Systems for programming cells to hematopoietic lineage and methods thereof

Abstract

Provided herein are systems that modulate gene expression, methods of use thereof, and cells, such as hematopoietic cells, engineered therefrom for the purpose of differentiating the cells.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 446,228, filed February 16, 2023, and U.S. Provisional Patent Application No. 63 / 620,361, filed January 12, 2024, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] Heterogeneous proteins and / or nucleic acid molecules can be used to elicit desired responses in cells. They can also modulate genes of interest (e.g., transgenic and / or endogenous genes) to program cells (e.g., differentiation, dedifferentiation). In some cases, endonuclease-based technologies (e.g., clustered regularly spaced 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, characterized by its versatility and readily available programmability, can be used to facilitate genome editing across different species. Summary of the Invention

[0003] This disclosure provides a method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: culturing the first plurality of cells in vitro in a culture medium substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL), wherein, within approximately 14 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%, wherein the second plurality of cells are CD45+.

[0004] This disclosure provides a method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: contacting the first plurality of cells with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially regulate the expression levels of a plurality of different target genes, wherein the plurality of gate units comprises: a first gate unit which is activated upon activation of the heterologous gene circuit to regulate the expression of the target genes; and a second gate unit which is activated upon activation of the first gate unit to regulate the expression of an additional target gene different from the target genes, such that, following regulation of the expression of the target genes, regulation of the expression of the additional target genes is induced, wherein the target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof, and wherein, upon contact, the plurality of gate units cooperate to achieve the conversion of the first plurality of cells into the second plurality of cells.

[0005] This disclosure provides a method for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), the method comprising: contacting the first plurality of cells with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially regulate the expression levels of multiple different target genes, wherein the plurality of gate units comprises: a first gate unit, which is activated upon activation of the heterologous gene circuit to regulate the expression of the target genes; and a second gate unit, which is activated upon activation of the first gate unit to regulate the expression of additional target genes different from the target genes, such that regulation of the expression of the additional target genes is induced following regulation of the expression of the target genes. The heterologous gene circuit is programmed such that: (x) the target gene includes one or more members selected from TBXT, TBX6, MIXL1 and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof; and / or (y) the target gene includes one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2 and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof, wherein, after the contact, the plurality of gate units cooperate to achieve the transition from the first plurality of cells to the second plurality of cells.

[0006] This disclosure provides a method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: contacting a target gene in the first plurality of cells with a heterologous gene regulatory portion to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, wherein the contact enables the conversion of the first plurality of cells into the second plurality of cells.

[0007] This disclosure provides a method for guiding the differentiation of multiple stem cells, the method comprising: contacting the multiple stem cells with a heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units cooperate to sequentially induce multiple distinct regulation of a target gene in the multiple stem cells, each of the multiple distinct regulation being necessary for the guidance of differentiation of the multiple stem cells but insufficient individually to achieve the guidance, wherein the multiple gate units comprise: (i) a first gate unit that is activated upon activation of the heterologous gene circuit to induce a first distinct regulation among the multiple distinct regulation; and (ii) a second gate unit that is activated upon activation of the heterologous gene circuit to induce a second distinct regulation among the multiple distinct regulation, wherein the second distinct regulation is induced after the first distinct regulation such that both the first distinct regulation and the second distinct regulation increase or decrease the expression and / or activity level of the target gene in the cells, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and wherein, upon contact, the multiple gate units cooperate to achieve the guidance of differentiation of the multiple stem cells.

[0008] This disclosure provides a system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the system comprising: a culture medium for in vitro culture of the first plurality of cells, wherein the culture medium is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL), wherein, within approximately 14 days after in vitro culture of the first plurality of cells in the culture medium, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%, wherein the second plurality of cells are CD45+.

