System for cell programming and method thereof

Abstract

Provided herein are systems that modulate gene expression, methods of use thereof, and engineered cells thereof for purposes of differentiating cells (e.g., immune cells).

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 311,122, filed on February 17, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Heterologous proteins and / or nucleic acid molecules can be used to cause a desired response in a cell. Heterologous proteins and / or nucleic acid molecules can regulate genes of interest (e.g., transgenic and / or endogenous genes) to program cells (e.g., differentiation, dedifferentiation). In some cases, endonuclease-based techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins or "CRISPR / Cas") have been used to manipulate polynucleotide sequences, their epigenetic modifications and / or their expression levels. For example, CRISPR / Cas technology can be characterized by its versatility and easy-to-program properties, and can be used to facilitate genome editing across different species. Summary of the invention

[0004] The present disclosure provides a method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous gene circuit comprising a plurality of gating units, wherein upon activation of the heterologous gene circuit, the plurality of gating units operate in concert to induce a plurality of different regulation of the target gene in a sequential manner, each of the plurality of different regulation being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprise: (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gating unit that is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the first different regulation and the second different regulation both enhance or both reduce the expression and / or activity level of the target gene in the cell, wherein upon contact, the plurality of gating units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell.

[0005] The present disclosure also provides a system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to induce a plurality of different regulation of the target gene in a sequential manner, each of the plurality of different regulation being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gate units comprise: (i) a first gate unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gate unit that is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that both the first different regulation and the second different regulation enhance or both enhance the expression and / or activity level of the target gene in the cell, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell.

[0006] The present disclosure also provides a method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to induce a plurality of different regulation of the target gene in a sequential manner, each of the plurality of different regulation being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gate units comprise: (i) a first gate unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation of the plurality of different regulation; and (ii) a second gate unit that is activatable to induce disruption of the already activated first gate unit, wherein the inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation such that the second different regulation attenuates the first different regulation, wherein, upon contact, the plurality of gate units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell.

[0007] The present disclosure also provides a system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to induce a plurality of different regulation of the target gene in a sequential manner, each of the plurality of different regulation being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gate units comprise: (i) a first gate unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation of the plurality of different regulation; and (ii) a second gate unit that is activatable to induce disruption of the already activated first gate unit, wherein the inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation such that the second different regulation attenuates the first different regulation, wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell.

[0008] Incorporation by reference

[0009] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as "Figures" and "FIG.") which set forth illustrative embodiments in which the principles of the present invention are utilized, in which:

[0011] Figure 1 The autologous T cell development pathway in the circuit model is shown.

[0012] Figure 2 Genes known to influence T cell differentiation at different steps during T cell development are shown.

[0013] Figure 3 Depicted is how the heterologous genetic circuit (HGC) acts on further T cell development at different stages compared to the theoretical functions of the autologous T cell development pathway.

[0014] Figure 4 Guide nucleic acid molecules (e.g., gRNAs) in accordance with the systems and methods of the present disclosure are described.

[0015] Figure 5A-Figure 5I An example of a heterologous genetic circuit (HGC) in accordance with the systems and methods of the present disclosure is illustrated. Figure 5A T cell HGC1 is depicted. Figure 5B T cell HGC2 is depicted. Figure 5C T cell HGC3 is depicted. Figure 5D T cell HGC4 is depicted. Figure 5E T cell HGC5 is depicted. Fig. 5F T cell HGC6 is depicted. Figure 5G T cell HGC7 is depicted. Figure 5H T cell HGC8 is depicted. Fig.5I T cell HGC9 is depicted.

[0016] Figure 6 Depicted are the intermediate T cell stages induced by HGCs overlaid on the conventional T cell development pathway used as a reference.

[0017] Figure 7 Schematic diagram of a heterologous gene circuit. The activation part starts the circuit and can activate the gate unit. The gate unit consists of the gate part and / or the gene regulation part.

[0018] Figure 8 An exemplary heterologous gene circuit is depicted.

[0019] Fig. 9 Scatter plots (ie, volcano plots) at days 3, 5, and 9 are shown to identify one or more heterologous gene circuits that differentiate induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells.

[0020] Figures 10A-10D Shown is the method used to generate Fig. 9 Example of a scatter plot of hematopoietic progenitor cell marker analysis data. Fig. 10A The cell marker CD34 is shown + result. Fig. 10B The cell marker CD43 is shown + result. Fig. 10C The cell marker CD45 is shown + result. Fig. 10D The cell marker CD34 is shown + / CD43- / CD45 + Combined results. Left and right points represent replicates for each condition. Center point represents mean.

[0021] Figures 11A-11D It was shown that after five days in culture, the heterologous gene circuit produced significantly more hematopoietic progenitor cells compared to directed differentiation. Fig.11A CD34 + and CD45 +Cell marker results for markers. Fig. 11B CD34 + 、CD43 + and CD45 + Additional cell marker results for markers. Fig. 11C Shows Fig. 11B Data summary. Fig.11D A summary of results from replicate experiments is shown. DETAILED DESCRIPTION

[0022] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may conceive of many variations, changes and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0023] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a door unit" includes a plurality of door units.

[0024] The term "about" or "approximately" generally means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" may mean within 1 standard deviation or greater than 1 standard deviation. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of a value, preferably within 5 times, and more preferably within 2 times. Where a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for that particular value.

[0025] The use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives. The term "and / or" should be understood to mean either or both of the alternatives.

[0026] definition

[0027] As used interchangeably herein, the terms "gene circuit," "biological circuit," or "circuit" generally refer to a collection of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) that are operably coupled (e.g., operated simultaneously, sequentially, etc.) according to a circuit design. The collection of molecular components can be capable of providing one or more specific outputs in a cell (e.g., regulation of one or more genes) in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs can be sufficient to trigger the molecular components of the gene circuit to provide one or more specific outputs. For example, a gene circuit can include one or more molecular switches (e.g., a switch) that can be activated by one or more inputs. Figure 7 ).

[0028] A gene circuit can be a controllable gene expression system comprising an assembly of biological parts that work together as a logical function (e.g., simultaneously, sequentially, etc.). A gene circuit can comprise a plurality of gate units, wherein at least one gate unit of the plurality of gate units can be activated by an activation part (e.g., a heterologous input to the cell) to activate other gate units of the plurality of gate units (e.g., simultaneously at once, sequentially in a cascaded manner, etc.) ( Figure 7 ). For example, at least one gate unit in the plurality of gate units can be activated by another gate unit in the plurality of gate units (e.g., directly or indirectly) to (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other gate unit in the plurality of gate units, and / or (ii) deactivate at least one other gate unit in the plurality of gate units, thereby collectively regulating the expression and / or activity level of one or more target genes in a desired manner, as predetermined by the design of the gene circuit ( Figure 7 As used herein, the terms "heterologous gene circuit", "HGC", "cellular algorithm", or "cell algorithm" may be used interchangeably.

[0029] As referred to herein, the term "gate unit" generally refers to a portion of a gene circuit that can control gene regulation by acting similar to a logic gate, where it can control information flow and allow the circuit to make multiple decisions at different points. More specifically, the term refers to a nucleic acid that encodes a genetic switch and a transcription / translation regulatory region or a series of regions on which a genetic switch acts. The input of a gate unit can be an activation portion and / or another gate unit. The output of a gate unit can be used to activate another gate unit, deactivate another gate unit, affect a target gene, and / or any combination of the above. For example, a gate unit can be composed of multiple gate portions and / or multiple gene regulatory portions ( Figure 7 )composition.

[0030] As mentioned herein, the term "activation portion" generally refers to a portion that can activate multiple gene circuits and / or multiple gate units. The activation portion can be a heterologous input to a cell. In some cases, the activation portion can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acids, polypeptides, polynucleotides, small molecules, light, or a combination thereof. For example, the activation portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., Cas protein) to bind to a polynucleotide sequence of an inactivated gate portion (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such a gate portion that can target one or more gene regulatory portions (e.g., inducing expression of another guide nucleic acid molecule functional form).

[0031] As mentioned herein, the term "gate portion" generally refers to a portion that can affect the function of the gene regulatory portion within the gate unit. The gate portion can activate and / or deactivate the gene regulatory portion. For example, the gate portion can regulate the expression of the gene regulatory portion by editing the nucleic acid sequence and thereby activating or deactivating the gene regulatory portion. For example, the gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of a gene regulatory portion (e.g., a plasmid encoding another guide nucleic acid molecule) to activate a gene regulatory portion that can target one or more endogenous genes of a cell (e.g., inducing the expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, the gate portion can activate and / or deactivate another gate unit ( Figure 7 ). For example, a gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of another gate portion that is inactivated (e.g., a plasmid encoding another guide nucleic acid molecule) to activate the other gate portion (e.g., induce expression of a functional form of another guide nucleic acid molecule). In another example, a gate portion can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of another gate portion that is activated (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate the other gate portion (e.g., reduce expression of a functional form of another guide nucleic acid molecule).

[0032] As used interchangeably herein, the terms "gene regulatory moiety" or "gene editing moiety" generally refer to a moiety that can regulate the expression and / or activity profile of a nucleic acid sequence or protein (whether exogenous or endogenous to a cell). Figure 7). For example, the gene editing portion can regulate the expression of a gene by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nucleases, TALEN, or siRNA). In some cases, the gene editing portion can regulate the expression of a gene by editing a genomic DNA sequence. In some cases, the gene editing portion can regulate the expression of a gene by editing an mRNA template. In some cases, editing a nucleic acid sequence can change the basic template for gene expression (e.g., an RNA targeting system inspired by CRISPR-Cas). Alternatively, the gene editing portion can inhibit the translation of a gene (e.g., Cas13).

[0033] Alternatively or additionally, the gene editing part may be able to regulate the expression or activity of the gene by specifically binding to a target sequence (or a target sequence within a gene) that is operably coupled to the gene, and regulate the production of mRNA from DNA (such as chromosomal DNA or cDNA). For example, the gene editing part may recruit or include at least one transcription factor that is bound to a specific DNA sequence, thereby controlling the transcription rate of genetic information from DNA to mRNA. The gene editing part itself can be bound to DNA and regulated transcription by physical obstruction, for example, preventing proteins (such as RNA polymerase and other associated proteins) from assembling on a DNA template. The gene editing part can regulate the expression of genes at the translation level, for example, by regulating the production of proteins from mRNA templates. In some cases, the gene editing part can regulate gene expression by affecting the stability of mRNA transcripts. In some cases, the gene editing part can regulate genes (such as Cas12) by epigenetic editing.

[0034] In some cases, the plasmid may encode a non-functional form of a gene editing portion. The plasmid may be activated to express a functional form of a gene editing portion, for example, via activation of a functional gate portion. For example, a gene editing portion may encode a non-functional form of a guide nucleic acid molecule that would otherwise be able to bind to a target gene of a cell. When a functional gate portion (e.g., another guide nucleic acid molecule complexed with a Cas protein) is bound to a plasmid, the plasmid may be edited to allow expression of a functional form of a gene editing portion (e.g., a functional form of a guide nucleic acid molecule that specifically binds to a target gene of a cell) to permit regulation of a target gene in a cell.

[0035] Therefore, the gene regulation part can include a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease such as a Cas protein). Alternatively or additionally, the gene regulation part can include an endonuclease or be operably coupled to an endonuclease. An endonuclease can be an enzyme that cuts a phosphodiester bond within a polynucleotide chain. An endonuclease can be included in a restriction endonuclease that cuts DNA at a specific site without damaging the base. Restriction endonucleases can include endonucleases type I, type II, type III, and type IV, which can further include subtypes. In some cases, the endonuclease can be Cas1, Cas2, Cas 3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12, Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (Cas14 or C2c10), Cas12g, Cas12h, Cas12i, Cas12k (C2c5), Cas 13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, Csn2. The endonuclease may be a dead endonuclease exhibiting reduced cleavage activity. For example, the endonuclease may be a nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0036] The gene regulation part can be a transcriptional regulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulation part can be a gene repressor complex comprising a dCas protein operably coupled to (e.g., fused to) a transcriptional repressor. Non-limiting examples of transcriptional repressors can include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX inhibitory domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2 D, JARID1A, RBP2, JARID1B / PLU-1, JARIDIC / SMCX, JARIDID / SMCY, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, M.Hhal, METI, DRM3, ZMET2, CMT1, CMT2, lamin A, and lamin B. Alternatively, the gene regulatory portion can be a gene activator complex comprising a dCas protein operably coupled to (e.g., fused to) a transcriptional activator. Non-limiting examples of transcriptional activators can include VP16, VP64, VP48, VP160, p65 subdomain, SET1A, SET1B, MLL1, MLL2, MLL3, MLL4, MLL5, ASH1, SYMD2, NSD1, JHDM2a, JHDM2b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, TET1 CD, TET1, DME, DML1, DML2, and ROS1.

[0037] In some cases, the gene regulatory portion has an enzymatic activity that modifies the target gene without cutting the target gene. The modification of the target gene can cause, for example, epigenetic modifications that can modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory portion can include, but are not limited to: nuclease activity (such as the nuclease activity provided by a restriction enzyme (e.g., Fok1 nuclease)); methyltransferase activity (such as the activity provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2) methyltransferase activity); demethylase activity (such as provided by a demethylase (e.g., ten-eleven translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1) demethylase activity), DNA repair activity, DNA damage activity, deamination activity (such as provided by a deaminase (e.g., cytosine The invention also includes but is not limited to deaminating activity provided by an indole deaminase such as APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity (such as integrase activity provided by an integrase and / or a resolvase (e.g., Gin convertase, such as a hyperactive mutant of Gin convertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.), transposase activity, recombinase activity (such as recombinase activity provided by a recombinase (e.g., the catalytic domain of Gin recombinase)), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0038] Unless otherwise specified or apparent from the context, as used interchangeably herein, the terms "polynucleotide," "oligonucleotide," or "nucleic acid" generally refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof, whether single-stranded, double-stranded, or multi-stranded. Polynucleotides may be exogenous or endogenous to a cell. Polynucleotides may be present in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may include one or more analogs (e.g., altered backbones, sugars, or nucleobases). In the presence of modifications, the nucleotide structure may be modified before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xenologous nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided nucleotides, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci defined by linkage analysis (locus), exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides (including cell-free DNA (cfDNA) and cell-free RNA (cfRNA)), nucleic acid probes and primers. The sequence of nucleotides may be interrupted by non-nucleotide components.

[0039] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) and their corresponding nucleotide sequences related to coding RNA transcripts. As used herein, the term includes intervening non-coding regions and regulatory regions for genomic DNA and may include 5' and 3' ends. In some uses, the term covers transcribed sequences, including 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons and introns. In some genes, the transcribed region will contain an "open reading frame" encoding a polypeptide. In some uses of the term, a "gene" only includes coding sequences (e.g., "open reading frames" or "coding regions") necessary for encoding polypeptides. In some cases, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. In some cases, the term "gene" includes not only transcribed sequences, but also non-transcribed regions, including upstream and downstream regulatory regions, enhancers and promoters. A gene may refer to an "endogenous gene" or a natural gene in its natural position in the genome of an organism. A gene may refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not usually found in host organisms but are introduced into host organisms by gene transfer. Non-natural genes can also refer to genes that are not in their natural location in the genome of an organism. Non-natural genes can also refer to naturally occurring nucleic acids or polypeptide sequences (e.g., non-natural sequences) that contain mutations, insertions, and / or deletions.

[0040] The term "expression" generally refers to one or more processes of polynucleotide transcription (such as transcription into mRNA or other RNA transcripts) from a DNA template and / or the process of transcribed mRNA being subsequently translated into peptides, polypeptides or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products". If a polynucleotide is derived from genomic DNA, expression in a eukaryotic cell can include the splicing of mRNA. With respect to expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or a polypeptide sequence relative to its expression level in a wild-type state, while "downregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or a polypeptide sequence relative to its expression in a wild-type state. The expression of a transfected gene can occur transiently or stably in a cell. During "transient expression", the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to transfected cells, the expression of the gene disappears over time. On the contrary, when a gene is co-transfected with another gene that gives a selective advantage to the transfected cell, the stable expression of the transfected gene can occur. Such a selective advantage may be resistance to a certain toxin presented to the cell.

[0041] As used interchangeably herein, the term "peptide", "polypeptide" or "protein" generally refers to a polymer of at least two amino acid residues connected by a peptide bond. This term does not mean a specific length of a polymer, nor is it intended to imply or distinguish whether a peptide is produced using recombinant technology, chemical or enzymatic synthesis or is naturally present. The term is applicable to naturally occurring amino acid polymers and amino acid polymers comprising at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structures (e.g., domains). The term also encompasses modified amino acid polymers, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other operation such as conjugation with labeled components. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including but not limited to modified amino acids and amino acid analogs. Modified amino acids may include natural amino acids and non-natural amino acids, which have been chemically modified to include groups or chemical moieties that are not naturally present on amino acids. Amino acid analogs may refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0042] As used interchangeably herein, with respect to polypeptides, the terms "derivative", "variant" or "fragment" generally refer to polypeptides that are related to wild-type polypeptides, for example, by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Derivatives, variants and fragments of polypeptides may comprise one or more amino acid variations (e.g., mutations, insertions and deletions), truncations, modifications or combinations thereof compared to wild-type polypeptides.

