9 was PCR-amplified from pAAV-EFS-CjCas9-eGFP-HIF1a (Addgene #137929) as two overlapping fragments using primers to create D8A and H559A mutations. These two CjdCas9 PCR fragments were then cloned into the SpdCas9 expression plasmid digested with BamHI and XbaI using NEBuilder HiFi DNA Assembly. This CjdCas9 expression plasmid was digested with BamHI and the PCR-amplified VP64 or VPR TADs were cloned in using NEBuilder HiFi DNA Assembly. HNH domain deleted SpdCas9 plasmids were generated using different primer sets designed to amplify the N-terminal and C-terminal portions of dCas9 excluding the HNH domain and resulting in either: no linker, a glycine-serine linker, or an XTEN16 linker, between HNH-deleted SpdCas9 fragments. These different PCR-amplified regions were cloned into the SpdCas9 expression plasmid digested with BamHI and XbaI using NEBuilder HiFi DNA Assembly. MCP-mCherry, MCP-MSN and MCP-p65-HSF1 were digested with NheI and a single strand oligonucleotide encoding the FLAG sequence was cloned onto the C-terminus of each respective fusion protein using NEBuilder HiFi DNA Assembly to enable facile detection via Western blotting. 1× 9aa TADs were designed and annealed as double strand oligos and then cloned into the BamHI/NheI-digested MCP-p65-HSF1 backbone plasmid (Addgene #61423) using T4 ligase (NEB). Heterotypic 2× 9aa TADs were generated by digesting MCP-1× 9aa TAD plasmids with either BamHI or NheI and then cloning single strand DNA encoding 1× 9aa TADs to the N- or C-termini using NEBuilder HiFi DNA Assembly. Heterotypic MCP-3× 9aa TADs were generated similarly by digesting MCP-2× 9aa TAD containing plasmids either with BamHI or NheI and then single strand DNA encoding 1× 9aa TADs were cloned to the N- or C-termini using NEBuilder HiFi DNA Assembly. Selected fusions between 3× 9aa TADs and eNRF2 were generated using gBlock (IDT) fragments and cloned into the BamHI/NheI-digested MCP-p65-HSF1 backbone plasmid (Addgene #61423) using NEBuilder HiFi DNA Assembly. To generate mini-DREAM compact single plasmid system, SpdCas9-HNH (no linker) deleted plasmid was digested with BamHI and then PCR amplified P2A self-cleaving sequence and MCP-eNRF2-3× 9aa TAD (eN3×9) was cloned using NEBuilder HiFi DNA Assembly. For dCas12a fusion proteins, SiT-Cas12a-Activ (Addgene #128136) was used. First, the inventors generated a nuclease dead (E993A) SiT-Cas12a backbone using PCR amplification and the inventors used this plasmid for subsequent C-terminal effector cloning using BamHI digestion and NEBuilder HiFi DNA Assembly. For E. coli Type I CRISPR systems, the Cas6-p300 plasmid (Addgene #106275) was digested with BamHI and then MSN and NMS domains were cloned in using NEBuilder HiFi DNA Assembly. Pae Type I Cascade plasmids encoding Csy1-Csy2 (Addgene #153942) and Csy3-VPR-Csy4 (Addgene #153943) were obtained from Addgene. The Csy3-VPR-Csy4 plasmid was digested with MluI (NEB) and BamHI (to remove the VPR TAD) and then the nucleoplasmin NLS followed by a linker sequence was added using NEBuilder HiFi DNA Assembly. Next, this Csy3-Csy4 plasmid was digested with AscI and either the MSN or NMS TADs were cloned onto the N-terminus of Csy3 NEBuilder HiFi DNA Assembly. ZF fusion proteins were generated by cloning PCR-amplified MSN, NMS, or VPR domains into the BsiWI and AscI digested ICAM1 targeting ZF-p300 plasmid10 using NEBuilder HiFi DNA Assembly. Similarly, TALE fusion proteins were created by cloning PCR-amplified MSN, NMS, or VPR domains into the BsiwI and AscI digested IL1RN targeting TALE plasmid backbone10 using NEBuilder HiFi DNA Assembly. pCXLE-dCas9VP192-T2A-EGFP-shP53 (Addgene #69535), GG-EBNA-OSK2M2L1-PP (Addgene #102898) and GG-EBNA-EEA-5guides-PGK-Puro (Addgene #102898) used for reprogramming experiments have been described previously17, 35. The PCR-amplified NMS domain was cloned into the sequentially digested (XhoI then SgrDI; to remove the VP192 domain) pCXLE-dCas9VP192-T2A-EGFP-shP53 backbone using NEBuilder HiFi DNA Assembly. TADs were directly fused to the C-terminus of dCas9 by digesting the dCas9-NLS-linker-BamHI-NLS-FLAG plasmid with BamHI and then cloning in PCR-amplified TADs using NEBuilder HiFi DNA Assembly. TADs were directly fused to the N-terminus to dCas9 by digesting the FLAG-NLS-MCS-linker-dCas9 plasmid with AgeI (NEB) and then cloning in PCR-amplified TADs using NEBuilder HiFi DNA Assembly. For constructs harboring both N- and C-terminal fusions, respective plasmids with TADs fused to the C-terminus of dCas9 were digested with AgeI and then PCR-amplified TADs were cloned onto the N-terminus of dCas9 using NEBuilder HiFi DNA Assembly. hSyn-AAV-EGFP (Addgene #50465) plasmid was used to generate different AAV based DNA constructs. For SpdCas9 cloning both EFGP and WPRE were removed using XbaI and XhoI and SpdCas9 and the modified smaller WPRE along with SV40 polyA signal (W3SL) were then cloned into this backbone using NEBuilder HiFi DNA Assembly. For expression of MS2-gRNA and hSyn-MCP-MSN from a single plasmid, both components were PCR amplified and cloned into an EGFP-removed hSyn-AAV-EGFP backbone using NEBuilder HiFi DNA Assembly. For All-In-One AAV backbone the M11 promoter36 was used to drive SaCas9 gRNA expression. The SCP137 and the EFS promoters were used to drive the expression of NMS-SadCas9. The efficient, smaller synthetic WPRE and polyadenylation signal CW3SA38 was utilized to maximize expression this size-limited context. Following cloning and sequence verification, 3 SaCas9 specific gRNAs targeting mouse Agrp gene were cloned into the all-in-one (AIO) vectors using Bbs1 restriction digestion. Following identification of the most efficacious gRNA (by transfecting into Neuro-2a cells), the SCP1 and EFS promoter driven SadCas9 based AIO plasmids were sequence verified by Plasmidsaurus. Sequence verified SpdCas9 and SCP1 and EFS promoter driven SadCas9 based AIO plasmids were sent to Charles River Laboratories for AAV8 production. Titers of different AAVs are included in source data.