[0009] This disclosure provides a system for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), the system comprising: a heterologous gene circuit containing a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units are configured to cooperate in sequentially regulating the expression levels of a plurality of different target genes, wherein the plurality of gate units include: a first gate unit, which is activated upon activation of the heterologous gene circuit to regulate the expression of the target genes; and a second gate unit, which is activated upon activation of the first gate unit to regulate expression of genes different from the target genes. The expression of additional target genes, such that regulation of the expression of said target genes, inducing regulation of the expression of said additional target genes, wherein said target genes include one or more members selected from TBXT, TBX6, MIXL1 and combinations thereof; and said additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2 and combinations thereof, and wherein, after the first plurality of cells are contacted with said heterologous gene circuit, the plurality of gate units cooperate to achieve said transition from the first plurality of cells to the second plurality of cells.

[0010] This disclosure provides a system for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), the system comprising: a heterogeneous gene circuit containing a plurality of gate units, wherein, upon activation of the heterogeneous gene circuit, the plurality of gate units are configured to cooperate in sequentially regulating the expression levels of multiple different target genes, wherein the plurality of gate units include: a first gate unit, which is activated upon activation of the heterogeneous gene circuit to regulate the expression of the target genes; and a second gate unit, which is activated upon activation of the first gate unit to regulate the expression of additional target genes different from the target genes, such that regulation of the expression of the additional target genes is induced after regulation of the expression of the target genes, wherein the heterogeneous gene circuit is The program is configured such that: (x) the target gene includes one or more members selected from TBXT, TBX6, MIXL1 and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof; and / or (y) the target gene includes one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2 and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof, wherein, after the first plurality of cells are contacted with the heterologous gene circuit, the plurality of gate units cooperate to achieve the transformation from the first plurality of cells to the second plurality of cells.

[0011] This disclosure provides a system for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), the system comprising: a heterologous gene regulatory portion exhibiting specific binding to a target gene in the first plurality of cells to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, wherein contact between the target gene and the heterologous gene regulatory portion enables the conversion of the first plurality of cells into the second plurality of cells.

[0012] This disclosure provides a system for guiding the differentiation of multiple stem cells, the system comprising: a heterogeneous gene circuit containing a plurality of gate units, wherein, upon activation of the heterogeneous gene circuit, the plurality of gate units cooperate to sequentially induce a plurality of different regulation of a target gene in the plurality of stem cells, each of the plurality of different regulation being necessary for the guidance of differentiation of the plurality of stem cells but insufficient individually to achieve the guidance, wherein the plurality of gate units include: (i) a first gate unit that is activated upon activation of the heterogeneous gene circuit to induce a first different regulation among the plurality of different regulation; and (ii) a second gate unit that is activated upon activation of the heterogeneous 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 and second different regulation increase or decrease the expression and / or activity level of the target gene in the cells, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and wherein, upon contact of the plurality of stem cells with the heterogeneous gene circuit, the plurality of gate units cooperate to achieve the guidance of differentiation of the plurality of stem cells.

[0013] This disclosure provides engineered cells comprising any of the systems disclosed herein. This disclosure also provides compositions comprising any of the systems disclosed herein and / or any of the engineered cells. Incorporation

[0014] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually identified as incorporated herein by reference. To the extent that any publication, patent, or patent application incorporated herein by reference conflicts with any disclosure contained herein, this specification is intended to supersede and / or give precedence to any such conflicting material. Attached Figure Description

[0015] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as the "Figures") illustrating exemplary embodiments in which the principles of the invention are utilized, in which: Figure 1 This is a schematic diagram of a heterologous gene circuit. Activation of the initiation circuit can also activate the gate unit. The gate unit consists of a gate portion and / or a gene regulatory portion.

[0016] Figure 2 An exemplary heterologous gene circuit is depicted.

[0017] Figure 3 Scatter plots (i.e., 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.

[0018] Figures 4A-4D It shows the method for generating Figure 3 Example of scatter plot analysis data for hematopoietic progenitor cell markers. Figure 4A The cell marker CD34 was shown. + The result. Figure 4B The cell marker CD43 was shown. + The result. Figure 4C The cell marker CD45 was shown. + The result. Figure 4D The cell marker CD34 was shown. + / CD43 - / CD45 + The combined result. The left and right points represent replicate samples for each condition. The center point represents the mean.