[0043] As used herein, with respect to polypeptide molecules (e.g., proteins), the terms "engineered", "chimeric" or "recombinant" generally refer to polypeptide molecules having heterologous amino acid sequences or altered amino acid sequences due to the application of genetic engineering techniques to nucleic acids encoding polypeptide molecules and cells or organisms expressing polypeptide molecules. As used herein, with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms "engineered" or "recombinant" generally refer to polynucleotide molecules having heterologous nucleic acid sequences or altered nucleic acid sequences due to the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning techniques; transfection, transformation and other gene transfer techniques; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, engineered or recombinant polynucleotides (e.g., genomic DNA sequences) can be modified or altered by gene editing portions.

[0044] Unless otherwise specified or apparent from the context, as used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotide may include synthetic nucleotides. Nucleotide may include synthetic nucleotide analogs. Nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof. Such derivatives may include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that impart nuclease resistance to nucleic acid molecules containing them. The term nucleotide as used herein may refer to dideoxyribonucleoside triphosphates (ddNTP) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates may include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may be unlabeled or detectably labeled by known techniques. Labeling may also be performed with quantum dots. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides may include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, which can be obtained from Perkin Elmer, Foster City, Calif.FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and fluorescein-15-2′-dATP available from Molecular Probes, Eugene, Oreg. Chromosome-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, Tetramethylrhodamine-6-UTP, Tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP and Texas Red-12-dUTP. Nucleotides can also be labeled or indicated by chemical modification. The chemically modified mononucleotide can be a biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0045] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional and / or biological unit of a living organism. A cell can be derived from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugar cane, pumpkin, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycopodiella, hornwort, liverwort, mosses), algae cells, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum splaying Patens, C. Agardh, etc.), algae (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), etc. Sometimes, cells are not derived from natural organisms (e.g., cells can be synthetically manufactured, sometimes referred to as artificial cells).

[0046] As used interchangeably herein, the terms "reprogramming", "dedifferentiation", "increasing cell potential" or "increasing developmental potential" generally refer to methods that increase the potential of a cell or dedifferentiate a cell to a lower differentiation state. For example, compared with the same cells in a non-reprogrammed state, cells with increased cell potential have more developmental plasticity (i.e., can be differentiated into more cell types). In other words, reprogrammed cells are lower than the same cell differentiation state in a non-reprogrammed state.

[0047] The term "differentiation" generally refers to the process by which non-specialized ("uncommitted") or less specialized cells acquire the characteristics of specialized cells (such as, for example, immune cells). Differentiated or differentiation-induced cells are cells that have acquired a more specialized ("committed") position within a cell lineage. The term "committed" generally refers to cells that have proceeded to a point in a differentiation pathway that, under normal circumstances, will continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, cannot differentiate into a different cell type or revert to a less differentiated cell type.

[0048] The term "pluripotent" generally refers to the ability of cells to form all pedigrees (i.e., embryoid bodies) of a body or somatic cells. For example, embryonic stem cells are pluripotent stem cell types that can form cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).Pluripotency can be a continuum of developmental potential ranging from incomplete or partially pluripotent cells (e.g., ectoderm stem cells) (which can not produce a complete organism) to more primitive, more potent cells (which can produce a complete organism) (e.g., embryonic stem cells).

[0049] The term "induced pluripotent stem cell" (iPSC) generally refers to a stem cell derived from a differentiated cell (e.g., a differentiated adult, neonatal or fetal cell) that has been induced or altered (i.e., reprogrammed) to be able to differentiate into all three germ layers or dermis: cells of mesoderm, endoderm and ectoderm tissues. The iPSC produced does not refer to cells found in nature. In some cases, iPSC can be engineered to directly differentiate into committed cells (e.g., natural killer (NK) cells. In some cases, iPSC can be engineered to first differentiate into tissue-specific stem cells (e.g., hematopoietic stem cells (HSC) or hematopoietic progenitor cells), which can be further induced to differentiate into committed cells (e.g., NK cells).

[0050] The term "embryonic stem cell" (ESC) generally refers to the cell of the naturally occurring pluripotent stem cell derived from the inner cell mass of an embryonic blastocyst. Embryonic stem cells are pluripotent and produce three primary germ layers during development: all derivatives of the ectoderm, endoderm, and mesoderm. In some cases, ESC can be engineered to directly differentiate into committed cells (e.g., NK cells). In some cases, ESC can be engineered to first differentiate into tissue-specific stem cells (e.g., HSC), which can be further induced to differentiate into committed cells (e.g., NK cells).

[0051] The term "isolated stem cell" generally refers to any type of stem cell disclosed herein (e.g., ESC, HSC, mesenchymal stem cell (MSC), etc.) isolated from a multicellular organism. For example, HSC can be isolated from the body of a mammal, such as a human body. In another example, embryonic stem cells can be isolated from an embryo.

[0052] The term "separated" generally refers to a cell or cell colony separated from its original environment. For example, the new environment of the separated cell is substantially free of at least one component when found in the environment of an "unseparated" reference cell. The separated cell can be a cell removed from some or all components when found in its natural environment, such as a cell separated from a tissue or biopsy sample. The term also includes cells removed from at least one, some or all components when finding a cell in a non-naturally occurring environment, such as, cells separated from cell culture or cell suspensions. Therefore, the separated cell is partially or completely separated from at least one component, including other substances, cells or cell colonies when it is found in nature or when it grows, stores or stores in a non-naturally occurring environment.

[0053] As used interchangeably herein, the terms "hematopoietic stem and progenitor cells", "hematopoietic stem cells", "hematopoietic progenitor cells" or "hematopoietic precursor cells" generally refer to cells that are stereotyped as hematopoietic lineages but can further hematopoietic differentiation (e.g., differentiate into NK cells), and include multipotent hematopoietic stem cells (hematoblasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells and lymphoid progenitor cells. Hematopoietic stem and progenitor cells (HSC) are multipotent stem cells that produce all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK cells). In some cases, HSC can be a CD34+ hematopoietic cell that can produce mature myeloid cells and lymphoid cell types (including T cells, NK cells and B cells).

[0054] The term "immune cell" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells may include NK cells, T cells, monocytes, innate lymphocytes, tumor infiltrating lymphocytes, macrophages, granulocytes, and the like.

[0055] Overview

[0056] Biological programming (such as cell programming) allows cells to be engineered to produce desired results. The results of cell programming can include inducing or preventing a wide range of common and / or new cell functions; the results can also include enhancing or inhibiting cell functions that have already occurred. Cell programming can be accomplished by using gene circuits. Cell programming can be accomplished by manipulating biomolecules (e.g., DNA). For example, due to the versatility and programmability of CRISPR or CRISPR / Cas systems, CRISPR or CRISPR / Cas systems have been used for genome editing across many species. Cell programming can affect endogenous or exogenous genes. Cell programming can be implemented to act in a time-dependent manner or a time-independent manner.

[0057] Gene circuits used in cellular programming can be used to control cascades of multiple desired expression and / or activity profiles of multiple genes in a cell. To allow for greater control of specific cellular outcomes, gene circuits can be multiplexed to create positive and / or negative feedback systems.

[0058] Although CRISPR / Cas system is widely used in gene editing, Cas is basically a single-turnover nuclease because it keeps binding to the double-stranded breaks it produces, and many regions of the genome are resistant to genome editing. The increased understanding of genome editing based on CRISPR / Cas has encouraged the development of cascade control systems to further utilize this technology for engineered cell development. By implementing a series of activatable gRNAs, genome editing can be more regulated from target site to target site in a temporal manner, sequential genome editing can be performed to work like a domino effect, and cells can be barcoded. However, this simple barcoding, which is usually done using exogenous fluorophores, does not allow multiple regulation of endogenous genes to achieve cell differentiation.

[0059] Therefore, there is still an unmet need for activatable multiplexed CRISPR / Cas systems and their use for editing target polynucleotides (e.g., the genome of a cell, particularly a eukaryotic cell), which use a cascade of gRNAs to form a gene circuit including a feedback loop to independently affect gene regulation and thereby cell fate determination. In view of the improved multiplexing capabilities through the use of internal positive and / or negative feedback loops, preprogrammed, activatable, and self-regulating gRNA cascade CRISPR / Cas systems are, for example, used in gene therapy, gene circuits, and / or complex cell fate determination and / or control.

[0060] The present disclosure provides systems and methods for engineering CRISPR / Cas9 systems, which include Cas endonucleases and homologous single guide RNA (sgRNA or gRNA) arrays, which have inactivation sequences in non-essential regions and are activatable to allow the system to be regulated and modified by using positive and negative feedback loops. The present disclosure also provides engineered cells that may contain any of the above systems or be capable of performing any of the above methods.

[0061] Systems and methods for engineering genetic circuits, cells containing the same, and methods of use thereof

[0062] Various aspects of the present disclosure provide systems for inducing desired expression and / or activity levels (or profiles thereof) of one or more target genes in cells. Various aspects of the present disclosure provide methods for inducing desired expression and / or activity levels (or profiles thereof) of one or more target genes in cells.

[0063] On the one hand, the present disclosure provides a system for inducing the desired expression and / or activity spectrum of a target gene in a cell. The system may include a heterologous gene circuit, which includes multiple gate units. Multiple gate units may include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more gate units. Multiple gate units may include at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2 or at most about 1 gate unit. The multiple gate units can be different (eg, comprise different polynucleotide sequences).

[0064] Heterogeneous gene circuits as disclosed herein can be operated with multiple gate units in series (e.g., multiple gate units are sequentially connected in an end-to-end manner to form a single path), multiple gate units in parallel (e.g., multiple gate units are cross-connected to each other to form, for example, two or more parallel paths), or a combination thereof.

[0065] Multiple gate units as disclosed herein can be operated in concert (e.g., as predetermined by the design of heterologous gene circuits) to induce results in cells. Results in cells can include cell functions (e.g., movement, reproduction; response to external stimuli, nutrient output, excretion, respiration, growth) and / or cell states (e.g., cell fate, differentiation, quiescence, programmed cell death). Such results can be determined in vitro, in vitro and / or in vivo. For example, results as disclosed herein can be determined in vitro by the following items: (i) measuring the expression level of a gene of interest by polymerase chain reaction (PCR) or Western blotting, (ii) via small molecules or antibody staining, (iii) cell sorting based on cell size, morphology and / or surface protein expression, (iv) using assays (e.g., cell proliferation assays, metabolic activity assays, cell killing assays) to measure phenotypic differentiation and cell function, (v) microscopy, and / or (iv) using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics and / or transcriptomics to screen for molecules and / or genetic differences.

[0066] The result in the cell can include the regulation of the target gene.The regulation of the target gene can include multiple different regulation of the target gene.Multiple gate units can each induce one of the multiple different regulation of the target gene, so that the collection of different regulation synergistically produces the final expression and / or activity spectrum of the target gene.At least two different regulation in multiple different regulation can all increase the expression and / or activity level of the target gene.At least two different regulation in multiple different regulation can all reduce the expression and / or activity level of the target gene.Alternately, the first different regulation in multiple different regulation can increase the expression and / or activity level of the target gene, and the second different regulation in multiple different regulation can reduce the expression and / or activity level of the target gene.In this case, the first different regulation can occur before the second different regulation or vice versa.Alternately, the different regulation (for example, the first and / or second regulation) of multiple different regulation can maintain the expression and / or activity level of the target gene at the expression and / or activity level before regulation.

[0067] In some cases, as disclosed herein, each of the multiple different regulation of the target gene may be necessary, but individually insufficient to achieve the desired expression and / or activity profile of the target gene. Thus, in the absence of any of the multiple different regulation of the target gene, the result (e.g., enhanced cellular function, induced cell state, etc.) in the cell induced by the multiple different regulation of the target gene may be impossible. Alternatively, the degree or measure of the result in the cell induced by the multiple different regulation of the target gene may be greater than the degree or measure of the result in the control cell induced by none, one or more but not all of the multiple different regulation of the target gene, and / or all of the multiple different regulation of the target gene occurring through a different sequential order of events.

[0068] The second door unit can be activated by the first door unit (e.g., directly or indirectly). For example, the second door unit can be directly activated by the first door unit. Alternatively, the second door unit can be activated by one or more additional door units that are activated by the first door unit (e.g., directly or indirectly). The one or more additional door units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more door units. One or more additional door units are at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 door unit. In yet another alternative, the second door unit can be activated via another part responsible for activating the first door unit (e.g., an activation part, a different door unit, etc.).

[0069] The second gate unit can be activatable to induce inactivation of the first gate unit that has been activated. The terms "inactivation" or "disruption" can be used interchangeably herein. Inactivation and as disclosed herein can be induced by generating a modification (e.g., a cleavage, such as a single-strand or double-strand break, and insertion-deletion (indel), etc.) to at least a portion of the first gate unit (e.g., the gate portion and / or the gene regulatory portion of the first gate unit), which is responsible for inducing the first different regulation of the target gene.

[0070] As disclosed herein, the inactivation of the gate portion and / or gene regulation portion by the first gate unit can be achieved by a system based on nuclease (e.g., CRISPR / Cas system).Alternately or additionally, inactivation can be achieved by using a transcription regulator system (e.g., transcription inhibitor).Endonuclease transcription regulator system (e.g., Cas inhibitor) can be used to achieve polynucleotide cutting (e.g., for inactivating the gate portion and / or gene regulation portion).Polynucleotide cutting can create nucleic acid modifications, such as single-strand breaks, double-strand breaks, insertions, deletions, or insertion-deletions (indel).Alternately or additionally, endonuclease transcription regulator system (e.g., Cas inhibitor) can be used to regulate target gene expression.

[0071] Alternatively, the second gating unit can be activatable to amplify or enhance the activation of an already activated first gating unit. The amplification or enhancement of the first gating unit can be induced by generating a modification (e.g., a cleavage, such as a single-strand or double-strand break, and an insertion-deletion, etc.) to at least a portion of the first gating unit (e.g., the gating portion and / or the gene regulatory portion of the first gating unit) that is responsible for inducing the first different regulation of the target gene.

[0072] In some cases, the guide nucleic acid molecule (gNA) (e.g., functional gNA) expressed by the second gate unit upon activation can create a modification to at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can modify the polynucleotide sequence of the first gate unit encoding the gNA (e.g., activatable gNA) or the promoter sequence of the first gate unit that is operably coupled to such gNA of the first gate unit. Such modifications can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to the target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0073] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a single-strand break in which there is a discontinuity in one nucleotide chain. Inactivation of a polynucleotide sequence or a target gene can be caused by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more single-strand breaks. In some cases, inactivation of a gene can be caused by at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 single-strand break.

[0074] In some cases, the gNA is at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 21 nucleotides, at least about 22 nucleotides, at least about 23 nucleotides, at least about 24 nucleotides, at least about 25 nucleotides, at least about 26 nucleotides, at least about 27 nucleotides, at least about 28 nucleotides, at least about 29 nucleotides, at least about 30 nucleotides, or longer in length.

[0075] In some cases, the systems and methods of the present disclosure can utilize at least two different endonucleases. The first endonuclease (e.g., the Cas protein not coupled to a transcriptional regulator) can be used in conjunction with a guide nucleic acid to induce the cutting of a target polynucleotide sequence of a gate portion or a gene regulation portion plasmid, thereby activating and / or deactivating the gate portion or the gene regulation portion, respectively. In addition, the second endonuclease (e.g., the Cas protein coupled to a transcriptional regulator) can be used in conjunction with another guide nucleic acid to be bound to a target gene (e.g., a target endogenous gene) of a cell, to regulate the expression and / or activity of the target gene, without producing cutting in the target gene.

[0076] In some cases, the disclosed systems and methods can utilize a single endonuclease system (e.g., Cas inhibitor) to achieve both (i) polynucleotide cutting (e.g., for activating / inactivating gate parts and / or gene regulation parts) and (ii) regulation of target gene expression. When using a single endonuclease transcription regulator system, a unique guide nucleic acid molecule (gNA) with different lengths of spacer sequences can be used to determine whether the single endonuclease transcription regulator system can (i) hybridize with a polynucleotide sequence to induce the Cas-mediated nuclease activity of the polynucleotide sequence, or (ii) hybridize with a target gene (e.g., genomic DNA) to regulate the expression and / or activity level of the target gene via the action of a transcription activator without mediating Cas nuclease activity, as desired by a single heterologous gene loop. For example, using gNAs with different lengths of spacer sequences that are bound to different targets can allow the second gate unit as provided herein to induce the inactivation of the first gate unit that has been activated and / or induce different regulation of the second target gene.