gRNA Design and Construction. All protospacer sequences for SpCas9 systems were designed using the Custom Alt-R® CRISPR-Cas9 guide RNA design tool (IDT). All gRNA protospacers were then phosphorylated, annealed, and cloned into chimeric U6 promoter containing sgRNA cloning plasmid (Addgene #47108) and/or an MS2 loop containing plasmid backbone (Addgene #61424) digested with Bbs1 and treated with alkaline phosphatase (Thermo) using T4 DNA ligase (NEB). The SaCas9 gRNA expression plasmid (pIBH072) was a kind gift from Charles Gersbach and was digested with BbsI or Bpil (NEB or Thermo, respectively) and treated with alkaline phosphatase and then annealed protospacer sequences were cloned in using T4 DNA ligase (NEB). gRNAs were cloned into the pU6-Cj-sgRNA expression plasmid (Addgene #89753) by digesting the vector backbone with BsmBI or Esp3I (NEB or Thermo, respectively), and then treating the digested plasmid with alkaline phosphatase, annealing phosphorylated gRNAs, and then cloning annealed gRNAs into the backbone using T4 DNA ligase. MS2-stem loop containing plasmids for SaCas9 and CjCas9 were designed as gBlocks (IDT) with an MS2-stem loop incorporated into the tetraloop region for both respective gRNA tracr sequences. crRNA expression plasmids for the Type I Eco Cascade system were generated by annealing synthetic DNA ultramers (IDT) containing direct repeats (DRs) and cloning these ultramers into the BbsI and SacI-digested SpCas9 sgRNA cloning plasmid (Addgene #47108) using NEBuilder HiFi DNA Assembly. crRNA expression plasmids for Pae Type I Cascade system were generated by annealing and then PCR-extending overlapping oligos (that also harbored a BsmBI or Esp3I cut site for facile crRNA array incorporation) into the sequentially BbsI (or Bpil) and SacI-digested SpCas9 sgRNA cloning plasmid (Addgene #47108) using NEBuilder HiFi DNA Assembly. crRNA expression plasmids for Cas12a systems were generated by annealing and then PCR-extending overlapping oligos (that also harbored a BsmBI or Esp3I cut site for facile crRNA array incorporation) into the sequentially BbsI (or Bpil) and SacI-digested SpCas9 sgRNA cloning plasmid (Addgene #47108) using NEBuilder HiFi DNA Assembly.

crRNA Array Cloning. crRNA arrays for AsCas12a and Type I CRISPR systems were designed in fragments as overlapping ssDNA oligos (IDT) and 2-4 oligo pairs were annealed. Oligos were designed with an Esp3I cut site at 3′ of the array for subsequent cloning steps. Equimolar amounts of oligos were mixed, phosphorylated, and annealed similar to the standardized gRNA/crRNA assembly protocol above. Phosphorylated and annealed arrays were then cloned into the respective Esp3I-digested and alkaline phosphatase treated crRNA cloning backbone (described above) using T4 DNA ligase (NEB). crRNA arrays were verified by Sanger sequencing. Correctly assembled 4-8 crRNA array expressing plasmids were then digested again with Esp3I and alkaline phosphatase treated to enable incorporation of subsequent arrays up to 20 crRNAs.

Lentiviral packaging. All lentiviral transfer and packaging plasmids were purified using the Endofree Plasmid Maxi Kit (Qiagen, 12362). Lentivirus was packaged as previously described34 with minor modifications. Briefly, HEK293T cells were seeded into 225 mm flasks and maintained in DMEM. OptiMem was used for transfection and Sodium butyrate was added to a final concentration of 4 mM. Lentivirus was then concentrated 100× using the Lenti-X concentrator (Takara Bio, 631232). Biological titration of lentivirus by QPCR was carried out as previously described39, with the following modifications. Volumes of 10, 5, 1, 0.1, 0.01, and 0 μl of concentrated lentiviral particles were reverse transduced into 5×104 HEK293T cells with 8 μg/mL polybrene (Millipore-Sigma, TR1003G) in 24 well format with media exchanged after 14 hrs of transduction. gDNA was extracted 96 hours post transduction using the DNeasy Blood & Tissue Kit (Qiagen, 69506). qPCR was performed using 67.5 ng of gDNA for each condition in 10 ul reactions using Luna Universal qPCR Master Mix (NEB, M3003E).

Western Blotting. Cells were lysed in RIPA buffer (Thermo Scientific, 89900) with 1× protease inhibitor cocktail (Thermo Scientific, 78442), lysates were cleared by centrifugation and protein quantitation was performed using the BCA method (Pierce, 23225). 15-30 μg of lysate were separated using precast 7.5% or 10% SDS-PAGE (Bio-Rad) and then transferred onto PVDF membranes using the Transblot-turbo system (Bio-Rad). Membranes were blocked using 5% BSA in 1×TBST and incubated overnight with primary antibody (anti-Cas9; Diagenode #C15200216, Anti-FLAG; Sigma-Aldrich #F1804, anti-β-Tubulin; Bio-Rad #12004166). Then membranes were washed with 1×TBST 3 times (10 mins each wash) and incubated with respective HRP-tagged secondary antibodies for 1 hr. Next membranes were washed with 1×TBST 3 times (10 mins each wash). Membranes were then incubated with ECL solution (BioRad #1705061) and imaged using a Chemidoc-MP system (BioRad). The β-tubulin antibody was tagged with Rhodamine (Bio-Rad #12004166) and was imaged using Rhodamine channel in Chemidoc-MP as per manufacturer's instruction.