[0019] Figures 5A-5D The results showed that, compared with directed differentiation, the heterologous gene circuit generated significantly more hematopoietic progenitor cells after five days of culture. Figure 5A CD34 is shown + and CD45 + Cellular marker results. Figure 5B CD34 is shown + CD43 + and CD45 + Additional cellular marker results for the markers. Figure 5C It shows Figure 5B Data summary. Figure 5D The results from the repeated experiments are summarized.

[0020] Figure 6The developmental cell lineage from pluripotent stem cells (iPSCs) to hematopoietic progenitor cells (HPCs) is depicted. The expression of cell surface markers (e.g., KDR, CD34, CD43, CD45) indicating cell state is shown above the cells. Genes encoding transcription factors are indicated by arrows that indicate the relative order of lineage progression activities. Exemplary heterologous gene circuits using such genes are depicted in the appendix table.

[0021] Figure 7 The design concept for how three or four TF sets should be distributed in a 10-step cascade is described. An interval paradigm is designed to account for future additions to subsequent steps, time-distributed gene activation steps, and to maximize the activation efficiency of heterologous gene circuits of endogenous genes.

[0022] Figures 8A-8D The effects of heterologous gene circuits (CGO###; described in Table 4) and control conditions (described in Table 4) on iPSC differentiation are shown at 7 days after introduction. Flow cytometry data show the effects on cell surface markers (KDR / CD309) indicating early stages of lineage differentiation. Figure 8D A summary of the percentage of KDR+ cells determined from flow cytometry data is shown.

[0023] Figures 9A-9D The effects of the heterologous gene circuit (CGO###; described in Table 4) and control conditions (described in Table 4) on iPSC differentiation are shown at 7 days after introduction. Flow cytometry data show the effects on cell surface markers (CD34) indicating intermediate stages of lineage differentiation. Figure 9D A summary of the percentage of KDR-CD34+ (hematopoietic endothelial) cells determined from flow cytometry data is presented.

[0024] Figures 10A-10D The effects of heterologous gene circuits (CGO###; described in Table 4) and control conditions (described in Table 4) on iPSC differentiation are shown at 7 days after introduction. Flow cytometry data show the effects on cell surface markers (KDR / CD309; CD34) indicating intermediate stages of lineage differentiation. Figure 10D A summary of the percentage of KDR-CD34+ (hematopoietic endothelial) cells determined from flow cytometry data is presented.

[0025] Figures 11A-11D The effects of heterologous gene circuits (CGO###; described in Table 4) and control conditions (described in Table 4) on iPSC differentiation are shown at 7 days after introduction. Flow cytometry data show the effects on cell surface markers (CD43; CD34) indicating HPC stages. Figure 11DA summary of the percentage of double-positive (CD43+CD34+) cells determined from flow cytometry data is presented.

[0026] Figures 12A-12D The effects of heterologous gene circuits (CGO###; described in Table 4) and control conditions (described in Table 4) on iPSC differentiation are shown at 7 days after introduction. Flow cytometry data show the effects on cell surface markers (CD45; CD34) indicating HPC stages. Figure 12D A summary of the percentage of double-positive (CD45+CD34+) cells determined from flow cytometry data is presented.

[0027] Figures 13A-13D This shows the results of the 7-day differentiation procedure (in Figures 9A-11D The total cell yield (described in Table 4) was calculated as follows: 3000 cells were seeded into an expansion medium containing DLL4 and rich in growth factors, 6 days later. The total cell number yield was shown as a result of the analysis of total CD45 in pan-hematopoiesis. Figure 13A HPC (CD34+CD45+); Figure 13B ), pedigree typing (CD34- CD45+; Figure 13C ) and lymphoid (CD7; Figure 13D Cell type.