[0077] As described above, the length of the spacer sequence of gNA can affect the ability of gNA to mediate Cas nuclease activity. In some cases, gNA with spacer sequences of different lengths can be used in the same heterologous gene circuit to affect different types of cutting, activation, inactivation and / or regulation of one or more target nucleic acids. In some cases, the gNA spacer sequence shorter than the threshold length (for example, about 16 nucleotides) can hinder the nuclease activity of Cas transcription regulators, while still mediating DNA binding for the transcriptional regulation of target genes. In some cases, the gNA spacer sequence shorter than at least about 25 nucleotides, at least about 20 nucleotides, at least about 19 nucleotides, at least about 18 nucleotides, at least about 17 nucleotides, at least about 16 nucleotides, at least about 15 nucleotides, at least about 15 nucleotides, at least about 14 nucleotides, at least about 13 nucleotides, at least about 12 nucleotides, at least about 11 nucleotides or at least about 10 nucleotides can hinder the nuclease activity of Cas protein, while still mediating DNA binding.

[0078] For example, a gNA comprising a spacer sequence of 20 nucleotides (e.g., a gNA encoded by a gate portion of a plasmid for targeting a gene regulatory portion) may be sufficient to promote the nuclease activity of an endonuclease (e.g., Cas or Cas transcriptional regulator fusion protein). Alternatively or additionally, a gNA comprising a spacer sequence of 14 nucleotides (e.g., a gNA encoded by a gene regulatory portion) may hybridize with DNA, but may not be long enough to mediate nuclease activity—it may only promote endonuclease binding to a homologous DNA sequence. Therefore, shorter gNAs may selectively allow transcriptional regulation of a target gene, although an endonuclease transcriptional regulator system (e.g., a Cas activator system, a Cas inhibitor system) is used without cutting the target gene.

[0079] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by a double-strand break in which discontinuities are present in both nucleotide chains. In some cases, inactivation of a gene can be caused by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more double-strand breaks. In some cases, inactivation of a polynucleotide sequence or a target gene can be caused by at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 double-strand break.

[0080] In some cases, the modification of the polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be caused by insertion-deletion (also referred to as insertion-deletion mutation). Insertion-deletion mutations can include frameshift or non-frameshift mutations. Insertion-deletion mutations can include point mutations (also referred to as base substitutions), in which only one base or base pair is modified. The length of the insertion-deletion mutation can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000 or more bases or base pairs. The length of the insertion-deletion mutation can include up to about 2000, up to about 1000, up to about 900, up to about 800, up to about 700, up to about 600, up to about 500, up to about 400, up to about 300, up to about 200, up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 base or base pair.

[0081] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can be achieved without cutting the polynucleotide sequence or the target gene. For example, a gene regulatory portion (e.g., a nucleic acid molecule and / or an endonuclease, such as a complex comprising CRISPR / Cas proteins and guide nucleic acid molecules) can be specifically bound to a polynucleotide sequence or a target gene so that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory portion may include a transcriptional repressor or a transcriptional activator as provided herein.

[0082] In some cases, the modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate part) or a target gene can inactivate a gene. For example, the modification of a polynucleotide sequence or a target gene can terminate the expression and / or activity level of the polynucleotide sequence or the target gene. Alternatively, the modification of a polynucleotide sequence or a target gene can reduce the expression and / or activity level of the polynucleotide sequence of the target gene. In some cases, the modification of a polynucleotide sequence or a target gene can increase the expression and / or activity level of the polynucleotide sequence or the target gene. Alternatively, the modification of a polynucleotide sequence or a target gene can maintain the expression and / or activity level of the polynucleotide sequence or the target gene.

[0083] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can comprise reducing the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. Modification of a polynucleotide sequence or a target gene may comprise reducing the expression and / or activity level of the polynucleotide sequence or the target gene by up to about 500%, up to about 400%, up to about 300%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, respectively. , up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1% or less.

[0084] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can comprise increasing the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000%, or more. Modification of a polynucleotide sequence or a target gene may comprise increasing the expression and / or activity level of the polynucleotide sequence or target gene by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 3000%, or more. 0%, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1% or less.

[0085] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can comprise reducing the expression and / or activity level of the polynucleotide sequence or target gene by at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, at least or up to about 4 fold, at least or up to about 5 fold, at least or up to about 6 fold, at least or up to about 7 fold, at least or up to about 8 fold, at least or up to about 9 fold, at least or up to about 10 fold, at least or up to about 11 fold, at least or up to about 12 fold, at least or up to about 13 fold, at least or up to about 14 fold, at least or up to about 15 fold, at least or up to about 16 fold, at least or up to about 17 fold, at least or up to about 18 fold, at least or up to about 19 fold, at least or up to about 20 fold, at least or up to about 21 fold, at least or up to about 23 fold, at least or up to about 24 fold, at least or up to about 25 fold, at least or up to about 26 fold, at least or up to about 27 fold, at least or up to about 28 fold, at least or up to about 29 fold, at least or up to about 30 fold, at least times, at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times. Modification of a polynucleotide sequence or a target gene may comprise reducing the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by up to or less than about 10,000 times, up to or less than about 5,000 times, up to or less than about 1,000 times, up to or less than about 500 times, up to or less than about 100 times, up to or less than about 90 times, up to or less than about 80 times, up to or less than about 70 times, up to or less than about 60 times, up to or less than about 50 times, up to or less than about 40 times, up to or less than about 30 times, up to or less than about 45 times, up to or less than about 50 times, up to or less than about 60 times, up to or less than about 70 times, up to or less than about 80 times, up to or less than about 90 times, up to or less than about 100 times, up to or less than about 110 times, up to or less than about 120 times, up to or less than about 130 times, up to or less than about 140 times, up to or less than about 150 times, up to or less than about 160 times, up to or less than about 170 times, up to or less than about 180 times, up to or less than about 190 times, up to or less than about 200 times, up to or less than about 210 times, up to or less than about 220 times, up to or less than about 230 times, up to or less than about 240 times, up to or less than about 250 times, up to or less than about 260 times, up to or less than about 270 times, up to or less than about 280 times, up to or less than about 290 times, up to or less than about 300 times, up to or less than about 310 times, up to or less At most or less than about 20 times, at most or less than about 10 times, at most or less than about 9 times, at most or less than about 8 times, at most or less than about 7 times, at most or less than about 6 times, at most or less than about 5 times, at most or less than about 4 times, at most or less than about 3 times, at most or less than about 2 times, at most or less than about 1 times, at most or less than about 0.9 times, at most or less than about 0.8 times, at most or less than about 0.7 times, at most or less than about 0.6 times, at most or less than about 0.5 times, at most or less than about 0.4 times, at most or less than about 0.3 times, at most or less than about 0.2 times, at most or less than about 0.1 times.

[0086] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit, such as a gate portion) or a target gene can comprise increasing the expression and / or activity level of the polynucleotide sequence or target gene, respectively, by at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, as compared to a control expression and / or activity level. , at least or up to about 4 times, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times. Modification of a polynucleotide sequence or target gene may comprise increasing the expression and / or activity level of the target gene by at most or less than about 10,000-fold, at most or less than about 5,000-fold, at most or less than about 1,000-fold, at most or less than about 500-fold, at most or less than about 100-fold, at most or less than about 90-fold, at most or less than about 80-fold, at most or less than about 70-fold, at most or less than about 60-fold, at most or less than about 50-fold, at most or less than about 40-fold, at most or less than about 30-fold, at most or less than about 20-fold, at most or less than about 35-fold, at most or less than about 40-fold, at most or less than about 50-fold, at most or less than about 60-fold, at most or less than about 70-fold, at most or less than about 80-fold, at most or less than about 90-fold, at most or less than about 100-fold, at most or less than about 110-fold, at most or less than about 120-fold, at most or less than about 130-fold, at most or less than about 140-fold, at most or less than about 150-fold, at most or less than about 160-fold, at most or less than about 170-fold, at most or less than about 180-fold, at most or less than about 190-fold, at most or less than about 200-fold, at most or less than about 210-fold, at most or less than about 220-fold, at most or less than about 230-fold, at most or less than about 240-fold, at most or less than about 250-fold, at most or less than about 260-fold, at most or less than about 270-fold, At most or less than about 10 times, at most or less than about 9 times, at most or less than about 8 times, at most or less than about 7 times, at most or less than about 6 times, at most or less than about 5 times, at most or less than about 4 times, at most or less than about 3 times, at most or less than about 2 times, at most or less than about 1 times, at most or less than about 0.9 times, at most or less than about 0.8 times, at most or less than about 0.7 times, at most or less than about 0.6 times, at most or less than about 0.5 times, at most or less than about 0.4 times, at most or less than about 0.3 times, at most or less than about 0.2 times, at most or less than about 0.1 times.

[0087] In some cases, (i) the change (e.g., enhancement or decrease) in the expression and / or activity level of a target gene upon multiple different modulations of the target gene in a sequential manner can be greater than (ii) any change in the expression and / or activity level of the target gene upon only one or none of the multiple different modulations, to the extent of at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%. , at least or up to about 19%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 35%, at least or up to about 40%, at least or up to about 45%, at least or up to about 50%, at least or up to about 55%, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300%.

[0088] In some cases, (i) the change (e.g., enhancement or decrease) in the expression and / or activity level of a target gene upon multiple different modulations of the target gene in a sequential manner can last for a longer duration than (ii) any change in the expression and / or activity level of the target gene upon only one or none of the multiple different modulations, to the extent of at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19%, at least or up to about 20%, at least or up to about 21%, at least or up to about 22%, at least or up to about 23%, at least or up to about 24%, at least or up to about 25%, at least or up to about 26%, at least or up to about 27%, at least or up to about 28%, at least or up to about 29%, at least or up to about 30%, at least or up to about 31%, at least or up to about 32%, at least or up to about 33%, at least or up to about 34%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about %, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 125%, at least or up to about 150%, at least or up to about 175%, at least or up to about 200%, at least or up to about 225%, at least or up to about 250%, at least or up to about 275%, or at least or up to about 300%.

[0089] In some cases, the expression and / or activity level of a target gene may be less (i) at multiple different regulation of the target gene in a sequential manner (e.g., including sequential disruption) than the expression and / or activity level of the target gene in only a first different regulation but not a second different regulation by at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19%, at least or up to about 20%, at least or up to about 21%, at least or up to about 22%, at least or up to about 23%, at least or up to about 24%, at least or up to about 25%, at least or up to about 26%, at least or up to about 27%, at least or up to about 28%, at least or up to about 29%, at least or up to about 30%, at least or up to about 31%, at least or up to about 32%, at least or up to about 33%, at least or up to about 34%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about 44%, at least or up to about 45%, at least %, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100%.

[0090] In some cases, the expression and / or activity level of a target gene may be shorter (i) at multiple different regulation of the target gene in a sequential manner (e.g., including sequential disruption) than (ii) at only a first different regulation but not a second different regulation, by at least or up to about 1%, at least or up to about 2%, at least or up to about 3%, at least or up to about 4%, at least or up to about 5%, at least or up to about 6%, at least or up to about 7%, at least or up to about 8%, at least or up to about 9%, at least or up to about 10%, at least or up to about 11%, at least or up to about 12%, at least or up to about 13%, at least or up to about 14%, at least or up to about 15%, at least or up to about 16%, at least or up to about 17%, at least or up to about 18%, at least or up to about 19%, at least or up to about 20%, at least or up to about 21%, at least or up to about 22%, at least or up to about 23%, at least or up to about 24%, at least or up to about 25%, at least or up to about 26%, at least or up to about 27%, at least or up to about 28%, at least or up to about 29%, at least or up to about 30%, at least or up to about 31%, at least or up to about 32%, at least or up to about 33%, at least or up to about 34%, at least or up to about 35%, at least or up to about 36%, at least or up to about 37%, at least or up to about 38%, at least or up to about 39%, at least or up to about 40%, at least or up to about 41%, at least or up to about 42%, at least or up to about 43%, at least or up to about 44%, at least or up to about 45%, at least or up to about 46 %, at least or up to about 90%, at least or up to about 91%, at least or up to about 92%, at least or up to about 93%, at least or up to about 94%, at least or up to about 95%, at least or up to about 96%, at least or up to about 97%, at least or up to about 98%, at least or up to about 99%, or substantially about 100%.

[0091] In some cases, the first gate unit and the second gate unit as provided herein can be activated to induce a first different regulation and a subsequent second different regulation of a common gene (e.g., expression and / or activity spectrum of the common gene). The first gate unit and the second gate unit can be activated sequentially (e.g., at different time points) to achieve the first and second different regulation of a common gene in a sequential manner. Alternatively, the first gate unit and the second gate unit can be activated simultaneously subsequently, and such gates can be preconfigured to still achieve the first and second different regulation of a common gene in such a sequential manner.

[0092] In some cases, activation of the plurality of gate units may be the result of a single activation of the heterogeneous gene circuit (e.g., by a single activation moiety at a single point in time). The plurality of gate units may include one of a first gate unit and a second gate that are preconfigured to be sequentially activated when the heterogeneous gene circuit is activated by a single activation. In some cases, one of the first and second gate units may be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by an additional activation moiety that is different from the activation moiety of the heterogeneous gene circuit (e.g., a different guide nucleic acid). The additional activation moiety may be a portion of the heterogeneous gene circuit that is only produced (e.g., expressed) when the heterogeneous gene circuit is activated. Alternatively or additionally, the first and second gate units may each be activated by a different activation moiety that is different from the activation moiety of the heterogeneous gene circuit. Such a different activation moiety may be a portion of the heterogeneous gene circuit that is only produced (e.g., expressed) when the heterogeneous gene circuit is activated.

[0093] In some embodiments of any of the systems disclosed herein, the gate unit can include a gate portion (e.g., at least 1, 2, 3, 4, 5 or more different gate portions) and / or a gene regulatory portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different gene regulatory portions). The gate portion as disclosed herein can include a guide nucleic acid molecule (gNA) (e.g., at least 1, 2, 3, 4, 5 or more gNAs). The gene regulatory portion as disclosed herein can include a gNA (e.g., at least 1, 2, 3, 4, 5 or more gNAs). The guide nucleic acid molecule as disclosed herein can include, but is not limited to, DNA, RNA, any analogs of the same, or any combination thereof. In some embodiments of any of the systems disclosed herein, the gate portion and / or the gene regulation portion can be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and the complex can be configured to or can bind to a target polynucleotide, for example, to regulate the expression and / or activity level of a target polynucleotide or another polynucleotide sequence operably coupled to the target polynucleotide. For example, the complex can regulate the expression and / or activity level of a gene comprising a target polynucleotide.

[0094] In some embodiments of any of the systems disclosed herein, the initial (or first) gate unit of the heterologous gene circuit as disclosed herein can be activated (e.g., directly activated) by an activation portion. The activation portion can directly bind to at least a portion of the initial gate unit to activate the initial gate unit, for example, thereby sequentially activating the heterologous gene circuit. Alternatively, the activation portion (e.g., electromagnetic energy) can activate the initial gate unit without directly binding to at least a portion of the initial gate unit. In some cases, the initial gate unit can include at least one gate portion and at least one gene regulatory portion. In some cases, the initial gate unit can include at least one gate portion, but may not include and need not include a gene regulatory portion. In some cases, the initial gate unit can include at least one gene regulatory portion, but may not include and need not include a gate portion (e.g., the activation portion can be configured to activate the starting gate unit and at least one additional gate unit).

[0095] In some embodiments of any of the systems disclosed herein, the gNA of the gate portion and / or the gene regulatory portion (e.g., the gNA encoded by the gate portion and / or the gene regulatory portion) can be an activatable gNA. The activatable gNA can be, but is not limited to, any of the following: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs of such, or any combination thereof. In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a cis-ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA can be self-cleavable to become non-functional (e.g., not configured to bind to a target gene), unless the gene encoding the activatable gNA is modified before the activatable gNA is expressed. In some embodiments, the activatable gNA molecule comprises a non-canonical transcription termination sequence (e.g., a polyX sequence, such as a polyU sequence or a polyT sequence), so that a functional gNA molecule is not expressed until the gene encoding the activatable gNA having a non-canonical transcription termination sequence can be modified (e.g., to remove some or all of the transcription termination sequence). Thus, in the absence of modification of the transcription termination sequence, non-functional variants of the gNA (e.g., non-functional fragments) can be expressed. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have an attached fluorescent label.