Quantitative Reverse-transcriptase PCR (QPCR). RNA (including pre-miRNA) was isolated using the RNeasy Plus mini kit (Qiagen #74136). 500-2000 ng of RNA (quantified using Nanodrop 3000C; Thermo Fisher) was used as a template for cDNA synthesis (Bio-Rad #1725038). cDNA was diluted 10× and 4.5 μL of diluted cDNA was used for each QPCR reaction in 10 μL reaction volume. Real-Time quantitative PCR was performed using SYBR Green mastermix (Bio-Rad #1725275) in the CFX96 Real-Time PCR system with a C1000 Thermal Cycler (Bio-Rad). Results are represented as fold change above control after normalization to GAPDH in all experiments using human cells. For murine cells, 18s IRNA was used for normalization. For CHO-K1 cells, GnbI was used for normalization. Undetectable samples were assigned a Ct value of 45 cycles.

Mature miRNA isolation and QPCR for miRNAs. Mature miRNA (miRNA) was isolated using the miRNA isolation kit (Qiagen #217084). 500 ng of isolated miRNA was polyadenylated using poly A polymerase (Quantabio #95107) in 10 μL reactions per sample and then used for cDNA synthesis using qScript Reverse Transcriptase and oligo-dT primers attached to unique adapter sequences to allow specific amplification of mature miRNA using QPCR in a total 20 μL reaction (Quantabio #95107). cDNA was diluted and 10 ng of miRNA cDNA was used for QPCR in a 25 μL reaction volume. PerfeCTa SYBR Green SuperMix (Quantabio #95053), miR-146a specific forward primer, and PerfeCTa universal reverse primer was used to perform QPCR. U6 snRNA was used for normalization.

Immunofluorescence Microscopy. Human foreskin fibroblasts (HFFs; CRL-2429, ATCC) and HFF-derived iPSCs were grown in Geltrex (Gibco, A1413302) coated 12-well plates and were fixed with 3.7% formaldehyde and then blocked with 3% BSA in 1×PBS for 1 hr at Room Temperature prior to imaging. Primary antibodies for SSEA-4 (CST #43782), TRA1-60 (CST #61220) and TRA1-81 (CST #83321) were diluted in 1% BSA in 1×PBS and incubated overnight at 4° C. The next day, cells were washed with 1×PBS, incubated with appropriate Alexaflour-488 conjugated secondary antibodies for 1 hr at Room Temperature and then washed again with 1×PBS. Cells were then incubated with DAPI (Invitrogen #D1306) containing PBS for 10m, washed with 1×PBS, and then imaged using a Nikon ECLIPSE Ti2 fluorescent microscope.

Fibroblast Reprogramming. HFFs were cultured in 1×DMEM supplemented with 1×Glutamax (Gibco, 35050061) for two passages before transfection with respective components. Cells were grown in 15 cm dishes (Corning), and detached using TrypLE select (Gibco, #12563011). Single cell suspensions were washed with complete media and then with 1×PBS. For each 1×106 cells, a total of 6 μg of endotoxin free plasmids (Macherey-Nagel, 740424; 2 μg CRISPR activator plasmid, 2 μg of pluripotency factor targeting gRNA plasmid, and 2 μg of EEA-motif targeting gRNA expression plasmids) were nucleofected using a 100 μL Neon transfection tip in R buffer using the following settings: 1650V, 10 ms, and 3 pulses. Nucleofected fibroblasts were then immediately transferred to Geltrex (Gibco) coated 10 cm cell culture dishes in prewarmed media. The next day media was exchanged. 4 days later, media was replaced with iPSC induction media17. Induction media was then exchanged every other day for 18 days. After 18 days iPSC colonies were counted, and colonies picked using sterile forceps and then transferred to Geltrex coated 12-well plates. iPSC colonies were maintained in complete E8 media and passaged as necessary using ReLeSR passaging reagent (Stem Cell Technology, #05872). RNA was isolated from iPSC clones using the RNeasy Plus mini kit (Qiagen #74136) and colonies were immunostained using indicated antibodies and counterstained with DAPI (Invitrogen) for nuclear visualization.

RNA Sequencing (RNA-seq). RNA-seq was performed in duplicate for each experimental condition. 72 hrs post-transfection RNA was isolated using the RNeasy Plus mini kit (Qiagen). RNA integrity was first assessed using a Bioanalyzer 2200 (Agilent) and then RNA-seq libraries were constructed using the TruSeq Stranded Total RNA Gold (Illumina, RS-122-2303). The qualities of RNA-seq libraries were verified using the Tape Station D1000 assay (Tape Station 2200, Agilent Technologies) and the concentration of RNA-seq libraries were checked again using real time PCR (QuantStudio 6 Flex Real time PCR System, Applied Biosystem). Libraries were normalized and pooled prior to sequencing. Sequencing was performed using an Illumina Hiseq 3000 with paired end 75 base pair reads. Reads were aligned to the human genome (hg38) Gencode Release 36 reference using STAR aligner (v2.7.3a). Transcript levels were quantified to the reference genome using a Bayesian approach. Normalization was done using counts per million (CPM) method. Differential expression was done using DESeq2 (v3.5) with default parameters. Genes were considered significantly differentially expressed based upon a fold change >2 or <−2 and an FDR <0.05.

9aa TAD Prediction. 9aa TADs were predicted using previously described software (world-wide-web at at.embnet.org/toolbox/9aatad/.) 40 using the “moderately stringent pattern” criteria and all “refinement criteria” and only TADs with 100% matches were then selected for evaluation in MCP fusion proteins.

Toxicity Assays. Cellular toxicity assays in primary T cells were performed 72 hours post-transduction using the Annexin V: PE Apoptosis Detection Kit (BD Biosciences, 559763). In brief, cells were stained with 7-AAD and Annexin V: PE according to the manufacturer's protocol. Stained cell fluorescence was measured using a Sony SA3800 spectral analyzer. EGFP positive single cells were gated and assessed for 7-AAD and Annexin V: PE fluorescence. All conditions were measured in biological triplicate and measured in technical duplicate. The toxicity of treatment groups was compared to the negative control (dCas9 alone), camptothecin (5 mM), and 65° C. heat shock were used as positive controls of apoptosis and membrane permeability respectively.

Data Analysis. All data used for statistical analysis had a minimum 3 biological replicates. Data are presented as mean±SEM Gene expression analyses were conducted using Student's t-tests (Two-tailed pair or multiple unpaired). Results were considered statistically significant when the P-value was <0.05. All bar graphs, error bars, and statistics were generated using GraphPad Prism v 9.0.