[0028] Figures 14A-14B The effects of the heterologous gene circuit (CGO###; described in Table 4) compared to the baseline protocol targeting CD34+CD45+CD43+ cells are shown. Figure 14A CGO178 using iPSC and transient plasmids is shown on day 7. Figure 14B The baseline protocol using transgenic iPSC cell lines at day 15 is shown. Detailed Implementation

[0029] Although various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0030] Unless the context clearly requires otherwise, the singular forms “a,” “an,” and “the” as used in this specification and claims include plural references. For example, the term “gate cell” includes multiple gate cells.

[0031] The terms “about” or “approximately” generally mean within an acceptable range of error for a particular value, as determined by a person skilled 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 practice in the art, “about” may mean within or greater than one 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 this application and claims, unless otherwise stated, the term “about” should be assumed to mean within an acceptable range of error for that particular value.

[0032] The use of alternatives (e.g., "or") should be understood to mean any one, both, or any combination of the alternatives. The term "and / or" should be understood to mean any one or both of the alternatives.

[0033] definition As used interchangeably herein, the terms “genetic circuit,” “biological circuit,” or “circuit” generally refer to a collection of molecular components (e.g., biological materials such as peptides and / or polynucleotides, non-biological materials, etc.) that are operatively coupled (e.g., simultaneously, sequentially, etc.) according to circuit design. The collection of molecular components may be able to provide one or more specific outputs in the 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 may be sufficient to trigger the molecular components of the genetic circuit to provide one or more specific outputs. For example, a genetic circuit may contain one or more molecular switches that can be activated by one or more inputs (…). Figure 1 ).

[0034] A gene circuit can be a controllable gene expression system comprising the assembly of biological parts that work together as logical functions (e.g., simultaneously, sequentially, etc.). A gene circuit can contain multiple gate units, wherein at least one of the gate units can be activated by an activation component (e.g., heterologous input to the cell) to activate other gate units (e.g., simultaneously at once, sequentially in a cascaded manner, etc.). Figure 1For example, at least one of a plurality of phylum members may be activated by another phylum member (e.g., directly or indirectly) to (i) regulate the expression or activity levels of one or more target genes, (ii) activate at least one of the other phylum members, and / or (ii) deactivate at least one of the other phylum members, thereby jointly regulating the expression and / or activity levels of one or more target genes in a desired manner, as predetermined by the design of the gene circuit. Figure 1 The terms “heterogeneous gene circuit”, “HGC”, “cellular algorithm”, or “cellgorithm” used in this article are interchangeable.

[0035] As mentioned herein, the term "gate unit" generally refers to a portion of a gene circuit that controls gene regulation by functioning similarly to a logic gate, where it controls the flow of information and allows the circuit to make multiplexed decisions at different points. More specifically, the term refers to nucleic acids encoding genetic switches and transcriptional / translational regulatory regions, or a series of regions on which genetic switches act. The input to a gate unit can be an activating part and / or another gate unit. The output of a gate unit can activate another gate unit, deactivate another gate unit, affect a target gene, and / or any combination thereof. For example, a gate unit can consist of multiple gate parts and / or multiple gene regulatory parts (…). Figure 1 )composition.