[0096] In some cases, the size of the polyT sequence is greater than or equal to a threshold length, wherein the threshold length is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence. Therefore, a plasmid (e.g., a gate portion or a gene regulatory portion) can encode an inactivated gNA comprising a polyT sequence greater than or equal to a threshold length, and editing such a plasmid to reduce the length of the polyT to less than the threshold length can allow the gNA to be expressed in its entirety without premature termination, thereby activating the gNA. In some cases, the polyT sequence comprises at least 5 Ts. In some cases, the polyT sequence comprises at least 7 Ts. In some cases, the polyT sequence comprises at least 8 Ts. In some cases, the polyT sequence comprises at least 10 Ts. In some cases, the polyT sequence comprises 5 to 15 Ts. In some cases, the polyT sequence comprises one or more additional nucleotides that are not T.

[0097] In some cases, the gene regulation portion (e.g., a guide nucleic acid and / or an endonuclease) can be configured to be bound to a target polynucleotide sequence that is operably coupled to a target gene in a cell. The target gene can include a coding polynucleotide sequence that encodes a target nucleic acid molecule or a target protein. The target polynucleotide sequence can be a part of the coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be a part of the coding polynucleotide sequence. For example, the target polynucleotide sequence can be upstream of the coding polynucleotide sequence (e.g., a part of the promoter of the coding polynucleotide sequence, such as a transcription start site (TSS).

[0098] A plurality of different adjustments of a target gene can be different (e.g., different degrees of change in the expression and / or activity level of a target gene). For example, the first adjustment applied by the first gene unit and the second adjustment applied by the second gate unit can differ by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%. The difference between the first adjustment and the second adjustment can be at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, or at most about 0.1%. Alternatively or additionally, the different regulation of the target genes may be substantially the same (eg, identical).

[0099] A plurality of different modulations may be individually sufficient to induce a desired change in the expression and / or activity level of a target gene. Alternatively, a different modulation may be individually insufficient to induce a desired change in the expression and / or activity level of a target gene.

[0100] The one or more target genes as disclosed herein may include one or more endogenous genes (eg, genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or a combination thereof.

[0101] One or more target genes as disclosed herein may include cell differentiation regulators, molecular function regulators, binding factors, fusogenic factors, protein folding chaperones, protein tags, RNA folding chaperones, cell signaling factors, immune response factors, sensory receptors, cell structural factors, protein binding factors, cargo receptors, catalytic factors, or small molecule sensors.

[0102] One or more target genes as disclosed herein may include cell differentiation regulatory factors, including growth factors, transcription factors, myogenic regulatory factors, immune cell regulatory factors, neuronal regulatory factors, stem cell differentiation factors, chondrogenic regulatory factors, osteogenic regulatory factors, aging factors, stemness factors (e.g., dedifferentiation factors), and the like.

[0103] In some cases, one or more target genes (e.g., one or more myogenic regulatory factors) can include Msgn1, Pou5f1, Pax7, Myog, Myf5, Myf6, Myof, Srf, Ccnd2, Fgf2, Sox2, Tbx6, Tbxt, Ctnnb1, Mycl, Bcl2l2, Bcl2l1, Bcl2a1, Myod1, Bcl2, Tdgf1, Pax3, Chrd, Ccnd3, Hgf, Ccnd1, Foxm1, Myc, Tgfb1, Mdm2, Mcl1, Igf1, Mef2c, Mrf4, MyHC, MCK, Six1, Six4, Hdac4, Notch1, Ezh2, p21, Myh1, Myh2, Myh4 and / or Smad4.

[0104] In some cases, one or more target genes (e.g., one or more immune cell regulatory factors) can include Mycn, Ptcra, Bcl11b, Hhex, Notch1, Tcf3, Rag2, Dtx1, Runx1, Hoxa9, Rag1, Spi1, Ets1, Id2, Bcl11a, Id3, Tcf7, Ikzf1, Tcf12, Runx3, Lmo2, Lef1, Gfi1, Lyl1, Aqp3, Meis1, Gata3, Hes1, St18, Nr4a1, C20orf100, Ikaros, Spib, Rorc, Tcf1, Myc, Ahr, Foxo1, Notch1, Notch3, IL-2, IL4, IL7, IL15 Ebf1, Pax5, Tal1, Myb, Erg, Hhex, E4pb4, Gfi1b, pTα, Tbx21 Ets1, Ets2, or Mef2c ( Figure 1-2 ). For example, one or more target genes for inducing T cell differentiation can be selected from Hes1, Gata3, Tcf1, Tcf3, Tcf7, Spi1, Lmo2, Ikzf1, Notch1, IL7, IL2, Runx1 and Bcl11b ( Figure 3). In another example, one or more target genes for inducing B cell differentiation can be selected from Ebf1, IL4, Tcf3, Foxo1, and Pax5. In another example, one or more target genes for inducing NK cell differentiation can be selected from E4bp4, Spi1, ZBTB16, Tbx21, IL7, IL15, and Notch1.

[0105] In some cases, one or more target genes (e.g., one or more stem cell differentiation factors) can include HoxB4, Notch1, Sox11, Sox17, Runx1, Gata2, Fli1, Erg, Wnt, Hnf1, Hnf3, Hnf4, Cdx2, and Lin28A.

[0106] In some cases, one or more target genes (e.g., one or more neuronal regulatory factors) can include Ezh2, Sox1, Sox 3, Sox4, Sox9, Sox10, Sox11, Sox21, Hes1, Dlk1, Ascl1, Ngn1, Ngn2, Lhx6, Zeb1, Zeb2, Smad7, Myt11, Pax6, Wnt7a, Nptx1, Nab2, Lhx6, Olig2, Nfia, Nfib, Dlx5, Nurr1, Gpx1, Otx2, Pbx1, Foxa2, Isl1, Phox2a, Phox2b, Pitx3, Brn3a, and Brn4.

[0107] In some cases, one or more target genes (e.g., one or more chondrogenic regulatory factors) can include Sox9, Gli3, Trps1, Nkx3.2, Runx2, Runx3, Smad1, Smad5, and Smad8.

[0108] In some cases, one or more target genes (e.g., one or more osteogenic regulatory factors) can include Fos, Crebbp, Hnf4a, Irf1, Irf9, Foxo4, Meis1, Egr1, Pou2f1, Anf281, Sp1, Esr1, Sox9, Znf6, Znf162, and Znf281.

[0109] In some cases, the one or more target genes (e.g., one or more senescence factors) can include p21, p53, Bax, Apaf1, Mdm2, E2f1, and Foxo3.

[0110] In some cases, one or more target genes (e.g., one or more stemness factors) can include Hes1, Hes5, Cbf1, Soc2, Hmga2, Olig2, Id2, Id4, Hesrl, Hesr2, Gli1, Gli2, Gli3, SoxB, and Bmi1.

[0111] In some cases, one or more target genes may include T-box transcription factors (TBX genes). TBX transcription factors are involved in development. T-box proteins have relatively large DNA binding domains. Non-limiting examples of TBX transcription factors may include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein).

[0112] In some cases, one or more target genes may include basic helix-loop-helix transcription factors (bHLH genes). bHLH transcription factors are involved in the regulation of the cell cycle and many other developmental processes. bHLH proteins have a basic helix-loop-helix protein structure. Non-limiting examples of bHLH transcription factors can include AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATOH1, ATOH7, ATOH8, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, EPAS1, FERD3L, FIGLA, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MX D3, MXD4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, NPAS4, OAF 1. OLIG1, OLIG2, OLIG3, PTF1A, SCL, SCXB, SIM1, SIM2, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL 2. TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1 and USF2.

[0113] In some cases, one or more target genes may include SRY-related cassette transcription factors (SOX genes). SOX transcription factors are involved in developmental regulation. Non-limiting examples of SOX transcription factors may include SOX1, SOX2, SOX3, SOX4, SOX5, SOX6, SOX7, SOX8, SOX9, SOX10, SOX11, SOX12, SOX13, SOX14, SOX15, SOX17, SOX18, SOX21, SOX30, and SRY.

[0114] In some cases, one or more target genes may include a SOX A group, which includes SRY. In some cases, one or more target genes may include a SOX B1 group, which includes SOX1, SOX2, and / or SOX3. In some cases, one or more target genes may include a SOX B2 group, which includes SOX14 and / or SOX21. In some cases, one or more target genes may include a SOX C group, which includes SOX4, SOX11, and / or SOX12. In some cases, one or more target genes may include a SOX D group, which includes SOX5, SOX6, and / or SOX13. In some cases, one or more target genes may include a SOX E group, which includes SOX8, SOX9, and / or SOX10. In some cases, one or more target genes may include a SOX F group, which includes SOX7, SOX 17, and / or SOX18. In some cases, one or more target genes may include a SOX G group, which includes SOX15. In some cases, one or more target genes may include a SOX H group, which includes SOX30.

[0115] In some cases, one or more target genes may include a forkhead box (FOX). FOX is a transcription factor that regulates the expression of genes involved in cell growth, proliferation, differentiation and lifespan. Some FOX genes can bind to chromatin during the cell differentiation process. Non-limiting examples of FOX genes may include FOXA, FOXB, FOXC, FOXD, FOXE, FOXF, FOXG, FOXH, FOXI, FOXJ, FOXK, FOXL, FOXM, FOXN, FOXO, FOXP, FOXQ, FOXR and FOXS.

[0116] In some cases, one or more target genes may include erythroblast transformation specific (ETS) genes. ETS is a transcription factor unique to animals and is involved in tissue development. Non-limiting examples of ETS genes may include ELF1, ELF2 (NERF) ELF4 (MEF) GABPα, ERG, FLI1, FEV, ERF (PE2), ETV3 (PE1), ELF3 (ESE1 / ESX), ELF5 (ESE2), ESE3 (EHF), ETS1, ETS2, SPDEF (PDEF / PSE), ETV4 (PEA3 / E1AF), ETV5 (ERM), ETV1 (ER81), ETV2 (ER71), SPI1 (PU.1), SPIB, SPIC, ELK1, ELK4 (SAP1), ELK3 (NET / SAP2), ETV6 (TEL) and ETV7 (TEL2).

[0117] In some cases, one or more target genes may include collagen. Collagen is a fibrous protein and is a major element of skin, bones, tendons, cartilage, blood vessels, and teeth. Collagen forms insoluble fibers with high tensile strength. Non-limiting examples of collagen genes may include COL1A1, COL1A2, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, COL6A3, COL6A4P1, COL6A4P2, COL6A5, COL6A6, COL7A1, COL8A1, COL8A2, COL9A1, COL9A2, COL9A3, COL10A1, COL11A1, COL11A2, COL12A1, COL13A1, COL14A1, COL15A1, COL16A1, COL17A1, COL18A1, COL19A1, COL20A1, COL21A1, COL22A1, COL23A1, COL24A1, COL25A1, COL26A1, COL27A1 and COL28A1.

[0118] In some cases, one or more target genes may include a homeobox gene. A homeobox gene is a gene that regulates, for example, a wide range of anatomical features in the early stages of embryonic development. The types of homeobox genes include HOX genes, LIM genes, PAX genes, POU genes, CERS genes, HNF genes, SINE genes, CUT genes, ZF genes, paraHOX genes, DLX genes, TALE genes, PRD genes, and NKL genes. Non-limiting examples of homeobox genes can include HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA13, HOXB1, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXB7, HOXB8, HOXB9, HOXB13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13, HOXD 1. HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX1, EVX1, EVX2, GBX1, GBX2, MEOX1, MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, PBX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LH X9, LMX1A, LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, P OU5F1P1, POU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LASS4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SI X4, SIX5, SIX6, ONECUT1, ONECUT2, ONECUT3, CUX1, CUX2, SATB1, SATB2, ADNP, ADNP2, TSHZ1, TSHZ2, TSHZ3, ZEB1, ZEB2, ZFHX2,ZFHX3, ZFHX4, ZHX1, HOMEZ, ALX1(CART1), ALX3, ALX4, ARGFX, ARX, DMBX1, DPRX, DRGX, DUXA, DUXB, DUX(1, 2, 3, 4, 4c, 5), ESX1, GSC, GSC2, HESX1, HOPX, ISX, LEUTX, MIXL1, NOBOX, OTP, OTX1, OTX2, CRX, PAX2, PAX3, PAX4, PAX5, PAX6, PAX7, PAX8, PHOX2A, PHOX2B, PITX1, PITX2, PITX3, PROP1, PRRX1, PRRX2, RAX, RAX2, RHOXF1, RHOXF2 / 2B, SEBOX , SHOX, SHOX2, TPRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX 2. TLX3, TSHZ1, TSHZ2, TSHZ3, VAX1, VAX2, VENTX, NKX2-1, NKX2-4, NKX2-2, NKX2-8, N KX3-1, NKX3-2, NKX2-3, NKX2-5, NKX2-6, HMX1, HMX2, HMX3, NKX6-1, NKX6-2 and NKX6-3. ,

[0119] In some cases, one or more target genes may include a GATA gene. A GATA gene is a transcription factor characterized by its ability to bind to the DNA sequence "GATA". Non-limiting examples of GATA genes may include GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0120] In some cases, the use of a heterologous gene circuit as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, mesogenic progenitor cells, hematopoietic progenitor cells) into hematopoietic cells, wherein at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the resulting cells produced by using a heterologous gene circuit as disclosed herein are target cell types. The target cell types can include T cells, natural killer cells, B cells, dendritic cells, macrophages, or other hematopoietic cells.

[0121] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, mesenchymal progenitor cells, hematopoietic progenitor cells) into immune cells (T cells, NK cells, etc.), for example, in the absence of one, two, or all of feeder cells, serum, and exogenous Notch. The use of heterologous gene circuits as disclosed herein can be used to differentiate from up to about 1x10 6 pcs, up to about 9x10 5 pcs, up to about 8x10 5 pcs, up to about 7x10 5 pcs, up to about 6x10 5 pcs, up to about 5x10 5 pcs, up to about 4x10 5 pcs, up to about 3x10 5 pcs, up to about 2x10 5 pcs, up to about 1x10 5 pcs, up to about 5x10 4 pcs, up to about 2x10 4 pcs, up to about 1x10 4 or fewer EMP cells (e.g., human EMP cells) produce at least about 1×10 4 At least about 2x10 4 At least about 5x10 4 At least about 1x10 5 At least about 2x10 5 At least about 5x10 5 At least about 1x10 6 At least about 2x10 6 At least about 5x10 6 At least about 1x10 7 At least about 2x10 7 At least about 5x10 7 At least about 1x10 8 At least about 2x10 8 At least about 5x10 8 At least about 1x10 9 At least about 2x10 9 At least about 5x10 9 At least about 1x10 10 At least about 2x10 10 At least about 5x10 10 At least about 1x10 15 At least about 2x10 15 At least about 5x10 15or more T cells.

[0122] The generation of such T cells by using a heterologous gene circuit as disclosed herein can be achieved within a span of at most about 60 days, at most about 55 days, at most about 50 days, at most about 45 days, at most about 40 days, at most about 35 days, at most about 30 days, at most about 25 days, at most about 20 days, at most about 15 days, at most about 10 days, at most about 5 days, or less. The resulting T cells generated by using a heterologous gene circuit as disclosed herein can induce at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or more of target cells (e.g., cancer cells) when the effector to target (E:T) ratio is determined (e.g., in vitro, in vivo) to be at least about 1:3, at least about 1:4, at least about 1:5, at least about 1:6, at least about 1:7, at least about 1:8, at least about 1:9, at least about 1:10, at least about 1:12, at least about 1:15, at least about 1:20, at least about 1:30, at least about 1:40, at least about 1:50 or less (e.g., an E:T ratio of 1:100 is less than an E:T ratio of 1:50). When injected into mice, the resulting T cells generated by using a heterologous gene circuit as disclosed herein can extend the lifespan of the mice by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more.

[0123] In some cases, the T cell produced by the method has a killing function that is superior to the T cell obtained via directed differentiation. The T cell produced using the provided method can have a killing function greater than the T cell obtained via directed differentiation at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400% or at least about 500% killing function. Alternatively or additionally, the T cell produced by the method can be a killing function equivalent to that of primary peripheral blood derived T cells.

[0124] In some cases, the T cells produced in addition to primary peripheral derived T cells have a killing ability similar to that of primary peripheral derived T cells, as measured by CAR activity. T cell types may include, but are not limited to, γ-δ T cells, NKT cells, and MAIT cells. In some cases, the use of heterologous gene circuits as disclosed herein can be used to obtain cells with non-natural phenotypes (e.g., non-T cells with killing ability).