Select TADs from MTFs can activate transcription from diverse endogenous human loci when recruited by dCas9. The inventors first isolated TADs from 7 different serum-responsive MTFs (YAP, YAP-S397A41, TAZ, SRF, MRTF-A, MRTF-B, and MYOCD) and analyzed their ability to activate transcription when recruited to human promoters using either N- or C-terminal fusion to Streptococcus pyogenes dCas9 (dCas9), SunTag-mediated recruitment14, or recruitment via a gRNA aptamer and fusion to the MCP protein15 (FIGS. 7a-g). TADs derived from MRTF-A, MRTF-B, or MYOCD displayed consistent transactivation potential across recruitment architectures. The inventors next compared the optimal recruitment strategies for MRTF-A and MRTF-B TADs because they were more potent than, or comparable to, the MYOCD TAD yet slightly smaller. These results demonstrated that TADs from MRTF-A and B functioned best when fused to the MCP protein and recruited via gRNA aptamers (FIGS. 8a-f), and further that this strategy could be used with pools or single gRNAs, and to activate enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs).

Although the NRF2-ECH homology domains 4 and 5 (Neh4 and Neh5, respectively) within the oxidative stress/antioxidant regulated NRF2 human MTF have been shown to activate gene expression in Gal4 systems27, the inventors observed that neither Neh4 nor Neh5 were capable of potent human gene activation when recruited to promoters in any dCas9-based architecture (FIGS. 9a-g). Therefore, the inventors constructed an engineered TAD called eNRF2, consisting of Neh4 and Neh5 separated by an extended glycine-serine linker and found that the eNRF2 TAD stimulated high levels of transactivation in all dCas9-based recruitment configurations (FIGS. 9a-g). Similar to the MRTF-A/B TADs, eNRF2 displayed optimal potency in the gRNA aptamer/MCP-based recruitment architecture and transactivated diverse human regulatory loci (FIGS. 10a-e). The inventors next tested whether TADs derived from one of 6 different cytokine regulated/JAK-STAT family MTFs (STAT1-6) could transactivate human genes but observed that single STAT TADs alone were incapable of potent transactivation regardless of dCas9-based recruitment context (FIGS. 11a-g). Nevertheless, these data demonstrate that TADs from human MTFs can transactivate human loci when recruited via dCas9 and that these TADs are amenable to protein engineering.

Combinations of TADs from MTFs can potently activate human genes when recruited by dCas9. STAT proteins typically activate gene expression in combination with co-factors42. Therefore, the inventors tested if TADs from different STAT proteins might synergize with other MTF TADs. The inventors built 24 different bipartite fusion proteins by linking each STAT TAD to the N- or C-terminus of either the MRTF-A or MRFT-B TAD and then assayed the relative transactivation potential of each bipartite fusion when recruited to the human OCT4 promoter using gRNA aptamer/MCP-based recruitment (FIGS. 12a-c). Each of these 24 fusions markedly outperformed TADs from MRTF-A/B or STAT TADs alone, and one bipartite TAD configuration (MRFT-A/STAT1) was comparable to MCP fused to the dCas9-SAM derived bipartite p65-HSF115 module. The inventors next investigated whether the eNRF2 TAD could further enhance the potency of the MRFT-A/STAT1 module by building tripartite fusions consisting of MRTF-A/STAT1/eNRF2 (MSN) or eNRF2/MRTF-A/STAT1 (NMS) TADs. Both MSN and NMS stimulated OCT4 mRNA synthesis to levels comparable to the state-of-the-art CRISPRa platforms (FIGS. 13a-b) when recruited to the OCT4 promoter using gRNA aptamers/MCP-based targeting. Surprisingly, this potency was not further enhanced by the direct fusion of other TADs to the C-terminus of dCas9 (FIG. 13c). Collectively, these data show that gRNA aptamer/MCP-based recruitment of the MSN or NMS modules—termed the CRISPR-dCas9 recruited enhanced activation module (DREAM) platform—can efficiently stimulate transcription without viral components. These results also demonstrate that natural and engineered human TADs can have non-obvious interactions when combinatorially recruited in bi- and tripartite fashions.

CRISPR-DREAM displays potent activation of endogenous promoters, is specific, and is robust across diverse mammalian cell types. To assess the relative transactivation potential of CRISPR-DREAM, the inventors first targeted the DREAM or SAM15 systems (FIGS. 1a-b), to different human promoters in HEK293T cells. All components for both the DREAM and SAM systems were well-expressed in HEK293T cells (FIG. 1c). At all promoters targeted using pools of gRNAs (n=15), DREAM was superior or comparable or to the SAM system (FIG. 1d and FIGS. 14a-m). Similarly, when human promoters were targeted using only single gRNAs (n=11), DREAM remained superior or comparable to the SAM system in all experiments (FIG. 1e and FIGS. 15a-i). Interestingly, this trend extended throughout ˜1 kb upstream of the transcription start sites (TSSs) surrounding human genes (FIGS. 16a-d). Collectively, these data demonstrate that, although the DREAM system is smaller than the SAM system, and is devoid of viral TADs, it displays superior or comparable transactivation potency in human cells.

To test the transcriptome-wide specificity of CRISPR-DREAM, the inventors used 4 gRNAs to target the DREAM or the SAM system to the HBG1/HBG2 locus in HEK293T cells and then performed RNA-seq (FIG. 1f). HBG1/HBG2 gene activation was specific and potent for both the CRISPR-DREAM and SAM systems relative to dCas9+MCP-mCherry control treated cells. However, DREAM activated substantially more HBG1/HBG2 transcription than the SAM system or dCas9-VPR9 (FIG. 1f and FIGS. 17a-e). The inventors also found that the DREAM system was significantly (P<0.05) more potent than the SAM system at all targeted genes when each system was combined with a pool of six gRNAs, each targeting a different gene (FIG. 1g). Additionally, the inventors evaluated the efficacy of the DREAM system across a battery of different human cell types, including a diverse panel of cancer cell lines (FIG. 1h and FIGS. 18a-f) as well as primary and/or karyotypically normal human cells (FIG. 1i and FIGS. 19a-d). Finally, the inventors tested the transactivation potency of the DREAM system in mammalian cell types widely used for disease modeling/biocompatibility applications and therapeutic production pipelines (NIH3T3 and CHO-K1 cells, respectively; FIGS. 20a-b. Across all experiments the DREAM system displayed highly potent transactivation. Overall, these data demonstrate that CRISPR-DREAM is robust, broadly potent, specific, and functionally compatible with diverse human and mammalian cell types.