[0036] As mentioned herein, the term "activation part" generally refers to a portion that can activate multiple gene circuits and / or multiple gate units. The activation part can be a heterologous input to the cell. In some cases, the activation part can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acids, peptides, polynucleotides, small molecules, light, or combinations thereof. For example, the activation part can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., Cas protein) to bind to a polynucleotide sequence of an inactivated gate part (e.g., a plasmid encoding another guide nucleic acid molecule) to activate such a gate part that can target one or more gene regulatory parts (e.g., induce the expression of another functional form of the guide nucleic acid molecule). As mentioned herein, the term "gate part" generally refers to a portion that can affect the function of a gene regulatory part within a gate unit. A gate part can activate and / or deactivate a gene regulatory part. For example, a gate part can regulate the expression of a gene regulatory part by editing a nucleic acid sequence and thereby activating or deactivating the gene regulatory part. For example, the phylum portion can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a multinucleotide sequence of a gene regulatory portion (e.g., a plasmid encoding another guide nucleic acid molecule) to activate the gene regulatory portion that can target one or more endogenous genes in the cell (e.g., induce the expression of a functional form of another guide nucleic acid molecule). Alternatively or additionally, the phylum portion can activate and / or deactivate another phylum unit of the gene circuit ( Figure 1 For example, the phylum moiety can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an inactivated phylum moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to activate that other phylum moiety (e.g., to induce the expression of a functional form of the other guide nucleic acid molecule). In another example, the phylum moiety can be a guide nucleic acid molecule that forms a complex with a nuclease (e.g., a Cas protein) to bind to a polynucleotide sequence of an activated phylum moiety (e.g., a plasmid encoding another guide nucleic acid molecule) to inactivate that other phylum moiety (e.g., to reduce the expression of a functional form of the other guide nucleic acid molecule).

[0037] As used interchangeably in this article, the terms "gene regulation portion" or "gene editing portion" generally refer to the portion that can regulate the expression and / or activity profile of nucleic acid sequences or proteins (whether exogenous or endogenous to the cell). Figure 1For example, gene editing can regulate gene expression by editing nucleic acid sequences (e.g., CRISPR-Cas, zinc finger nucleases, TALENs, or siRNA). In some cases, gene editing can regulate gene expression by editing genomic DNA sequences. In others, it can regulate gene expression by editing mRNA templates. In still others, editing nucleic acid sequences can alter the underlying template used for gene expression (e.g., CRISPR-Cas-inspired RNA targeting systems). Alternatively, gene editing can suppress gene translation (e.g., Cas13).

[0038] Alternatively or additionally, gene-editing portions can regulate gene expression or activity by specifically binding to a target sequence (or a target sequence within a gene) operatively coupled to the gene, and regulate the production of mRNA from DNA (such as chromosomal DNA or cDNA). For example, a gene-editing portion can recruit or contain at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. The gene-editing portion itself can bind to DNA and regulate transcription by physically hindering it, for example, by preventing the assembly of proteins (such as RNA polymerases and other associated proteins) on a DNA template. Gene-editing portions can regulate gene expression at the translational level, for example, by regulating the production of proteins from an mRNA template. In some cases, gene-editing portions can regulate gene expression by affecting the stability of mRNA transcripts. In some cases, gene-editing portions can regulate genes (e.g., Cas12) through epigenetic editing.

[0039] In some cases, plasmids can encode non-functional forms of gene-editing portions. Plasmids can be activated to express functional forms of the gene-editing portions, for example, via activation of functional gate portions. For instance, a gene-editing portion can encode a non-functional form of a guide nucleic acid molecule that would otherwise be able to bind to a target gene in the cell. When a functional gate portion (e.g., another guide nucleic acid molecule complexed with a Cas protein) is bound to the plasmid, the plasmid can be edited to allow expression of the functional form of the gene-editing portion (e.g., a functional form of a guide nucleic acid molecule that specifically binds to a target gene in the cell), thereby permitting the regulation of the target gene in the cell.

[0040] Therefore, the gene regulatory portion may contain 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 regulatory portion may contain or be operatively coupled to an endonuclease. An endonuclease may be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. Endonucleases may be restriction endonucleases that cleave DNA at specific sites without damaging bases. Restriction endonucleases may include endonucleases type I, II, III, and IV, which may further include subtypes. In some cases, the endonuclease can be Cas1, Cas2, Cas3, 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), Cas13 (C2c2), Cas13b, Cas13c, Cas13d, Cas13x.1, Cse1, Cse2, Csy1, Csy2, Csy3, Csm2, Cmr5, Csx10, Csx11, Csf1, or Csn2. Endonucleases can be dead endonucleases that exhibit reduced cleavage activity. For example, endonucleases can be nuclease-inactivated Cas, such as dCas (e.g., dCas9).