[0125] In some cases, the use of heterologous gene circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitors (EMP) cells, mesenchymal progenitors, hematopoietic progenitors) can induce stem cells to differentiate into immune cells in the absence of feeder cells. In some cases, the use of heterologous gene circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitors (EMP) cells, mesenchymal progenitors, hematopoietic progenitors) can induce stem cells to differentiate into immune cells in the absence of serum. In some cases, the use of heterologous gene circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitors (EMP) cells, mesenchymal progenitors, hematopoietic progenitors) can induce stem cells to differentiate into immune cells in the absence of exogenous Notch. In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitors (EMP) cells, mesenchymal progenitors, hematopoietic progenitors) into T cells, for example, in the absence of one or both of embryoid bodies and serum. The resulting T cells produced by using a heterologous gene circuit as disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population. In some cases, the use of a heterologous gene circuit as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, mesenchymal progenitor cells, hematopoietic progenitor cells) into natural killer cells, for example, in the absence of one or both of embryoid bodies and serum. The resulting natural killer cells produced by using a heterologous gene circuit as disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population.

[0126] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, mesenchymal progenitor cells, hematopoietic progenitor cells) into B cells. The resulting B cells produced by using heterologous gene circuits as disclosed herein are at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population after differentiation.

[0127] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, mesenchymal progenitor cells, hematopoietic progenitor cells) into dendritic cells. The resulting dendritic cells produced by using heterologous gene circuits as disclosed herein are at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the total resulting cell population after differentiation.

[0128] In some cases, the use of heterologous gene circuits as disclosed herein can be used to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitors (EMP) cells, mesenchymal progenitor cells, hematopoietic progenitor cells) into macrophages. The resulting macrophages produced by using heterologous gene circuits as disclosed herein are similar to the natural cell state in terms of their ability to detect, engulf and destroy harmful cells, as measured by their ability to act as a surrogate for CAR or by their ability to kill cancerous cells.

[0129] In some cases, at least two different adjustments including a first adjustment and a second adjustment can be made to the target gene. The timing of the first adjustment and the second adjustment can be controlled (e.g., as predetermined by the design of the heterologous gene circuit). For example, the initiation of the second adjustment (e.g., by at least a portion of the second gating unit, such as the second gene regulation portion) can be at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds, at least about 5 seconds, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes ... The invention may occur in the following manner: the present invention may occur in the following manner: at least about 1 minute, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days. Initiation of the second regulation (e.g., by at least a portion of the second gating unit, such as the second gene regulatory portion) can be at most about 10 days, at most about 9 days, at most about 8 days, at most about 7 days, at most about 6 days, at most about 5 days, at most about 4 days, at most about 3 days, at most about 2 days, at most about 1 day, at most about 20 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about 2 days, at most about 1 day, at most about 20 hours, at most about 10 hours, at most about 9 hours, at most about 8 hours, at most about 7 hours, at most about 6 hours, at most about 5 hours, at most about 4 hours, at most about 3 hours, at most about At most, about 2 hours, at most about 1 hour, at most about 50 minutes, at most about 40 minutes, at most about 30 minutes, at most about 20 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6 minutes, at most about 5 minutes, at most about 4 minutes, at most about 3 minutes, at most about 2 minutes, at most about 1 minute, at most about 50 seconds, at most about 40 seconds, at most about 30 seconds, at most about 20 seconds, at most about 10 seconds, at most about 9 seconds, at most about 8 seconds, at most about 7 seconds, at most about 6 seconds, at most about 5 seconds, at most about 4 seconds, at most about 3 seconds, at most about 2 seconds, or at most about 1 second occurs.

[0130] In some cases, the number of gate units that need to be activated (e.g., sequentially activated) between activation of a first regulation by a first gate unit and subsequent activation of a second regulation by a second gate unit can at least partially determine (e.g., substantially determine) the timing between the first regulation and the second regulation. Upon activation of a first regulation of a target gene by a first gate unit, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more additional gate units may need to be activated (e.g., sequentially activated) to activate a second gate unit for inducing a second regulation. Upon activation of a first regulation of a target gene by a first gate unit, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1 additional gate units may need to be activated (e.g., sequentially) to activate a second gate unit for inducing a second regulation.

[0131] The results of the cell can include the regulation of multiple target genes. For example, the results can include the regulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more target genes. The results can include the regulation of at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2 or at most about 1 target gene. At least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50 or more modulations may be performed on each gene disclosed herein. Up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1 modulation may be performed on each gene disclosed herein. One or more modulations of a target gene (e.g., an endogenous gene) as induced by a heterologous genetic circuit of the present disclosure can be artificial modulations (or heterologous modulations) that otherwise would not occur in the cell in the absence of (i) the heterologous genetic circuit and / or (ii) the activating portion of the heterologous genetic circuit.

[0132] The plurality of gate units may be operated sequentially (e.g., each of the plurality of gate units is activated in a sequential manner). For example, a gate unit in the plurality is activated to activate a subsequent gate unit in the plurality. The sequential operation of the gate units may be linear. Alternatively, the sequential operation of the gate units may be sent back to each other as input to form a loop. For example, the plurality of gate units may cause a feedback loop, such as a positive feedback loop or a negative feedback loop.

[0133] In some embodiments of any of the systems disclosed herein, the first gating unit can include a first gene regulatory portion that can be activatable to exhibit specific binding to the target gene to induce a first different regulation. Alternatively or additionally, the first gating unit can include a first gene regulatory portion that can be activatable to exhibit non-specific binding to the target gene to induce a first different regulation.

[0134] The first differential modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. The first differential modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 150%, at most about 160%, at most about 170%, at most about 180%, at most about 190%, at most about 200%, at most about 210%, at most about 220%, at most about 230%, at most about 240%, at most about 250%, at most about 260%, at most about 270%, at most about 280%, at most about 290%, at most about 300%, at most about 300%, at most about 310%, at most about 310%, at most about 320%, at most about 330%, at most about 340%, at most about 350%, at most about 360%, at most about 370%, at most about 380%, at most about 390%, at most about 400%, at most about 410%, at most about 420%, at most about 430%, at most about 440%, at most about 450%, at most about 460%, at most about 480%, at most about 490%, at most about 500%, at most about About 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2%, up to about 0.1% or less.

[0135] As compared to a control expression and / or activity level, a first differential modulation as disclosed herein (e.g., induced by a first gating unit) can induce a change (e.g., an increase or decrease) in the expression and / or activity level of a target gene by at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, at least or up to about 4 fold, at least or up to about 5 fold, at least or up to about 6 fold, at least or up to about 7 fold, at least or up to about 8 fold, at least or up to about 9 fold, at least or up to about 10 fold, at least or up to about 11 fold, at least or up to about 12 fold, at least or up to about 13 fold, at least or up to about 14 fold, at least or up to about 15 fold, at least or up to about 16 fold, at least or up to about 17 fold, at least or up to about 18 fold, at least or up to about 19 fold, at least or up to about 20 fold, at least or up to about 21 fold, at least or up to about 23 fold, at least or up to about 24 fold, at least or up to about 25 fold, at least or up to about 26 fold, at least or up to about 27 fold, at least or up to about 28 fold, at least or up to about 29 fold, at least or up to or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times. Compared to the control expression and / or activity level, the first differential modulation can induce a change (e.g., an increase or decrease) in the expression and / or activity level of the target gene by at most or less than about 10,000 times, at most or less than about 5,000 times, at most or less than about 1,000 times, at most or less than about 500 times, at most or less than about 100 times, at most or less than about 90 times, at most or less than about 80 times, at most or less than about 70 times, at most or less than about 60 times, at most or less than about 50 times, at most or less than about 40 times, at most or less than about 30 times, at most or less than about 20 times. , at most or less than about 10 times, at most or less than about 9 times, at most or less than about 8 times, at most or less than about 7 times, at most or less than about 6 times, at most or less than about 5 times, at most or less than about 4 times, at most or less than about 3 times, at most or less than about 2 times, at most or less than about 1 times, at most or less than about 0.9 times, at most or less than about 0.8 times, at most or less than about 0.7 times, at most or less than about 0.6 times, at most or less than about 0.5 times, at most or less than about 0.4 times, at most or less than about 0.3 times, at most or less than about 0.2 times, at most or less than about 0.1 times.

[0136] Subsequently, a second different modulation as disclosed herein (e.g., induced by a second gating unit) can induce an additional change (e.g., an increase, decrease, or selective attenuation) in the expression and / or activity level of the target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 101%, at least about 112%, at least about 123%, at least about 130%, at least about 131%, at least about 132%, at least about 143%, at least about 154%, at least about 155%, at least about 160%, at least about 171%, at least about 181%, at least about 191%, at least about 202%, at least about 213%, at least about 224%, at least about 235%, at least about 247%, at least about 256%, at least about 268%, at least about 271%, at least about 281%, at least about 291%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, or at least about 1,000,000%. The second different modulation can induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene by at most about 1,000,000%, at most about 100,000%, at most about 9,000%, at most about 8,000%, at most about 7,000%, at most about 6,000%, at most about 5,000%, at most about 4,000%, at most about 3,000%, at most about 2,000%, at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 3000%, at most about 5000%, at most about 6000%, at most about 7000%, at most about 8000%, at most about 9000%, at most about 10000%, at most about 15000%, at most about 16000%, at most about 17000%, at most about 18000%, at most about 19000%, at most about 20000%, at most about 25 ... %, up to about 200%, up to about 100%, up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, up to about 0.9%, up to about 0.8%, up to about 0.7%, up to about 0.6%, up to about 0.5%, up to about 0.4%, up to about 0.3%, up to about 0.2% or up to about 0.1%.

[0137] The additional change via the second different regulation can induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene compared to the control expression and / or activity level by at least or up to about 0.1 fold, at least or up to about 0.2 fold, at least or up to about 0.3 fold, at least or up to about 0.4 fold, at least or up to about 0.5 fold, at least or up to about 0.6 fold, at least or up to about 0.7 fold, at least or up to about 0.8 fold, at least or up to about 0.9 fold, at least or up to about 1 fold, at least or up to about 2 fold, at least or up to about 3 fold, at least or up to about 4 fold, at least or up to about 5 times, at least or up to about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times. The second different modulation may induce an additional change (e.g., increase or decrease) in the expression and / or activity level of the target gene by up to or less than about 10,000-fold, up to or less than about 5,000-fold, up to or less than about 1,000-fold, up to or less than about 500-fold, up to or less than about 100-fold, up to or less than about 90-fold, up to or less than about 80-fold, up to or less than about 70-fold, up to or less than about 60-fold, up to or less than about 50-fold, up to or less than about 40-fold, up to or less than about 30-fold, up to or less than about 20-fold, or up to or less than about 50-fold, up to or less than about 60-fold, up to or less than about 70-fold, up to or less than about 80-fold, up to or less than about 90-fold, up to or less than about 100-fold, up to or less than about 110-fold, up to or less than about 120-fold, up to or less than about 130-fold, up to or less than about 140-fold, up to or less than about 150-fold, up to or less than about 160-fold, up to or less than about 170-fold, up to or less than about 180-fold, up to or less than about 190-fold, up to or less than about 200-fold, up to or less than about 210-fold, up to or less than about 230-fold, up to or less than about 240-fold, up to or less than about 250-fold, up to or less than about 260-fold, up to or less than about 270-fold, up to or less than about 280-fold, up to or less than about 290-fold, up to or times, at most or less than about 10 times, at most or less than about 9 times, at most or less than about 8 times, at most or less than about 7 times, at most or less than about 6 times, at most or less than about 5 times, at most or less than about 4 times, at most or less than about 3 times, at most or less than about 2 times, at most or less than about 1 times, at most or less than about 0.9 times, at most or less than about 0.8 times, at most or less than about 0.7 times, at most or less than about 0.6 times, at most or less than about 0.5 times, at most or less than about 0.4 times, at most or less than about 0.3 times, at most or less than about 0.2 times, at most or less than about 0.1 times.

[0138] When the expression and / or activity level of the target gene reaches a target level via the action of a first differential regulation (eg, by the design of a heterologous gene circuit), an additional change via a second differential regulation may occur.

[0139] Compared to the control expression and / or activity level, when the expression and / or activity level of the target gene is changed (e.g., increased or decreased) by at least or more than about 0.1 times, at least or more than about 0.2 times, at least or more than about 0.3 times, at least or more than about 0.4 times, at least or more than about 0.5 times, at least or more than about 0.6 times, at least or more than about 0.7 times, at least or more than about 0.8 times, at least or more than about 0.9 times, at least or more than about 1 times, at least or more than about 2 times, at least or more than about 3 times, at least or more than about 4 times, at least or more than about 5 times, at least or more than about 6 times, at least or more than about 7 times, at least or more than about 8 times, at least or more than about 9 times, at least or more than about 10 times, at least or more than about 11 times, at least or more than about 12 times, at least or more than about 13 times, at least or more than about 14 times, at least or more than about 15 times, at least or more than about 16 times, at least or more than about 17 times, at least or more than about 18 times, at least or more than about 19 times, at least or more than about 20 times, at least or more than about 21 times, at least or more than about 22 times, at least or more than about 23 times, at least or more than about 24 times, at least or more than about 25 times, at least or more than about 26 times, at least or more than about 27 times, at least or more than about 28 times, at least or more than about 29 times, at least or more than about 30 times, at least or more than about 31 times, at least or more than about 32 times, at least or more than about 33 times, at least or more than about 34 times, at least An additional change via a second different modulation may occur when the amount of the peptide is increased by about 6 times, at least or up to about 7 times, at least or up to about 8 times, at least or up to about 9 times, at least or up to about 10 times, at least or up to about 20 times, at least or up to about 30 times, at least or up to about 40 times, at least or up to about 50 times, at least or up to about 60 times, at least or up to about 70 times, at least or up to about 80 times, at least or up to about 90 times, at least or up to about 100 times, at least or up to about 500 times, at least or up to about 1,000 times, at least or up to about 5,000 times, or at least or up to about 10,000 times. When the expression and / or activity level of the target gene is altered (e.g., increased or decreased) by at most or less than about 10,000 times, at most or less than about 5,000 times, at most or less than about 1,000 times, at most or less than about 500 times, at most or less than about 100 times, at most or less than about 90 times, at most or less than about 80 times, at most or less than about 70 times, at most or less than about 60 times, at most or less than about 50 times, at most or less than about 40 times, at most or less than about 30 times, at most or less than about 20 times, at most or less than about 1 An additional change via a second different adjustment may occur when the amount of the second different adjustment is at most or less than about 0.0 times, at most or less than about 9 times, at most or less than about 8 times, at most or less than about 7 times, at most or less than about 6 times, at most or less than about 5 times, at most or less than about 4 times, at most or less than about 3 times, at most or less than about 2 times, at most or less than about 1 times, at most or less than about 0.9 times, at most or less than about 0.8 times, at most or less than about 0.7 times, at most or less than about 0.6 times, at most or less than about 0.5 times, at most or less than about 0.4 times, at most or less than about 0.3 times, at most or less than about 0.2 times, at most or less than about 0.1 times.

[0140] Alternatively or additionally, a second different modulation as disclosed herein (e.g., induced by a second gating unit) can induce a change (e.g., an increase or decrease) in the expression and / or activity level of an additional target gene by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 101%, at least about 112%, at least about 123%, at least about 130%, at least about 131%, at least about 132%, at least about 143%, at least about 154%, at least about 155%, at least about 160%, at least about 171%, at least about 181%, at least about 191%, at least about 202%, at least about 213%, at least about 224%, at least about 235%, at least about 247%, at least about 256%, at least about 268%, at least about 271%, at least about 281%, at least about 291%, At least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000% or at least about 1,000,000%. The second differential modulation may induce a change (e.g., an increase or decrease) in the expression and / or activity level of the additional target gene by at most about 1,000,000%, at most about 100,000%, at most about 9,000%, at most about 8,000%, at most about 7,000%, at most about 6,000%, at most about 5,000%, at most about 4,000%, at most about 3,000%, at most about 2,000%, at most about 1,000%, at most about 900%, at most about 800%, at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 3000%, at most about 500%, at most about 600%, at most about 700%, at most about 800%, at most about 900%, at most about 1000%, at most about 1500%, at most about 1600%, at most about 1700%, at most about 1800%, at most about 1900%, at most about 2000%, at most about 25 ... At most about 1%, at most about 2%, at most about 3%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 15%, at most about 16%, at most about 17%, at most about 18%, at most about 20%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34%, at most about 36%, at most about 37%, at most about 38%, at most about 39%, at most about 40%, at most about 51%, at most about 52%, at most about 53%, at most about 54%, at most about 55%, at most about 56%, at most about 57%, at most about 58%, at most about 59%, at most about 60%, at most about 61%, at most about 62%, at most about 63%, at most about 64%, at most about 65%, at most about 66%, at most about 67%, at most about 68%, at most about 69%, at most about 70%, at most about 71%, at most about 72%, at most about 73%, at most about 74%, at most about 75%, at most about 76%, at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about 88%, at most about 89%, at most about 81%, at most about 8

[0141] Cells (e.g., initial cells to be modified into engineered cells as disclosed herein, final cell products produced from engineered cells as disclosed herein, etc.) can include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurse cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, duct cells, odontoblasts, cementoblasts, glial cells or interstitial cells.