CRISPR-DREAM efficiently catalyzes RNA synthesis from noncoding genomic regulatory elements. Since CRISPR-DREAM efficiently and robustly activated mRNAs when targeted to promoter regions, the inventors next tested whether the DREAM system could also activate transcription from distal human regulatory elements (i.e., enhancers) and other non-coding transcripts (i.e., enhancer RNAs; eRNAs, long noncoding RNAs; lncRNAs, and microRNAs; miRNAs). The inventors first targeted the DREAM or SAM systems to the OCT4 distal enhancer (DE)43 and found that the DREAM system significantly (P<0.05) upregulated OCT4 expression relative to the SAM system when targeted to the DE (FIG. 2a). Similar results were observed when targeting the DREAM system to the DRR enhancer44 upstream of the MYOD gene (FIG. 21a). The inventors also targeted the DREAM system to the human HS2 enhancer45, 46 and observed that the DREAM system induced expression from the downstream HBE, HBG, and HBD genes (FIG. 2b). The inventors further observed transactivation of the SOCS1 gene when the DREAM system was targeted to either of two different intragenic SOCS1 enhancers; one located ˜15 kb, and the other ˜50 kb downstream of the SOCS1 TSS (FIG. 2c). Together these data demonstrate that CRISPR-DREAM can stimulate human gene expression when targeted to different classes of enhancers (those regulating a single-gene, multiple genes, or intragenic enhancers) embedded within native chromatin.

The inventors next tested whether CRISPR-DREAM could activate eRNAs when targeted to endogenous human enhancers. When targeted to the NET1 enhancer, the DREAM system activated eRNA transcription (FIG. 2d), consistent with other reports47. Moreover, when the DREAM system was targeted to the bidirectionally transcribed KLK3 and TFF1 enhancers, the inventors observed substantial upregulation of eRNAs in both the sense and antisense directions (FIGS. 2e-f). Similar results were obtained when targeting the human FKBP5 and GREB1 enhancers (FIGS. 21b-c). CRISPR-DREAM also stimulated the production of endogenous lncRNAs when targeted to the CCAT1, GRASLND, HOTAIR, or MALAT1 loci (FIGS. 2g-h, FIGS. 21d-e). Finally, the inventors found that the DREAM system activated miRNA-146a expression when targeted to the miRNA-146a promoter (FIG. 2i). Taken together, these data show that CRISPR-DREAM can robustly transactivate regulatory regions spanning diverse classes of the human transcriptome.

Smaller, orthogonal CRISPR-DREAM platforms enable expanded genomic targeting beyond NGG PAM sites. To enhance the versatility of CRISPR-DREAM beyond SpdCas9 and to expand targeting to non-NGG PAM sites, the inventors selected the two smallest naturally occurring orthogonal Cas9 proteins; SadCas9 (1,096aa) and CjdCas9 (1,027aa) for further analyses (FIG. 3a, FIG. 3d). The inventors used SaCas9-specific gRNAs harboring MS2 loops48 to compare the potency between the SadCas9-DREAM and SAM systems in HEK293T cells. SadCas9-DREAM was significantly (P<0.05) more potent than SadCas9-SAM when targeted to either the HBG1 or TTN promoters (FIG. 3b). The inventors also found that SadCas9-DREAM outperformed or was comparable to SadCas9-VPR when targeted to these loci (FIG. 3c). CjdCas9-based transcriptional activation platforms have also recently been developed using viral TADs (miniCAFE)49; however, gRNA-based recruitment of transcriptional modulators using CjdCas9 has not been described. Therefore, the inventors engineered the CjCas9 gRNA scaffold to incorporate an MS2 loop within the tetraloop of the CjCas9 gRNA scaffold (FIG. 22c). The inventors used this MS2-modified CjCas9 gRNA to generate CjdCas9-DREAM and compared the potency between CjdCas9-DREAM, CjdCas9-SAM, and the miniCAFE systems at the HBG1 or TTN promoters (FIGS. 3e-f) in HEK293T cells. At all targeted sites, CjdCas9-DREAM outperformed or was comparable to the CjdCas9-SAM or miniCAFE systems. The inventors also observed high levels of transactivation using SadCas9-DREAM and CjdCas9-DREAM in a different human cell line (FIGS. 22a, 22b, 22d, and 22e). These data demonstrate that DREAM is not only compatible with other orthogonal dCas9 targeting systems, but that it displays superior performance at most tested promoters.

Generation and validation of a compact mini-DREAM system. The inventors next sought to reduce the sizes of the CRISPR-DREAM components. The inventors first investigated whether individual TADs could be minimized while still retaining the transactivation potency when recruited by dCas9. The inventors focused on individual TADs from MTFs that displayed transactivation potential (i.e., MRTF-A, MRTF-B, and MYCOD proteins, FIGS. 7a-g, FIG. 8a-f). 9aa TADs have been shown to synthetically activate transcription previously using GAL4 systems40, 50. Therefore, the inventors used predictive software40 to identify 9aa TADs in MRTF-A, MRTF-B, and MYCOD proteins, and recruited these TADs to human loci using dCas9 and MCP-MS2 fusions in single, bipartite, and tripartite formats (FIG. 23a-j). Interestingly, the inventors observed that only tripartite combinations of 9aa TADs were able to robustly activate endogenous gene expression, and to varying degrees (FIG. 23f). The inventors selected one tripartite 9aa combination (3× 9aa TAD; MRTF-B.3+MYOCD.1+MYOCD.3) for further analysis (FIG. 3g). This 3× 9aa TAD activated HBG1, TTN, and CD34 gene expression when recruited to corresponding promoters using dCas9 (FIG. 3h; FIG. 23g). The inventors also found that this 3× 9aa TAD combination could activate gene expression via a single gRNA, and moreover could transactivate other endogenous regulatory loci (FIGS. 23h-j). These results suggest that combinations of 9aa TADs can be used as minimal functional units to transactivate endogenous human loci when recruited via dCas9.