[0041] The gene regulatory portion can be a transcriptional regulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory portion can be a gene repressor complex comprising a dCas protein operatively coupled to (e.g., fused to) a transcriptional repressor. Non-limiting examples of transcriptional repressors may include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repressor 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 may be a gene activator complex comprising a dCas protein operatively coupled to (e.g., fused to) a transcription activator. Non-restricted examples of transcriptional activators may 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, TET1CD, TET1, DME, DML1, DML2, and ROS1.

[0042] In some cases, the gene regulatory portion possesses enzymatic activity that modifies the target gene without cleaving it. Modification of the target gene can induce epigenetic modifications, such as those that modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory portion include, but are not limited to: nuclease activity (such as that provided by restriction enzymes (e.g., Fokl nucleases); methyltransferase activity (such as that provided by methyltransferases (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)); and demethylase activity (such as that provided by demethylases (e.g., deca-11 translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS). 1) Provided demethylase activity), DNA repair activity, DNA damage activity, deamination activity (such as deamination activity provided by deaminases (e.g., cytosine deaminases such as APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity (such as integrase activity provided by integrase and / or dissociation enzymes (e.g., Gin convertase, such as the hyperactive mutant of Gin convertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 dissociation enzyme, etc.), transposase activity, recombinase activity (such as recombinase activity provided by recombinase (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylation enzyme activity.

[0043] 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, ribonucleotides, or analogs, and whether single-stranded, double-stranded, or multi-stranded. Polynucleotides can be exogenous or endogenous to the cell. Polynucleotides can exist in a cell-free environment. Polynucleotides can be genes or fragments thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides can include one or more analogs (e.g., modified backbones, sugars, or nucleobases). Where modifications are present, the nucleotide structure can be modified before or after polymer assembly. Some non-limiting examples of analogues include: 5-bromouracil, peptide nucleic acids, heteronucleic acids, morpholino derivatives, locked nucleic acids, glycol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-denitro-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogues, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine nucleoside, brassinoside, and woyoside. Non-restrictive examples of polynucleotides include coding or non-coding regions of genes or gene segments, loci (locus) as defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched-chain 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.

[0044] The term "gene" generally refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) that encode RNA transcripts and their corresponding nucleotide sequences. As used herein with respect to genomic DNA, the term includes intercalated non-coding regions as well as regulatory regions and may include 5′ and 3′ ends. In some uses, the term encompasses 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, "gene" contains only the coding sequence necessary to encode a polypeptide (e.g., "open reading frame" or "coding region"). In some cases, genes do not encode polypeptides, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. 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 can refer to an "endogenous gene" or a natural gene located in its native position within an organism's genome. A gene can also refer to an "exogenous gene" or a non-natural gene. Non-natural genes can refer to genes that are not normally found in a host organism but are introduced into the host organism through gene transfer. Non-natural genes can also refer to genes in an organism's genome that are not in their natural location. Furthermore, non-natural genes can refer to naturally occurring nucleic acid or polypeptide sequences that contain mutations, insertions, and / or deletions (e.g., non-natural sequences).

[0045] The term "sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid similarity between two polynucleotide or polypeptide sequences. Techniques used to determine sequence identity typically involve determining the nucleotide sequence of the polynucleotide and / or the amino acid sequence it encodes, and comparing these sequences to the second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percentage of identity." Whether it's a nucleic acid or amino acid sequence, the percentage of identity between two sequences is the exact number of matches between the two sequences being compared divided by the length of the longer sequence and multiplied by 100. The percentage of identity can also be determined, for example, by comparing sequence information using advanced BLAST computer programs, including version 2.2.9 available from the National Institutes of Health. The BLAST procedure is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and is discussed in Altschul et al., J. Mol. Biol., 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993); and Altschul et al., NucleicAcids Res., 25:3389-3402 (1997). This procedure can be used to determine full-length sequence identity of compared proteins. Default parameters are provided to optimize short query sequence searches in the blastp procedure (e.g., using the blastp procedure). The program also allows the use of SEG filters to filter out segments of the query sequence as determined by the SEG procedure of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993). The desired degree of sequence identity ranges from approximately 50% to 100% and integer values ​​in between. Generally, this disclosure covers sequences that have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with any sequence provided herein.