[0142] Non-limiting examples of such cells may include lymphoid cells, such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, T helper cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., US20080241194); myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / hypersegmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, thrombocytes / megakaryocytes, dendritic cells; cells from the endocrine system, including thyroid cells (thyroid epithelial cells, parafollicular cells), parathyroid cells (parathyroid chief cells, eosinophils), adrenal cells (chromaffin cells), pineal cells (pinealocytes); cells of the nervous system, including glial cells (astrocytes, microglia), magnocellular neurosecretory cells, astrocytes, Boettcher cells (Boettcher cells).cells) and pituitary cells (gonadotropins, adrenocorticotropic hormone cells, thyrotropin cells, somatic cells, and lactotrophs); cells of the respiratory system, including pneumocytes (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, and dust cells; cells of the circulatory system, including cardiomyocytes and pericytes; cells of the digestive system, including gastric cells (chief cells, peripheral cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, and S cells. cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, liver cells (hepatocytes, Kupffer cells), cartilage / bone / muscle; bone cells, including osteoblasts, osteocytes, osteoclasts, tooth cells (cementoblasts, ameloblasts); cartilage cells, including chondroblasts, chondrocytes; skin cells, including hair cells, keratinocytes, melanocytes (nevus cells); muscle cells, including myocytes; urinary system cells, including podocytes, juxtaglomerular cells, intraglomerular mesangial cells / extraglomerular mesangial cells cells, renal proximal tubule brush border cells, macula densa cells; reproductive system cells, including sperm, Sertoli cells, Leydig cells, oocytes; and other cells, including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), fingernail and toenail keratinocytes, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinous hair stem cells, keratinous hair root sheath cells, hair root sheath cells of Huxley layer, hair root sheath cells of Henle layer cells, outer root sheath cells, hair matrix cells (stem cells), wet stratified barrier epithelial cells, surface epithelial cells of the stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urothelial cells (lining the urethra, bladder and urinary tract), exocrine epithelial cells, salivary mucous cells (rich in polysaccharide secretion), salivary serous cells (rich in glycoprotein enzyme secretion), von Ebb cells in the tongueEbner's) gland cells (cleans taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), cerumen gland cells in the ear (wax secretion), eccrine sweat gland dark cells (glycoprotein secretion), eccrine sweat gland light cells (small molecule secretion). Apocrine sweat gland cells (smell secretion, sex hormone sensitivity), eyelid cells (specialized sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Bowman's gland cells in the nose (washing the olfactory epithelium), Brunner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting seminal components, including fructose for motile sperm), prostate cells (secreting seminal components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), Littre's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), separate goblet cells of the respiratory and digestive tracts (mucus secretion), mucous cells of the stomach lining (mucus secretion), gastric gland zymogen cells (pepsinogen secretion), gastric gland oxyntic cells (hydrochloric acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), small intestine Paneth cells (lysozyme secretion), II of the lung Type I pneumocytes (surfactant secretion), Clara cells of the lung, hormone secreting cells, anterior pituitary cells, somatic cells, prolactin cells (Lactotropes), thyrotropin, gonadotropin cells, adrenocorticotroph cells, intermediate pituitary cells, magnocellular neurosecretory cells, cells of the intestinal and respiratory tract, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophils, adrenal cells, chromaffin cells, interstitial cells of the testis, theca cells of the follicle, luteal cells of the ruptured follicle, granulosa luteal cells, theca luteal cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney cells (Kidney), type I pneumocytes (lining of the air spaces of the lungs air space), pancreatic duct cells (follicular cells), non-striated duct cells (sweat glands, salivary glands, mammary glands, etc.), duct cells (spermatic vesicles, prostate, etc.), epithelial cells lining closed internal body cavities, ciliated cells with propulsion function, extracellular matrix secreting cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells)cell), megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), Langerhans cells of the epidermis, osteoclasts (in bone), dendritic cells (in lymphoid tissue), microglia (in the central nervous system), neutrophils, eosinophils, basophils, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, stem cells and committed progenitor cells of the blood and immune system (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory sensor cells (Sensory transducer cells), autonomic neuronal cells, sensory organ and peripheral neuronal supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oocytes (Oogonium / Oocyte), spermatocytes, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testes), thymic epithelial cells, interstitial cells and interstitial kidney cells.

[0143] Stem cells may include induced pluripotent stem cells (iPSC), multipotent stem cells (PSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), erythroid myeloid progenitor (EMP) cells, mesenchymal progenitor cells, hematopoietic stem cells (HSC), hematopoietic progenitor cells, muscle stem cells, neural stem cells, epithelial stem cells, epidermal stem cells, mammary stem cells, intestinal stem cells, neural crest stem cells or testicular stem cells.

[0144] Various aspects of the present disclosure provide engineered cells programmed to induce a desired expression and / or activity level (or profile thereof) of one or more target genes in the cell.

[0145] In some embodiments, the engineered cells of the present disclosure (e.g., engineered muscle cells, engineered immune cells) can be generated from isolated stem cells (e.g., isolated ESCs, iPSCs, EMPs, etc.). As disclosed herein, heterologous gene circuits and / or components thereof (e.g., gating units, gating portions, activation portions, etc.) can be introduced during any stage (or cell state) between and including (a) isolated stem cells and (b) their differentiated immune cell state (e.g., a terminally differentiated immune cell state, such as a terminally differentiated T cell).

[0146] In some cases, the engineered cell T cell can be derived from an EMP, and the heterologous gene circuit and / or its components (e.g., heterologous gating units, heterologous activation parts, heterologous gating parts, etc.) can be introduced into the cell in (A) an EMP state, (B) a hematopoietic stem cell state, (C) a T cell state, or (D) any other intermediate cell state. During one of (A), (B), (C), or (D), the heterologous gene circuit and / or its components (e.g., heterologous gating units, heterologous activation parts, heterologous gating parts, etc.) can be introduced into the cell once. Alternatively, during two, three, or all of (A), (B), (C), or (D), the heterologous gene circuit and / or its components (e.g., heterologous gating units, heterologous activation parts, heterologous gating parts, etc.) can be introduced into the cell multiple times.

[0147] The engineered cells (e.g., engineered T cells) disclosed herein can be used for (e.g., administered to) treat subjects in need. The subject may suffer from or may be suspected of having a condition, such as a disease (e.g., cancer). Cells (e.g., stem cells or differentiated cells) can be obtained from a subject, and such cells can be cultured in vitro and genetically modified to produce any subject engineered cells (e.g., any immune cells) as disclosed herein. Subsequently, engineered immune cells can be administered to the subject for adaptive immunotherapy. Therefore, engineered cells can be autologous for subjects in need. Alternatively, engineered cells can be allogeneic for a subject (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0148] The cells or constructs disclosed herein can be used in (e.g., administered to) pharmaceutical preparations. The pharmaceutical preparations can further comprise additional therapeutic agents. Additional therapeutic agents can include chemotherapeutic agents, immunosuppressants, and / or antibiotic agents.

[0149] Chemotherapeutic agents, also known as anti-tumor agents, are types of cancer treatments used to directly or indirectly inhibit the growth and proliferation of cancer cells. Chemotherapeutic agents can be alkylating agents (e.g., oxazaphosphorine, nitrogen mustard, imidazotetrazine, nitrosourea, alkyl sulfonate, hydrazine or platinum-based agents), antimetabolites (e.g., antifolates, pyrimidine antagonists, purine antagonists or ribonuclease reductase inhibitors), topoisomerase inhibitors (e.g., topoisomerase I inhibitors or topoisomerase II inhibitors), antibiotics (e.g., bleomycin, actinomycin D, anthracycline or mitomycin), mitotic inhibitors (e.g., vinca alkaloids, taxanes or non-taxane microtubule inhibitors), protein kinase inhibitors (e.g., tyrosine kinase inhibitors, MEK inhibitors, CDK inhibitors), proteasome inhibitors or PARP inhibitors.

[0150] Immunosuppressants are agents that reduce immune responses. Non-limiting examples of immunosuppressants can include steroids (e.g., prednisone, methylprednisolone, dexamethasone), colchicine, hydroxychloroquine, sulfasalazine, dapsone, methotrexate, mycophenolate mofetil, azathioprine, anti-IL-1 biologics, anti-TNF biologics, anti-IL-6 biologics, B cell growth factors, T cells, cytokines or JAK inhibitors of targeted biologics.

[0151] Antibiotics are agents that destroy or inhibit the growth of microorganisms. Non-limiting examples of antibiotics can include tetracycline, oxytetracycline, metacycline, doxycycline, minocycline, erythromycin, lincomycin, penicillin G, clindamycin, kanamycin, chloramphenicol, neomycin, streptomycin, norfloxacin, ciprofloxacin, ofloxacin, grepafloxacin, levofloxacin, sparfloxacin, ampicillin, carbenicillin, methicillin, cephalosporin, vancomycin, bacitracin, gentamicin, fusidic acid, ciprofloxacin and other quinolones, erythromycin, gentamicin, sulfonamides, trimethoprim, dapsone, isoniazid, teicoplanin, avopacin, synercid, vitamin virginiamycin, piperacillin, ticarcillin, cefepime, cefpirome, rifampicin, pyrazinamide, enrofloxacin, amikacin, netilmicin, imipenem, meropenem, linezolid, cefuroxime, ceftriaxone, cefadroxil, cefazolin, ceftazidime, cefotaxime, roxithromycin, cefaclor, cephalexin, cefoxitin, amoxicillin, co-amoxiclav, mupirocin, cloxacillin, and co-trimoxazole.

[0152] The pharmaceutical preparation may further comprise an excipient. The excipient may be a buffer, a carrier, a stabilizer, a solubilizer, a filler, a preservative, a diluent, a vehicle, a detergent, a salt, a peptide, a surfactant, an oligosaccharide, an amino acid, an adjuvant, a carbohydrate and / or a bulking agent.

[0153] The engineered cells disclosed herein can be administered to a subject before, simultaneously with, or after activation of a heterologous gene circuit in an engineered stem cell. For example, the engineered cells can be activated after being administered into a subject, for example, by administering to the subject an activator of a heterologous gene circuit.

[0154] A subject can be treated (e.g., administered) with a population of engineered cells (e.g., engineered T cells) of the disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses. Alternatively or additionally, a subject can be treated (e.g., administered) with a population of engineered cells (e.g., engineered T cells) of the present disclosure for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years, 80 years, 90 years, or 100 years.

[0155] Any of the methods disclosed herein can be utilized to treat a target cell, target tissue, target condition, or target disease in a subject.

[0156] Non-limiting examples of target tissues may include cells that can be obtained from a subject, such as immune cells (e.g., lymphocytes including T cells and NK cells). Non-limiting examples of objects include humans, dogs, cats, mice, rats, and transgenic species thereof. Examples of samples from objects from which cells can be derived include, but are not limited to, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive juices, tears, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, eye fluid, sweat, mucus, cerumen, oil, glandular secretions, cerebrospinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washes, cerebrospinal fluid, tissue, throat swabs, biopsies, fetal fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excreta or body tissues.

[0157] The target disease of the subject can be cancer or a tumor. Non-limiting examples of cancer can include cells of cancer, including acanthoma, acinar cell carcinoma, acoustic neuroma, acral freckle melanoma, acrohidrosis, acute eosinophilic leukemia, acute lymphocytic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, odontogenic adenomatous tumor, adrenocortical carcinoma, adult T cell leukemia, aggressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, adult ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary site, carcinosarcoma, Castleman's disease Disease), embryonal tumors of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, chronic neutrophilic leukemia, clear cell tumors, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease (Degosdisease), dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial carcinoma, endometrial carcinoma, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer , Ganglioglioma, Ganglioglioma, Gastric Cancer, Gastric Lymphoma, Gastrointestinal Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal Stromal Tumor, Germ Cell Tumor, Germ Cell Tumor, Gestational Choriocarcinoma, Gestational Trophoblastic Tumor, Giant Cell Tumor of Bone, Glioblastoma Multiforme, Glioma, Gliomatosis Cerebri, Glomus Tumor, Glucagonoma, Gonadoblastoma, Granular Cell Tumor, Hairy Cell Leukemia, Hairy Cell Leukemia, Head and Neck Cancer, Head and Neck Cancer, Heart Cancer, Hemangioblastoma, Hemangiopericytoma, Angiosarcoma, Hematological Malignancies, Hepatocellular Carcinoma, Hepatosplenic T-Cell Lymphoma, Hereditary Breast and Ovarian Cancer Syndrome, Hodgkin Lymphoma (Hodgkin Lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, lentigo maligna, melanoma, leukemia, leukemia, lip and oral cancer, liposarcoma, lung cancer, luteal tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoid leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant triton tumor), MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid carcinoma, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary ...Primary), metastatic urothelial carcinoma, mixed Mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasm, neurilemmoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular tumors oncology), oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, superior sulcus tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation), pinealoblastoma, pituicytoma, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polyembryony, precursor T-lymphocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sezary syndrome Syndrome), signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestinal cancer, soft tissue sarcoma, somatostatinoma, sooty wartswart), spinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, advanced lymphoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, urogenital tumors (Urogenital neoplasm), uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia In some embodiments, the targeted cancer cells represent a subpopulation within a cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a cancer of the hematopoietic lineage, such as a lymphoma. The antigen may be a tumor-associated antigen.

[0158] The present disclosure also provides a composition comprising an engineered gene circuit as disclosed herein. The composition may further comprise an actuator of a heterologous gene circuit. The present disclosure also provides a kit comprising the composition. The kit may further comprise an activator of a heterologous gene circuit. The activator may be in the same composition as the engineered cell. Alternatively or additionally, the activator may be in a composition different and separate from the engineered cell.

[0159] Example

[0160] Example 1: Differentiation of T cells

[0161] In this prophetic example, the CRISPR all-at-once approach was tested against different heterologous genetic circuits (HGCs) to show that HGCs outperform the CRISPR all-at-once approach.

[0162] Figure 4 Nine gRNAs (E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3, and Bcl11b) used in the CRISPR all-in-one approach are shown. In the CRISPR all-in-one approach, gRNAs are added to cells simultaneously by nucleofection. Cells are incubated until T cell differentiation is complete.

[0163] Figure 5A-Figure 5I Shown are the T cell HGCs (T cell HGC1-9) used in these experiments. In the HGC method, the activation part of the HGC is added to the cells by nuclear transfection. The cells are incubated until T cell differentiation is complete.

[0164] Figure 5A An example of an engineered cell in contact with a T cell HGC1 is shown. This engineered cell can be activated to exhibit expression and activity profiles of multiple genes associated with T cell differentiation. T cell HGC1 contains four gate units, which include four different steps. Gate unit 1 of HGC1 activates genes E2A, Ikaros, Runx1, Lmo2, and PU1 and activates gate unit 2. Gate unit 2 of HGC1 only activates gate unit 3 and acts as a holding stage, used as a filler to help development timing. Gate unit 3 of HGC1 activates genes Hes1, Tcf7, Gata3, and Bcl11b; in addition, it both activates gate unit 4 and negatively regulates earlier activated genes Lmo2 and PU1. Figure 6 Describe how four cell algorithm steps work together to achieve differentiation from starting cells to T cells. Gate unit 1 of T cell HGC1 can be activated by injection, and the injection includes a CRISPR sequence customized for gate unit 1 activated. Once the first gate unit is activated, the cell algorithm continues without any external activation until the heterologous gene circuit is completed (e.g., gate unit 4 / step 4). The result is an engineered T cell. Other cell algorithms not shown herein work by causing the third gate unit to strengthen the expression and activity spectrum of the gene regulated by the first gate unit.

[0165] A control cell colony was produced using a standard T cell culture method. iPSCs were formed into embryoid bodies and grown in a culture medium containing BMP-4, VEGF and bFGF for 12 days. On the 12th day, CD34+ cells were purified using magnetic beads and plated on a DLL4 coated plate. Cells were grown for 21 days in a culture medium containing TPO, IL-7, SDF, FLT3L and SCF. On the 21st day, cells were stimulated with CD3 antibodies in a culture medium containing Il-7, IL-2, DXM to allow the proliferation of mature T cells. Another control T cell colony was collected and purified from a mouse blood sample.

[0166] The T cells produced by HGC can grow with an amount greater than that of the T cells produced using the CRISPR integrated method. The T cells produced by HGC show normal signal transduction, gene regulation and proliferation control compared with the inherent T cells collected from blood samples. In addition, unlike the cells cultured in the culture medium, the T cells produced by HGC can not lose cytotoxic activity over time, with high killing efficiency (for example, lower effector to target ratio), longer persistence and better control and interaction with APC. By producing CD4 cells, the ratio of CD4+ and CD8+ cells can also be controlled, and the persistence and ability of cells interacting with other killer immune cells like NK cells can be affected.