The inventors next combined the 3× 9aa TAD with the engineered NRF2 TAD (eNRF2) in four different combinations to generate a small, yet potent transactivation module called eN3×9 (FIGS. 24a-b). Notably, minimized Cas9 proteins that retain DNA binding activity have also been recently created51, 52. Therefore, the inventors next evaluated the relative transactivation capabilities among a panel of minimized, HNH-deleted, dCas9 variants in tandem with MCP-MSN and found that an HNH-deleted variant without a linker between two RuvC domains was optimal, albeit with slight protein expression decreases (FIGS. 25a-b). The inventors further validated this linker-less, HNH-deleted CRISPR-DREAM variant at multiple human promoters and other regulatory elements (FIGS. 25c-h) and then combined this minimized dCas9 with MCP-eN3×9 to generate the mini-DREAM system (FIG. 3i). The mini-DREAM system transactivated HBG1, TTN, and IL1RN gene expression when recruited to corresponding promoters (FIG. 3j; FIG. 26a). The inventors also found that the mini-DREAM system could activate endogenous promoters via a single gRNA (FIGS. 26b-c), and could activate downstream gene expression when targeted to an upstream enhancer (FIG. 26d). Finally, the inventors evaluated whether the minimized components of the mini-DREAM system were functional when delivered within a single vector (FIG. 3k) and found that this compact, single vector mini-DREAM system retained transactivation potential when targeted to human promoters using pooled (FIG. 31; FIG. 26e-g), or a single gRNA (FIG. 26h). Overall, these data show that the components of the CRISPR DREAM system can be minimized to fit within a single vector delivery framework while retaining functionality.

The MSN and NMS effector domains are robust across programmable DNA binding platforms. The inventors next tested the potency of tripartite MSN and NMS effectors when fused the to dCas9 in different architectures and observed that both effectors could activate gene expression when fused to the N- or C-terminus of dCas9 (FIGS. 27a-e) or when recruited via the Sun-Tag14 architecture (FIGS. 28a-c). Interestingly, in contrast to MCP-mediated recruitment (FIGS. 13a-c), additional TADs were observed to improve performance in direct fusion architectures (FIG. 27a, FIG. 27c). In the SunTag architecture, the NMS domain was superior to other benchmarked effector domains, such as VP6414, VPR53, and p65-HSF154 (FIGS. 28a-c). To maximize the potential use of the MSN/NMS effector domains and explore their versatility, the inventors next tested whether each was capable of gene activation when fused to TALE or ZF scaffolds (FIG. 4a, FIG. 4d). Both effectors strongly transactivated IL1RN using a single TALE fusion protein (FIG. 29) or a pool of 4 TALE fusion proteins targeted to the IL1RN promoter (FIG. 4a). Similarly, both effectors activated ICAM1 expression using a single synthetic ZF fusion protein targeted to the ICAM1 promoter (FIG. 4b). These data demonstrate that the MSN and NMS effectors are compatible with diverse programmable DNA binding scaffolds beyond Type II CRISPR/Cas systems.

Transcriptional activators have recently been shown to modulate the expression of endogenous human loci when recruited by Type I CRISPR systems55. Therefore, to evaluate whether MSN and/or NMS were functional beyond Type II CRISPR systems, the inventors fused each to the Cas6 component of the E. coli Type I CRISPR Cascade (Eco-Cascade) system (FIG. 4c). These data showed that Cas6-MSN (or NMS) performed comparably to the Cas6-p300 system when targeted to a spectrum of human promoters (FIG. 4d; Figd. 30a-d). The inventors also observed that the Cas6-MSN (or NMS) systems could activate eRNAs from when targeted to the endogenous NET1 enhancer (FIG. 30e). One advantage of CRISPR Cascade is that the system can process its own crRNA arrays, which can enable multiplexed targeting to the human genome. Previous reports have leveraged this capability to simultaneously activate two human genes55. The inventors found that when Cas6 was fused to MSN, the CRISPR Cascade system could simultaneously activate up to six human genes when corresponding crRNAs were co-delivered in an arrayed format (FIG. 4e; FIG. 30f). The inventors also found that these transactivation capabilities were extensible to another Type I CRISPR system; Pae-Cascade56 (FIGS. 30g-i). In sum, these data show that the MSN and NMS effectors are robust and directly compatible with programmable DNA binding platforms beyond Type II CRISPR systems without any additional engineering.

The NMS effector enables superior multiplexed gene activation when fused to dCas12a. The CRISPR/Cas12a system has attracted significant attention because the platform is smaller than SpCas9, and because Cas12a can process its own crRNA arrays in human cells57. This feature has been leveraged for both multiplexed genome editing and multiplexed transcriptional control18. Therefore, the inventors next investigated the potency of the tripartite MSN and NMS effectors when they were directly fused to dCas12a (FIG. 4f). The inventors selected the AsdCas12a variant for this analysis because AsdCas12a (hereafter dCas12a) has been shown to activate human genes when fused to transcriptional effectors18. These results demonstrated that both dCas12a-MSN and dCas12a-NMS were able to induce gene expression when targeted to different human promoters using pooled or single crRNAs (FIG. 4g, FIG. 4h, FIGS. 31a-e). dCas12a-NMS was generally superior to dCas12a-MSN and to the previously described dCas12a-Activ system18 at the loci tested here. These data demonstrate that the NMS and MSN effectors domains are potent transactivation modules when combined with the dCas12a targeting system in human cells.

The inventors next tested the extent to which dCas12a-MSN/NMS could be used in conjunction with crRNA arrays for multiplexed endogenous gene activation. The inventors cloned 8 previously described crRNAs18 (targeting the ASCL1, ILIR2, IL1B or ZFP42 promoters) into a single plasmid in an array format and then transfected this vector into HEK293T cells with either dCas12a control, dCas12a-MSN, dCas12a-NMS, or the dCas12a-Activ system. Again, these data demonstrated that dCas12a-NMS was superior or comparable to dCas12a-Activ, even in multiplex settings (FIG. 31f). Finally, to evaluate if dCas12-NMS could simultaneously activate multiple genes on a larger scale, the inventors cloned 20 full-length (20 bp) crRNAs targeting 16 different loci into a single array (FIG. 31g). This array was designed to enable simultaneous targeting of several classes of human regulatory elements; including 13 different promoters, 2 different enhancers (one intrageneric; SOCS1, and one driving eRNA output; NET1), and one lncRNA (GRASLND). When this crRNA array was transfected into HEK293T cells along with dCas12a-NMS, RNA synthesis was robustly stimulated from all 16 loci (FIG. 4i). To the inventors' knowledge this is the most loci that have been targeted simultaneously using CRISPR systems, demonstrating the versatility and utility of the engineered NMS effector in combination with dCas12a. dCas9-NMS permits efficient reprogramming of human fibroblasts in vitro.