[0046] The term "expression" generally refers to one or more processes of transcription (such as transcription into mRNA or other RNA transcripts) of polynucleotides from a DNA template and / or the subsequent translation of transcribed mRNA into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include mRNA splicing. In terms of expression, "upregulation" generally refers to an increase in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA, such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state. Transfected gene expression can occur transiently or stably in the cell. During "transient expression," the transfected gene does not transfer to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression disappears over time. Conversely, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selective advantage to the transfected cell. Such a selective advantage could be resistance to a certain toxin presented to the cell.

[0047] As used interchangeably herein, the terms “peptide,” “polypeptide,” or “protein” generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not imply a specific length of polymer, nor is it intended to suggest or distinguish whether a peptide is produced using recombinant technology, chemical or enzymatic synthesis, or is naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers containing at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acid chains. 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 covers modified amino acid polymers, for example, through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation 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 can include both natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties not naturally present on the amino acid. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids.

[0048] As used interchangeably herein, with respect to polypeptides, the terms “derivative,” “variant,” or “fragment” generally refer to a polypeptide that is associated with a wild-type polypeptide, for example, by its 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 contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to wild-type polypeptides.

[0049] As used herein, with respect to polypeptide molecules (e.g., proteins), the terms "engineered," "chimeric," or "recombinant" generally refer to polypeptide molecules that have heterologous or altered amino acid sequences due to the application of genetic engineering techniques to the nucleic acids encoding the polypeptide molecule and to the cells or organisms expressing the polypeptide molecule. As used herein, with respect to polynucleotide molecules (e.g., DNA or RNA molecules), the terms "engineered" or "recombinant" generally refer to polynucleotide molecules that have heterologous 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) may be modified or altered by gene editing.

[0050] Unless otherwise specified or apparent from the context, as used herein, the term "nucleotide" generally refers to a combination of a base-sugar-phosphate ester. A nucleotide can include synthetic nucleotides. A nucleotide can include synthetic nucleotide analogs. A nucleotide can be a monomeric unit of a nucleic acid sequence, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term nucleotide can include ribonucleoside triphosphate 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 can include, for example, [αS]dATP, 7-denitro-dGTP, and 7-denitro-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. 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 using known techniques. Labeling may also be performed using quantum dots. Detectable labeling may include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labeling of nucleotides may include, but is 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 may 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 are available 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 are 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 are available from Boehringer Mannheim, Indianapolis, Ind.; and fluorescein-15-2′-dATP are available from Molecular Probes, Eugene, The chromosome-tagged nucleotides obtained by Oreg. include 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 chemically modified for labeling or marking. Chemically modified mononucleotides can be biotin-dNTPs. Some non-limiting examples of biotinylated dNTPs may 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).

[0051] 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. Cells can originate from any organism having one or more cells. Some non-limiting examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaea 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, sugarcane, squash, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, lycophytes, hornwort, liverwort, mosses), algal cells (e.g., *Botrytis cinerea*), and cells from algae (e.g., *Botrytis cinerea*). Botryococcus braunii ), Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii ), Microcystis thuringiensis ( Nannochloropsis gaditana Chlorella proteoglycans () Chlorella pyrenoidosa Sargassum fusiforme ( ) Sargassum patens , C. Agardh Cells can be derived from various organisms, including algae (e.g., kelp), fungal cells (e.g., yeast cells, mushroom cells), animal cells, cells from invertebrates (e.g., fruit flies, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), and cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, cells are not derived from natural organisms (e.g., cells can be synthetically manufactured, sometimes called artificial cells).