[0167] It should be understood that the various aspects of the present invention can be understood individually, collectively or in combination with each other. The various aspects of the present invention described herein can be applied to any specific application disclosed herein. The composition of matter comprising a compound of any formula disclosed herein in the composition part of the present disclosure can be used in the method part including the use and production method disclosed herein, or vice versa.

[0168] Example 2: Differentiation of hematopoietic progenitor cells

[0169] In this example, induced pluripotent stem cells (iPSCs) were differentiated into hematopoietic progenitor cells using multiple heterologous genetic circuits (HGCs).

[0170] iPSC maintenance

[0171] Complete Essential 8 for maintaining dissociated iPSCs (Gibco, CatA18945) on vitronectin (Gibco, Cat# A31804) coated flasks TM Flex Medium (Gibco, catalog #A2858501) or StemFlex TM Culture medium (Gibco, catalog #A3349401) and passaged using Versene (Gibco, catalog #15040066) to maintain 50%-90% confluence to elevate cells while maintaining cell clumps.

[0172] Nucleofection

[0173] On day 0 of the experiment, ACCUTASE TM iPSC cells were collected as a single cell suspension using the Stemcell Technologies (Catalog #07920). Cells were resuspended in a P3 primary cell Nucleofector at 1.2e9 cells / ml. TMSolution and nucleofection was performed at 8e5 cells per well in a 96-well nucleocuvette (Lonza, catalog # V4SP-3096) on Lonza's 4D Nucleofector with a 96-well shuttle (catalog # AAF-1003B / S) using optimized pulse codes.

[0174] Cells are nuclear transfected with plasmids encoding Cas9-VPR, core cascade and gene targeting spacers.Cas9-VPR is a Cas transcription regulator system, depending on the specific gNA used, the system can be used for cutting (for example, gate part plasmid, gene regulation part plasmid) and non-cutting gene regulation (for example, CRISPR activation of target endogenous gene, CRISPR inhibition of target endogenous gene).The activatable gNA used is only a polyT continuous sequence (tract), with a constant core cascade for performing four sequential steps.According to the sequence in Table 1, nucleotide gNA spacers are used to target genome. Figure 8 The gene targets used in each heterologous gene circuit cascade are described. Steps refer to the order in which genes are expected to be expressed as a result of Cas9-VPR-mediated targeting and activation.

[0175] Table 1: Spacer sequences

[0176]

[0177]

[0178]

[0179] Cell culture

[0180] 1.8e5 nucleofected cells per well were cultured in 24-well tissue culture plates treated with anti-adhesion wash solution (Stemcell Technologies, catalog #07010) and the plates were rotated at 100 RPM in a humidified 5% CO2 tissue culture incubator under standard normoxic conditions with 500ul per well. Day 0 culture medium included: StemPro-34 (Gibco, catalog #10639011), 1X penicillin-streptomycin (Gibco catalog #15140122), 2mL Glutamax (Gibco, catalog #35050061), 50ug / ml ascorbic acid (Santa Cruz Biotechnology, catalog #sc-39430), 4mM 1-thioglycerol (Sigma-Aldrich, catalog #M1753), 1X insulin-transferrin-selenium (ITS-G) (Gibco, catalog #41400045) and 10mM Y-27632 (Bio-Techne catalog #1254), 50ng / ml rhBMP4 (Peprotec, catalog #120-05ET), 50ng / ml FGF-2 (Peprotech, catalog #3718-FB) and 10uM CHIR99021 (Bio-Techne, catalog #4423).

[0181] On day 1, the cultures underwent a complete media change with StemPro-34 (Gibco, catalog #10639011), IX Penicillin-Streptomycin (Gibco catalog #15140122), 2 mL Glutamax (Gibco, catalog #35050061), 50 ug / ml ascorbic acid (Santa Cruz Biotechnology, catalog #sc-39430), 4 mM 1-thioglycerol (Sigma-Aldrich, catalog #M1753), IX Insulin-Transferrin-Selenium (ITS-G) (Gibco, catalog #41400045), 50 ng / ml rhBMP4 (Peprotec, catalog #120-05ET), 50 ng / ml FGF-2 (Peprotech, catalog #3718-FB), and 50 ng / ml VEGF-165 (Peprotech, catalog #100-20). On day 2, 6uM SB431542 (Selleckchem, catalog #101762-616) was added to the culture. On day 4, the cultures underwent a media change with StemPro-34 (Gibco, catalog #10639011), IX Penicillin-Streptomycin (Gibco catalog #15140122), 2 mL Glutamax (Gibco, catalog #35050061), 50 ug / ml ascorbic acid (Santa Cruz Biotechnology, catalog #sc-39430), 4 mM 1-thioglycerol (Sigma-Aldrich, catalog #M1753), IX Insulin-Transferrin-Selenium (ITS-G) (Gibco, catalog #41400045), 50 ng / ml FGF-2 (Peprotech, catalog #3718-FB), 50 ng / ml VEGF-165 (Peprotech, catalog #100-20), and 50 ng / ml SCF (Peprotech, catalog #300-07).

[0182] Flow cytometry

[0183] On days 3 and 5, cultures were analyzed by flow cytometry on a Penteon (Agilent, catalog #2010284AA). Embryoid bodies were harvested into 96 deep well plates (Nest, catalog #503162) and incubated at 37°C in 500 ml of TrypLE TM Express enzyme (Gibco, catalog #12605028) for 20-30 minutes, followed by mechanical disruption by pipetting. TMThe Express enzyme was quenched with DMEM-F12 (Stemcell Technologies, catalog #36254). The cells were incubated with Zombie Aqua TM Fixable (Biolegend, catalog #423102, 1:2000) was stained for 20 min, washed with 1X DPBS, 2% FBS, 0.02% NaN3, and blocked with human IgG (Lee Biosolutions catalog #340-21) for 10 min on ice. Cells were stained on ice for 30 minutes with PDGFR-BV421 BD Bioscienes catalog #562799, 1:400), CD73-BV785 (Biolegend, catalog #344028, 1:400), CD144 / VE-Cadherin-PE (Biolegend, catalog #348505, 1:100), CD34-PE-Dazz (Biolegend, catalog #343534, 1:400), CD309-PE-Cy7 (Biolegend, catalog #359912, 1:100), CD43-APC (Biolegend, catalog #343206, 1:800), CD45-AF700 (Biolegend, catalog #304024, 1:200). Cells were washed with 1X DPBS, 2% FBS, 0.02% NaN3. Cells were resuspended in 1X DPBS, 2% FBS, 0.02% NaN3 for analysis. Data were analyzed using FlowJo TM Analysis was performed using BD Biosciences v10.8 software (BD Life Sciences). Dead cells and doublets were excluded from the analysis using Zombie Aqua and doublet discrimination gates.

[0184] Aggregate flow cytometry data to form Figures 10A-10D , which depicts the relative frequency of cells among appropriately sized, viable, singlet cells at day 5 of culture. Scatter plots were made at different time points to compare the magnitude of increased hematopoietic gene expression induced by HGC relative to the control ( Fig. 9 ) visualization.

[0185] Example results of flow cytometry analysis are shown in Figure 11A-11B , which depicts the frequency of cells expressing the indicated surface markers in live singlet cells at day 5 of culture compared to no DNA controls for HSC (Cell Algorithm) #7 and #12, which underwent nucleofection in the absence of exogenous DNA. A summary graph is shown in Figure 11C-Figure 11D Shown in.

[0186] Implementation

[0187] The following non-limiting embodiments provide illustrative examples of the invention but do not limit the scope of the invention.

[0188] Embodiment 1. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising:

[0189] contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises:

[0190] (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and

[0191] (ii) a second gating unit, which is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the first different regulation and the second different regulation both enhance or both reduce the expression and / or activity level of the target gene in the cell,

[0192] wherein, upon said contacting, said plurality of gate units operate in concert to achieve said desired expression and / or activity profile of said target gene in said cell,

[0193] Optionally among them:

[0194] (1) The method further comprises contacting the cell with an activation moiety to activate the heterologous gene circuit; and / or

[0195] (2)(i) the first gate unit comprises a first gene regulatory portion, the first gene regulatory portion is activated upon activation of the first gate unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and

[0196] (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene; and / or

[0197] (3) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a substantially identical polynucleotide sequence of the target gene; and / or

[0198] (4) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene; and / or

[0199] (5) the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion,

[0200] Optionally among them:

[0201] (i) the first gate unit further comprises a first gate portion, which is activated upon activation of the first gate unit to induce: (a) activation of the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activation of the second gate portion via specific binding of the first gate portion to the second gate portion; and / or

[0202] (ii) the activation portion is capable of inducing: (a) activation of the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) activation of the second gating portion; and / or

[0203] (6) both the first differential regulation and the second differential regulation enhance the expression and / or activity level of the target gene; and / or

[0204] (7) both the first differential regulation and the second differential regulation reduce the expression and / or activity level of the target gene; and / or

[0205] (8) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gating unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or

[0206] (9) The second gating unit can be activated to regulate the expression and / or activity profile of another target gene, and wherein the first gating unit is not configured to regulate the expression and / or activity profile of the additional target gene.

[0207] Embodiment 2. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising:

[0208] contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises:

[0209] (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and

[0210] (ii) a second gating unit that is activatable to induce disruption of the already activated first gating unit, wherein inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the second different regulation attenuates the first different regulation,

[0211] wherein, upon said contacting, said plurality of gate units operate in concert to achieve said desired expression and / or activity profile of said target gene in said cell,

[0212] Optionally among them:

[0213] (1) The method further comprises contacting the cell with an activation moiety to activate the heterologous gene circuit; and / or

[0214] (2)(i) the first gate unit comprises a first gene regulatory portion, the first gene regulatory portion is activated upon activation of the first gate unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and

[0215] (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene; and / or

[0216] (3) the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion,

[0217] Optionally among them:

[0218] (i) the first gating unit further comprises a first gating portion, the first gating portion being activated upon activation of the first gating unit to induce: (a) activation of the first gene regulatory portion via specific binding of the first gating portion to the first gene regulatory portion, and (b) activation of the second gating portion via specific binding of the first gating portion to the second gating portion,

[0219] Further optionally wherein:

[0220] (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces disruption of the first gate unit via disruption of the first gate portion and / or the first gene regulatory portion; and / or

[0221] (B) the disruption comprises modifying a polynucleotide sequence encoding the first gate portion and / or the first gene regulatory portion; and / or

[0222] (ii) the activation portion is capable of inducing: (a) activation of the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) activation of the second gating portion,

[0223] Further optionally wherein:

[0224] (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces disruption of the first gate unit via disruption of the first gene regulatory portion; and / or

[0225] (B) the disruption comprises modifying a polynucleotide sequence encoding the regulatory portion of the first gene; and / or

[0226] (4) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gating unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or

[0227] (5) the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene; and / or

[0228] (6) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a substantially identical polynucleotide sequence of the target gene; and / or

[0229] (7) The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene.

[0230] Embodiment 3. The method of embodiment 1 or embodiment 2, further optionally wherein:

[0231] (1) the target gene comprises multiple target genes; and / or

[0232] (2) the second different regulation induces the selective regulation of one of the multiple target genes but does not induce the selective regulation of another of the multiple target genes; and / or

[0233] (3) the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous gene circuit to thereby activate the heterologous gene circuit; and / or

[0234] (4) each of the first gate unit, the first gate part, the first gene regulatory part, the second gate unit, the second gate part and / or the second gene regulatory part comprises an activatable gNA,

[0235] wherein upon activation of the gNA, the gNA forms a complex with an endonuclease; and / or

[0236] (5) (i) the activatable gNA comprises a self-cleaving gNA, and / or (ii) the activatable gNA comprises a non-canonical termination sequence; and / or

[0237] (6) The nuclease comprises a CRISPR / Cas protein,

[0238] Optionally wherein the CRISPR / Cas protein is operably coupled to a gene activator or a gene repressor; and / or

[0239] (7) inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation; and / or

[0240] (8) The activation part consists of a single activation part; and / or

[0241] (9) upon activation of the second gating unit, the second gating unit is further capable of regulating the expression and / or activity of an additional target gene in the cell; and / or

[0242] (10) the second gating unit comprises an additional gene regulatory portion, which is activated upon activation of the second gating unit to induce regulation of the expression and / or activity of the additional target gene via specific binding of the additional gene regulatory portion to the additional target gene; and / or

[0243] (11) the target gene is endogenous to the cell; and / or

[0244] (12) the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type; and / or

[0245] (13) the target cell type exhibits a target phenotype; and / or

[0246] (14) the target cell type is a tissue-specific cell, optionally wherein the tissue-specific cell is selected from immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells and epithelial cells; and / or

[0247] (15) the target phenotype includes (i) expression or activity level of a target protein, (ii) average cell size, and / or (iii) a desired function; and / or

[0248] (16) The target gene encodes a cell differentiation regulatory factor; and / or

[0249] (17) The cell differentiation regulatory factor comprises a growth factor; and / or

[0250] (18) The cell differentiation regulatory factor comprises a transcription factor; and / or

[0251] (19) The cell differentiation regulatory factors include immune cell regulatory factors, and the immune cell regulatory factors include E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3 and Bcl11b; and / or

[0252] (20) The cell differentiation regulatory factors include T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), homeobox proteins, forkhead boxes (FOX), SRY-related HMG boxes (SOX) and / or GATA proteins; and / or

[0253] (20) The cell is a stem cell selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells and epithelial stem cells.

[0254] Embodiment 4. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising:

[0255] A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises:

[0256] (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and

[0257] (ii) a second gating unit, which is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the first different regulation and the second different regulation both enhance or both reduce the expression and / or activity level of the target gene in the cell,

[0258] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell,

[0259] Optionally among them:

[0260] (1) the system further comprises an activation part for activating the heterologous gene circuit; and / or

[0261] (2)(i) the first gate unit comprises a first gene regulatory portion, the first gene regulatory portion is activated upon activation of the first gate unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and

[0262] (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene; and / or

[0263] (3) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a substantially identical polynucleotide sequence of the target gene; and / or

[0264] (4) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene; and / or

[0265] (5) the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion,

[0266] Optionally among them:

[0267] (i) the first gate unit further comprises a first gate portion, which is activated upon activation of the first gate unit to induce: (a) activation of the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activation of the second gate portion via specific binding of the first gate portion to the second gate portion; and / or

[0268] (ii) the activation portion is capable of inducing: (a) activation of the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) activation of the second gating portion; and / or

[0269] (6) both the first differential regulation and the second differential regulation enhance the expression and / or activity level of the target gene; and / or

[0270] (7) both the first differential regulation and the second differential regulation reduce the expression and / or activity level of the target gene; and / or

[0271] (8) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gating unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or

[0272] (9) The second gating unit can be activated to regulate the expression and / or activity profile of another target gene, and wherein the first gating unit is not configured to regulate the expression and / or activity profile of the additional target gene.

[0273] Embodiment 5. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising:

[0274] A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises:

[0275] (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and

[0276] (ii) a second gating unit that is activatable to induce disruption of the already activated first gating unit, wherein inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the second different regulation attenuates the first different regulation,

[0277] wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in concert to achieve the desired expression and / or activity profile of the target gene in the cell,

[0278] Optionally among them:

[0279] (1) the system further comprises an activation part for activating the heterologous gene circuit; and / or

[0280] (2)(i) the first gate unit comprises a first gene regulatory portion, the first gene regulatory portion is activated upon activation of the first gate unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and

[0281] (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene; and / or

[0282] (3) the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion,

[0283] Optionally among them:

[0284] (i) the first gating unit further comprises a first gating portion, the first gating portion being activated upon activation of the first gating unit to induce: (a) activation of the first gene regulatory portion via specific binding of the first gating portion to the first gene regulatory portion, and (b) activation of the second gating portion via specific binding of the first gating portion to the second gating portion,

[0285] Further optionally wherein:

[0286] (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces disruption of the first gate unit via disruption of the first gate portion and / or the first gene regulatory portion; and / or

[0287] (B) the disruption comprises modifying a polynucleotide sequence encoding the first gate portion and / or the first gene regulatory portion; and / or

[0288] (ii) the activation portion is capable of inducing: (a) activation of the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) activation of the second gating portion,

[0289] Further optionally wherein:

[0290] (A) upon activation of the second gene regulatory portion, the second gene regulatory portion induces disruption of the first gate unit via disruption of the first gene regulatory portion; and / or

[0291] (B) the disruption comprises modifying a polynucleotide sequence encoding the regulatory portion of the first gene; and / or

[0292] (4) the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gating unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene; and / or

[0293] (5) the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene; and / or

[0294] (6) the first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a substantially identical polynucleotide sequence of the target gene; and / or

[0295] (7) The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene.