CRISPRa systems using repeated portions of the alpha herpesvirus VP16 TAD (dCas9-VP192) have been used to efficiently reprogram human foreskin fibroblasts (HFFs) into induced pluripotent stem cells (iPSCs)17. To evaluate the functional capabilities of the inventors' engineered human transactivation modules, the inventors fused the NMS domain directly to the C-terminus of dCas9 (dCas9-NMS) and tested its ability to reprogram HFFs. The inventors used a direct dCas9 fusion architecture so that the inventors could leverage gRNAs previously optimized for this reprogramming strategy and to better compare dCas9-NMS with the corresponding state of the art (dCas9-VP192)17. The inventors used the NMS effector as opposed to MSN, as NMS displayed more potency than MSN when directly fused to dCas9 (FIG. 27a). The inventors targeted dCas9-NMS (or dCas9-VP192) to endogenous loci using the 15 gRNAs previously optimized to reprogram HFFs to pluripotency with the dCas9-VP192 system. Using this approach, the inventors observed morphological changes beginning by 8 days post-nucleofection (FIG. 5a) and efficient reprogramming by 16 days post-nucleofection, although to a lesser extent than when using dCas9-VP192 (FIG. 32a).

The inventors picked and expanded iPSC colonies and then measured the expression of pluripotency and mesenchymal genes ˜40 days post-nucleofection. The inventors found that genes typically associated with pluripotency (OCT4, SOX2, NANOG, LIN28A, REX1, CDH1, and FGF4)58, 59 were highly expressed in colonies derived from HFFs nucleofected with the gRNA cocktail and dCas9-NMS or dCas-VP192 (FIG. 5b; FIGS. 32b-f). Conversely, the inventors observed that genes typically associated with fibroblast/mesenchymal cell identity (THY1, ZEB1, ZEB2, TWIST, and SNAIL2)58, 59 were poorly expressed in colonies derived from HFFs nucleofected with the gRNA cocktail and dCas9-NMS or dCas-VP192 (FIG. 5c; FIGS. 32g-i). Finally, the inventors assessed the expression of pluripotency associated markers (SSEA-4, TRA-1-81 and TRA-1-60)60 and found that all were highly expressed in iPSC colonies derived from HFFs nucleofected with the gRNA cocktail and either dCas9-NMS or dCas-VP192 (FIG. 5d, FIG. 5e; FIG. 32j). These data show that engineered transactivation modules sourced from human MTFs can be used to efficiently reprogram complex cell phenotypes, including cell lineage.

The MSN, NMS, and eN3×9 transactivation modules are well tolerated and effective in clinically useful primary human cell types. The recent development of CRISPRa tools has enabled new therapeutic opportunities6, 61. However, it has been shown that in some cases, CRISPRa tools harboring viral TADs can be poorly tolerated, and even toxic12, 62-64 This prompted us to test the relative expression and efficacy of the human MTF derived multipartite TADs MSN, NMS, and eN3×9 tools in comparison to the viral multipartite TAD VPR in therapeutically relevant human primary cells. The inventors selected primary human umbilical cord MSCs and primary T cells for analysis. Lentiviral transduction was selected to ensure high levels of payload delivery. Interestingly, the inventors observed that lentiviral titers were influenced by fused TAD, with MCP fused to eN3×9 consistently generating the highest titers (FIGS. 33a-b). The inventors next transduced MSCs using an MOI of ˜10.0 for all conditions and observed variable expression levels among MCP fusions proteins at 72 hours post transduction using both microscopy and flow cytometry (FIG. 6a) despite using equal amounts of lentivirus. For instance, although MCP-eN3×9 and MCP-NMS displayed high levels expression via microscopy, MCP-VPR and MCP-MSN were relatively poorly expressed. Similarly, the inventors also tested the expression levels of these MCP fusions in primary T cells using lentiviral transduction at an MOI of ˜5.0 and observed that MCP-eN3×9 displayed the highest expression levels 72 hours post transduction, while MCP-VPR showed the lowest expression (FIG. 6b).

The inventors next assessed the gene activation capabilities of these MCP-TAD fusions in primary MSCs and T cells. In MSCs, eN3×9 outperformed all other effectors, and VPR showed the lowest potency when targeted to the TTN promoter (FIG. 6c). In primary T cells each TAD activated CARD9 expression to relatively similar and modest levels when targeted to the CARD9 promoter (FIG. 6d). However, in primary T cells the inventors observed that the human MTF derived multipartite TADs resulted in dramatically better T cell viability than the viral multipartite TAD VPR (FIGS. 34a-b). Collectively these data demonstrate that the human MTF derived multipartite MSN, NMS, and eN3×9 TADs are as or more potent than the VPR TAD, while also maintaining similar or superior expression levels in therapeutically relevant human primary cells. Notably, MSN, NMS, and eN3×9 are also much smaller than the VPR TAD, and in the case of primary T cells, are also much less cytotoxic.

Dual and all-in-one AAV mediated delivery of CRISPR-DREAM and SadCas9-NMS systems efficiently activates gene expression in primary neurons. AAV mediated delivery has emerged as a powerful method to deliver therapeutic payloads in vitro65 and in vivo66. However, due to strict payload limitations, the delivery of CRISPRa tools using AAV has been limited to dual AAV systems and/or the use of viral TADs67, 68. To assess the transcriptional activation potential of the compact CRISPR-DREAM components in combination with AAV mediated delivery, the inventors targeted the murine Agrp gene, which modulates food intake behavior and obesity69, 70, as a proof of concept. The inventors first tested 15 individual gRNAs targeting a ˜1 kb window upstream of the Agrp promoter in Neuro-2a cells to identify a top performing gRNA (FIGS. 35a-b). Based on this result, the inventors constructed a dual AAV delivery system, wherein one AAV expressed dCas9, and the other AAV expressed the top performing Agrp-targeting gRNA along with MCP-MSN (FIG. 6e). Both recombinant AAVs (and an EGFP control AAV) used the AAV8 serotype capsid to ensure efficient neuronal transduction71 (FIG. 35e). In dual AAV-transduced (dCas9 and gRNA/MCP-MSN, respectively) primary murine neurons, the inventors observed high levels of Agrp activation (FIG. 6f).

Encouraged by this result using a dual AAV strategy, the inventors next designed two different all-in-one (AIO) AAV approaches (FIG. 6g). These designs leveraged the M11 promoter to express a gRNA, and either the SCP1 or EFS promoter to drive the expression of NMS fused to the N-terminus of SadCas9. NMS was prioritized over MSN as it showed higher potency when fused to N-terminus of dCas9 (FIGS. 28a-c). To further reduce packaging size, the inventors also selected compact engineered WPRE and PolyA38 tail elements in these construct designs. After selecting a top performing Agrp-targeting SadCas9 gRNA in Neuro-2A cells (FIGS. 35g-h), the inventors made recombinant AAVs (using serotype AAV8) and delivered these AIO AAVs to primary murine neurons. In both cases, the inventors observed significant (P value <0.05) transcriptional upregulation of Agrp, with the EFS promoter harboring vector displaying superiority to the SCP promoter harboring vector (FIG. 6h). These data demonstrate that the compact components of the CRISPR-DREAM retain high transactivation potency when delivered using either dual or AIO AAV modalities.

Here, the inventors harnessed the programmability and versatility of different dCas9-based recruitment architectures (direct fusion, gRNA-aptamer, and SunTag-based) to optimize the transcriptional output of TADs derived from natural human TFs. The inventors leveraged these insights to build superior and widely applicable transactivation modules that are portable across all modern synthetic DNA binding platforms, and that can activate the expression of diverse classes endogenous RNAs. The inventors selected mechanosensitive TFs (MTFs) for biomolecular building blocks because they naturally display rapid and potent gene activation at target loci, can interact with diverse transcriptional co-factors across different human cell types, and because their corresponding TADs are relatively small72-74. The inventors not only identified and validated the transactivation potential of TADs sourced from individual MTFs, but the inventors also established the optimal TAD sequence compositions and combinations for use across different synthetic DNA binding platforms, including Type I, II and V CRISPR systems, TALE proteins, and ZF proteins.

Our study also revealed that for MTFs, tripartite fusions using TADs from MRTA-A (M), STAT1 (S), and NRF2 (N) in one of two different combinations (either MSN or NMS) consistently resulted in the most potent human gene activation across different DNA binding platforms. Interestingly, each of these components has been shown to interact with key transcriptional co-factors. For example, individual TADs from MRTF-A, STAT1, NRF2 can directly interact with endogenous p30029, 75. Moreover, the Neh4 and Neh5 TADs from NRF2 can also cooperatively recruit endogenous CBP for transcriptional activity27, 76. Therefore, the inventors suspect that the potency of the MSN and NMS tripartite effector proteins is likely related to their robust capacity to recruit the powerful and ubiquitous endogenous transcriptional modulators p300 and/or CBP, which is likely positively impacted by their direct tripartite fusion.

Additionally, this study demonstrated that the superior transactivation capabilities of the CRISPR/dCas9-recruited enhanced activation module (DREAM) system—consisting of dCas9 and a gRNA-aptamer recruited MCP-MSN fusion—are not reliant upon the direct fusion(s) of any other proteins (viral or otherwise) to dCas9, in contrast to the SAM system which relies upon dCas9-VP6415. The inventors used this advantage to combine the MCP-MSN module with HNH domain deleted dCas9 variants51, 52, which exhibited similar potencies to full-size dCas9 variants. To further reduce the size of CRISPR-DREAM, the inventors built a minimal transactivation module (eN3×9; 96aa) by evaluating the potency of a suite of 9aa TADs from MTFs and by next combining the most potent variants with the small eNRF2 TAD. The inventors then combined the minimized eN3×9 transactivation module with an HNH domain deleted dCas9 variant in two-vector (mini-DREAM) and single-vector (mini-DREAM compact) delivery architectures, which retained potent transactivation capabilities.

The inventors also integrated the MSN and NMS effectors with the Type I CRISPR/Cascade and Type II dCas12a platforms to enable superior multiplexed endogenous activation of human genes. This multiplexing capability holds tremendous promise for reshaping endogenous cellular pathways and/or engineering complex transcriptional networks. dCas9-based transcription factors harboring viral TADs have also been used for directed differentiation and cellular reprogramming9, 17, 77, 78. Here, the inventors showed that the inventors could reprogram human fibroblasts into iPSCs using dCas9 directly fused to the NMS transcriptional effector with similar gene expression profiles, times to conversion, and morphological characteristics compared to iPSCs derived using dCas9 fused to viral TADs17. However, dCas9-NMS resulted in slightly fewer iPSC colonies than dCas9-VP192, which the inventors attribute to the reprogramming framework tested here being optimized for use with dCas9-VP192.

The inventors also demonstrated that the MSN and NMS effectors were compatible with dual and all-in-one (AIO) AAV vectors. Additionally, the AIO AAV vector design, which combines the short SCP1 promoter, the short M11 gRNA promoter and the compact CW3SA modified WPRE/poly A tail elements, holds tremendous potential for future delivery architectures. Similarly, the potency of AIO AAV vectors encoding NMS-SadCas9 empower researchers with a new streamlined modality to induce endogenous gene expression in vivo that could be used within animal models or clinical settings. Finally, the inventors found that the NMS, MSN, and eN3×9 TADs were well-expressed and potent in therapeutically important human cells. Although the tripartite VPR TAD contains the potent VP64 and RTA viral elements, in the inventors' primary cell experiments VPR showed the lowest expression levels and gene activation potencies. In contrast, the hypercompact eN3×9 TAD was well expressed in both MSCs and T cells. In MSCs eN3×9 was also extremely potent, however in T cells, gene activation efficacy was modest for all activators tested. Nevertheless, MSN, NMS, and eN3×9 TADs were substantially less toxic compared to the VPR TAD in T cells. Further analyses at other target sites and over longer time courses will likely be useful for optimized therapeutic use cases.

In summary, the inventors have used the rational redesign of natural human TADs to build synthetic transactivation modules that enable consistent and potent performance across programmable DNA binding platforms, mammalian cell types, and genomic regulatory loci embedded within human chromatin. Although the inventors used MTFs as sources of TADs here, the inventors' work establishes a framework that could be used with practically any natural or engineered TF and/or chromatin modifier in future efforts. The potency, small size, versatility, capacity for multiplexing, and the lack of viral components associated with the newly engineered MSN, NMS, and eN3×9 TADs and CRISPR-DREAM systems developed here could be valuable tools for fundamental and biomedical applications requiring potent and predictable activation of endogenous eukaryotic transcription.