[0296] Embodiment 6. The system of embodiment 4 or embodiment 5, further optionally wherein:

[0297] (1) the target gene comprises multiple target genes; and / or

[0298] (2) the second different regulation induces the selective regulation of one of the multiple target genes but does not induce the selective regulation of another of the multiple target genes; and / or

[0299] (3) the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous gene circuit to thereby activate the heterologous gene circuit; and / or

[0300] (4) each of the first gate unit, the first gate part, the first gene regulatory part, the second gate unit, the second gate part and / or the second gene regulatory part comprises an activatable gNA,

[0301] wherein upon activation of the gNA, the gNA forms a complex with an endonuclease; and / or

[0302] (5) (i) the activatable gNA comprises a self-cleaving gNA, and / or (ii) the activatable gNA comprises a non-canonical termination sequence; and / or

[0303] (6) The nuclease comprises a CRISPR / Cas protein,

[0304] Optionally wherein the CRISPR / Cas protein is operably coupled to a gene activator or a gene repressor; and / or

[0305] (7) inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation; and / or

[0306] (8) The activation part consists of a single activation part; and / or

[0307] (9) upon activation of the second gating unit, the second gating unit is further capable of regulating the expression and / or activity of an additional target gene in the cell; and / or

[0308] (10) the second gating unit comprises an additional gene regulatory portion, which is activated upon activation of the second gating unit to induce regulation of the expression and / or activity of the additional target gene via specific binding of the additional gene regulatory portion to the additional target gene; and / or

[0309] (11) the target gene is endogenous to the cell; and / or

[0310] (12) the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type; and / or

[0311] (13) the target cell type exhibits a target phenotype; and / or

[0312] (14) the target cell type is a tissue-specific cell, optionally wherein the tissue-specific cell is selected from immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells and epithelial cells; and / or

[0313] (15) the target phenotype includes (i) expression or activity level of a target protein, (ii) average cell size, and / or (iii) a desired function; and / or

[0314] (16) The target gene encodes a cell differentiation regulatory factor; and / or

[0315] (17) The cell differentiation regulatory factor comprises a growth factor; and / or

[0316] (18) The cell differentiation regulatory factor comprises a transcription factor; and / or

[0317] (19) The cell differentiation regulatory factors include immune cell regulatory factors, and the immune cell regulatory factors include E2A, Ikaros, Runx1, Lmo2, PU.1, Hes1, Tcf7, Gata3 and Bcl11b; and / or

[0318] (20) The cell differentiation regulatory factors include T-box transcription factors (TBX), basic helix-loop-helix transcription factors (bHLH), homeobox proteins, forkhead boxes (FOX), SRY-related HMG boxes (SOX) and / or GATA proteins; and / or

[0319] (20) The cell is a stem cell selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), neural stem cells and epithelial stem cells.

[0320] Embodiment 7. An engineered cell comprising the system of any one of embodiments 4-6.

[0321] Embodiment 8. A composition comprising the system of any one of embodiments 4-6 or the engineered cell of embodiment 7,

[0322] Optionally among them:

[0323] (1) The composition further comprises a separate therapeutic agent,

[0324] Further optionally wherein the single therapeutic agent comprises a chemotherapeutic agent, an immunosuppressant agent and / or an antibiotic agent; and / or

[0325] (2) the composition further comprises the activation part; and / or

[0326] (3) The composition comprises the engineered cells, and wherein the composition is substantially free of the activation moiety.

[0327] The systems and methods of the present disclosure may be combined with or modified by other systems and methods, such as those described in International Patent Application No. PCT / US2018 / 052211, which is incorporated herein by reference in its entirety.

[0328] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific embodiments provided in this specification. Although the present invention has been described with reference to the above specific description, the description and explanation of the embodiments herein are not intended to be interpreted in a restrictive sense. Without departing from the present invention, those skilled in the art will now think of various variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be used to practice the present invention. Therefore, it is envisioned that the present invention should also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises: (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gating unit, which is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the first different regulation and the second different regulation both enhance or both reduce the expression and / or activity level of the target gene in the cell, Wherein, upon said contacting, said plurality of gate units operate in coordination to achieve said desired expression and / or activity profile of said target gene in said cell.

2. The method of claim 1, further comprising contacting the cell with an activating moiety to activate the heterologous gene circuit.

3. The method of claim 2, wherein the activation portion consists of a single activation portion.

4. The method of claim 2, wherein the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous genetic circuit to thereby activate the heterologous genetic circuit.

5. The method of claim 1, wherein both the first differential regulation and the second differential regulation enhance the expression and / or activity level of the target gene.

6. The method of claim 1, wherein both the first differential regulation and the second differential regulation reduce the expression and / or activity level of the target gene.

7. The method of claim 1, wherein the first gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gating unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene.

8. The method of claim 1, wherein the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

9. The method of claim 1, wherein the target gene comprises a plurality of target genes.

10. The method of claim 1, wherein inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation.

11. The method of claim 1, wherein the target gene is endogenous to the cell.

12. The method of claim 1, wherein the target gene encodes a cell differentiation regulatory factor.

13. The method of claim 12, wherein the cell differentiation regulatory factor comprises a transcription factor or a growth factor.

14. The method of claim 1, wherein the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type.

15. The method of claim 1, wherein the cells are stem cells.

16. The method of claim 15, wherein the stem cell is selected from the group consisting of induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells and epithelial stem cells.

17. The method of claim 1, wherein: (i) the first gating unit comprises a first gene regulatory portion, which is activated upon activation of the first gating unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene.

18. The method of claim 17, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to a substantially identical polynucleotide sequence of the target gene.

19. The method of claim 17, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene.

20. The method of claim 17, wherein the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion.

21. The method of claim 20, wherein the first door unit further comprises a first door portion, the first door portion being activated upon activation of the first door unit to induce: (a) activating the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activating the second gate portion via specific binding of the first gate portion to the second gate portion.

22. The method of claim 20, wherein the activating moiety is capable of inducing: (a) activating the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) Activating the second door portion.

23. The method of any one of the preceding claims, wherein each of the first gate unit, the first gate part, the first gene regulatory part, the second gate, the second gate part and / or the second gene regulatory part comprises an activatable gNA, in, Upon activation of the gNA, the gNA forms a complex with the endonuclease.

24. The method of claim 23, wherein the activatable gNA comprises a non-canonical stop sequence.

25. The method of any one of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein.

26. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises: (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gating unit, which is activatable upon activation of the heterologous gene circuit to induce a second different regulation among the plurality of different regulation, wherein the second different regulation is induced after the first different regulation, such that the first different regulation and the second different regulation both enhance or both reduce the expression and / or activity level of the target gene in the cell, Wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in coordination to achieve the desired expression and / or activity profile of the target gene in the cell.

27. The system of claim 26, further comprising an activation portion for activating the heterologous gene circuit.

28. The system of claim 27, wherein the activation portion consists of a single activation portion.

29. The system of claim 27, wherein the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous genetic circuit to thereby activate the heterologous genetic circuit.

30. The system of claim 27, wherein both the first differential regulation and the second differential regulation enhance the expression and / or activity level of the target gene.

31. The system of claim 27, wherein both the first differential regulation and the second differential regulation reduce the expression and / or activity level of the target gene.

32. The system of claim 27, wherein the first gate unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gate unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene.

33. The system of claim 27, wherein the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

34. The system of claim 27, wherein the target gene comprises a plurality of target genes.

35. The system of claim 27, wherein inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation.

36. The system of claim 27, wherein the target gene is endogenous to the cell.

37. The system of claim 27, wherein the target gene encodes a cell differentiation regulator.

38. The system of claim 37, wherein the cell differentiation regulatory factor comprises a transcription factor or a growth factor.

39. The system of claim 27, wherein the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type.

40. The system of claim 27, wherein the cells are stem cells.

41. The system of claim 40, wherein the stem cell is selected from the group consisting of induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells, and epithelial stem cells.

42. The system of claim 27, wherein: (i) the first gating unit comprises a first gene regulatory portion, which is activated upon activation of the first gating unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene.

43. The system of claim 42, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to a substantially identical polynucleotide sequence of the target gene.

44. The system of claim 42, wherein the first gene regulatory portion and the second gene regulatory portion exhibit complementarity to different polynucleotide sequences of the target gene.

45. The system of claim 42, wherein the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion.

46. ​​The system of claim 45, wherein the first door unit further comprises a first door portion, the first door portion being activated upon activation of the first door unit to induce: (a) activating the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activating the second gate portion via specific binding of the first gate portion to the second gate portion.

47. The system of claim 45, wherein the activating moiety is capable of inducing: (a) activating the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) Activating the second door portion.

48. The system of any of the preceding claims, wherein each of the first gate unit, the first gate part, the first gene regulatory part, the second gate, the second gate part and / or the second gene regulatory part comprises an activatable gNA, in, Upon activation of the gNA, the gNA forms a complex with the endonuclease.

49. The system of claim 48, wherein the activatable gNA comprises a non-canonical stop sequence.

50. The system of any of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein.

51. A method for inducing a desired expression and / or activity profile of a target gene in a cell, the method comprising: contacting the cell with a heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises: (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gating unit activatable to induce disruption of the already activated first gating unit, wherein the inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation such that the second different regulation attenuates the first different regulation, Wherein, upon said contacting, said plurality of gate units operate in coordination to achieve said desired expression and / or activity profile of said target gene in said cell.

52. The method of claim 51, further comprising contacting the cell with an activating moiety to activate the heterologous gene circuit.

53. The method of claim 52, wherein the activation moiety consists of a single activation moiety.

54. The method of claim 52, wherein the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous genetic circuit to thereby activate the heterologous genetic circuit.

55. The method of claim 51, wherein the first differential modulation comprises increasing the expression and / or activity level of the target gene, and the second modulation comprises decreasing the expression and / or activity level of the target gene.

56. The method of claim 51, wherein the first differential modulation comprises reducing the expression and / or activity level of the target gene, and the second modulation comprises increasing the expression and / or activity level of the target gene.

57. A method as claimed in claim 51, wherein the first gate unit is activatable to regulate the expression and / or activity profile of an additional target gene, and wherein the second gate unit is activatable to selectively induce the second different regulation of the target gene without regulating the expression and / or activity profile of the additional target gene.

58. The method of claim 51, wherein the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

59. The method of claim 51, wherein the second different modulation is configured to reverse at least about 30%, at least about 60%, at least about 90%, or substantially all of the first different modulation.

60. The method of claim 51, wherein inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation.

61. The method of claim 51, wherein the target gene is endogenous to the cell.

62. The method of claim 51, wherein the target gene encodes a cell differentiation regulator.

63. The method of claim 62, wherein the cell differentiation regulatory factor comprises a transcription factor or a growth factor.

64. The method of claim 51, wherein the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type.

65. The method of claim 51, wherein the cell is a stem cell.

66. The method of claim 65, wherein the stem cell is selected from the group consisting of induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells, and epithelial stem cells.

67. The method of claim 51, wherein: (i) the first gating unit comprises a first gene regulatory portion, which is activated upon activation of the first gating unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene.

68. The method of claim 67, wherein the second gate unit further comprises a second gate portion, which is activated upon activation of the second unit to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion.

69. The method of claim 68, wherein the first door unit further comprises a first door portion, the first door portion being activated upon activation of the first door unit to induce: (a) activating the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activating the second gate portion via specific binding of the first gate portion to the second gate portion.

70. The method of claim 69, wherein the activating moiety is capable of inducing: (a) activating the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) Activating the second door portion.

71. The method of any one of the preceding claims, wherein: Upon activation of the second gene regulatory part, the second gene regulatory part induces disruption of the first gate unit via disruption of the first gate part and / or the first gene regulatory part.

72. The method of any of the preceding claims, wherein the disruption comprises modifying a polynucleotide sequence encoding the first gate portion and / or the first gene regulatory portion.

73. The method of any of the preceding claims, wherein each of the first gate unit, the first gate part, the first gene regulatory part, the second gate, the second gate part and / or the second gene regulatory part comprises an activatable gNA, in, Upon activation of the gNA, the gNA forms a complex with the endonuclease.

74. The method of claim 73, wherein the activatable gNA comprises a non-canonical stop sequence.

75. The method of any of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein.

76. A system for inducing a desired expression and / or activity profile of a target gene in a cell, the system comprising: A heterologous genetic circuit comprising a plurality of gating units, wherein upon activation of the heterologous genetic circuit, the plurality of gating units operate in concert to induce a plurality of different modulations of the target gene in a sequential manner, each of the plurality of different modulations being necessary but individually insufficient to achieve the desired expression and / or activity profile of the target gene, wherein the plurality of gating units comprises: (i) a first gating unit that is activatable upon activation of the heterologous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gating unit activatable to induce disruption of the already activated first gating unit, wherein the inactivation induces a second different regulation of the plurality of different regulation, wherein the second different regulation is induced after the first different regulation such that the second different regulation attenuates the first different regulation, Wherein, upon activation of the heterologous gene circuit, the plurality of gate units operate in coordination to achieve the desired expression and / or activity profile of the target gene in the cell.

77. The system of claim 76, further comprising an activation portion for activating the heterologous gene circuit.

78. The system of claim 77, wherein the activation portion consists of a single activation portion.

79. The system of claim 77, wherein the activation moiety comprises a guide nucleic acid (gNA) capable of forming a complex with an endonuclease, wherein the gNA is capable of binding to at least a portion of the heterologous genetic circuit to thereby activate the heterologous genetic circuit.

80. The system of claim 76, wherein the first differential modulation comprises increasing the expression and / or activity level of the target gene, and the second modulation comprises decreasing the expression and / or activity level of the target gene.

81. The system of claim 76, wherein the first differential modulation comprises decreasing the expression and / or activity level of the target gene, and the second modulation comprises increasing the expression and / or activity level of the target gene.

82. The system of claim 76, wherein the first gate unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the second gate unit is activatable to selectively induce the second different regulation of the target gene without modulating the expression and / or activity profile of the additional target gene.

83. The system of claim 76, wherein the second gating unit is activatable to modulate the expression and / or activity profile of an additional target gene, and wherein the first gating unit is not configured to modulate the expression and / or activity profile of the additional target gene.

84. The system of claim 76, wherein the second different modulation is configured to reverse at least about 30%, at least about 60%, at least about 90%, or substantially all of the first different modulation.

85. The system of claim 76, wherein inducing the second different regulation is configured to occur at least about 5 minutes, at least about 1 hour, at least about 6 hours, or at least about 12 hours after inducing the first different regulation.

86. The system of claim 76, wherein the target gene is endogenous to the cell.

87. The system of claim 76, wherein the target gene encodes a cell differentiation regulator.

88. The system of claim 87, wherein the cell differentiation regulatory factor comprises a transcription factor or a growth factor.

89. The system of claim 76, wherein the desired expression and / or activity profile of the target gene induces differentiation of the cell toward a target cell type.

90. The system of claim 76, wherein the cells are stem cells.

91. The system of claim 90, wherein the stem cell is selected from the group consisting of induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells, and epithelial stem cells.

92. The system of claim 76, wherein: (i) the first gating unit comprises a first gene regulatory portion, which is activated upon activation of the first gating unit to induce the first different regulation via specific binding of the first gene regulatory portion to the target gene, and (ii) the second gating unit comprises a second gene regulatory portion, which is activated upon activation of the second gating unit to induce the second different regulation via specific binding of the first gene regulatory portion to the target gene.

93. The system of claim 92, wherein the second gate unit further comprises a second gate portion, which is activated when the second unit is activated to induce activation of the second gene regulatory portion via specific binding of the second gate portion to the second gene regulatory portion.

94. The system of claim 93, wherein the first door unit further comprises a first door portion, the first door portion being activated upon activation of the first door unit to induce: (a) activating the first gene regulatory portion via specific binding of the first gate portion to the first gene regulatory portion, and (b) activating the second gate portion via specific binding of the first gate portion to the second gate portion.

95. The system of claim 94, wherein the activating moiety is capable of inducing: (a) activating the first gene regulatory portion via specific binding of the activation portion to the first gene regulatory portion, and (b) Activating the second door portion.

96. A system as claimed in any preceding claim, wherein: Upon activation of the second gene regulatory part, the second gene regulatory part induces disruption of the first gate unit via disruption of the first gate part and / or the first gene regulatory part.

97. The system of any of the preceding claims, wherein the disruption comprises modifying a polynucleotide sequence encoding the first gate portion and / or the first gene regulatory portion.

98. The system of any of the preceding claims, wherein each of the first gate unit, the first gate portion, the first gene regulatory portion, the second gate, the second gate portion and / or the second gene regulatory portion comprises an activatable gNA, in, Upon activation of the gNA, the gNA forms a complex with the endonuclease.

99. The system of claim 98, wherein the activatable gNA comprises a non-canonical stop sequence.

100. The system of any of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein.

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