[0052] As used interchangeably in this text, the terms “reprogramming,” “dedifferentiation,” “increasing cellular potential,” or “increasing developmental potential” generally refer to methods that increase cellular potential or dedifferentiate cells to a lower differentiation state. For example, cells with increased cellular potential have greater developmental plasticity (i.e., they can differentiate into more cell types) compared to the same cells in a non-reprogrammed state. In other words, reprogrammed cells are at a lower differentiation state than the same cells in a non-reprogrammed state.

[0053] The term "differentiation" generally refers to the process by which undifferentiated ("undifferentiated") 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 reached a more specialized ("differentiated") position within a cell lineage. The term "differentiation" generally refers to cells that, under normal circumstances, will continue to differentiate into a specific cell type or a subset of cell types, and under normal circumstances, cannot differentiate into different cell types or revert to a less differentiated cell type.

[0054] The term "pluripotent" generally refers to the ability of a cell to form all lineages (i.e., embryonic bodies) of a somatic or body cell. For example, embryonic stem cells are a type of pluripotent stem cell that is capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm). Pluripotency can be a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., ectoderm stem cells) (which cannot produce a complete organism) to more primitive, more pluripotent cells (which can produce a complete organism) (e.g., embryonic stem cells).

[0055] The term "induced pluripotent stem cells" (iPSCs) generally refers to stem cells derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or altered (i.e., reprogrammed) to differentiate into tissues capable of differentiating into all three germ layers or dermal layers: mesoderm, endoderm, and ectoderm. The resulting iPSCs do not refer to cells found in nature. In some cases, iPSCs can be engineered to directly differentiate into morphological cells (e.g., natural killer (NK) cells). In other cases, iPSCs can be engineered to first differentiate into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs)), which can then be further induced to differentiate into morphological cells (e.g., NK cells).

[0056] The term "embryonic stem cell" (ESC) generally refers to naturally occurring pluripotent stem cells derived from the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, produce all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. In some cases, ESCs can be engineered to directly differentiate into morphological cells (e.g., NK cells). In other cases, ESCs can be engineered to first differentiate into tissue-specific stem cells (e.g., HSCs), which can then be further induced to differentiate into morphological cells (e.g., NK cells).

[0057] The term "isolated stem cells" generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs), etc.) isolated from a multicellular organism. For example, HSCs can be isolated from the body of a mammal such as a human. In another example, embryonic stem cells can be isolated from an embryo.

[0058] The term "isolated" generally refers to cells or groups of cells that have been separated from their original environment. For example, the new environment of isolated cells is substantially free of at least one component that would have been present in an environment containing "unisolated" reference cells. Isolated cells can be cells from which some or all of the components have been removed when they were found in their natural environment, such as cells isolated from tissue or biopsy samples. The term also includes cells from which at least one, some, or all of the components have been removed when they were found in a non-natural environment, such as cells isolated from cell cultures or cell suspensions. Thus, isolated cells are cells that have been partially or completely separated from at least one component, including other substances, cells, or groups of cells that were found in nature or that were grown, stored, or stored in a non-natural environment.

[0059] The term "hematopoietic lineage," when applied to cells or cell populations, generally refers to cells derived from (e.g., differentiated from) mesodermal cells (e.g., derived from pluripotent stem cells), and includes, for example, hematopoietic endothelial cells (HE), pre-hematopoietic stem cells (HSCs), HSCs, and hematopoietic progenitor cells (e.g., multipotent progenitor cells, lymphoid progenitor cells, early thymic progenitor cells, pre-T cell progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, myeloid progenitor cells, etc.). In some cases, hematopoietic progenitor cells or HSCs may be characterized as CD34+ (e.g., compared to pluripotent cells). In some cases, hematopoietic progenitor cells or HSCs may be characterized as CD34+CD45+ or CD34+CD43-CD45+ (e.g., compared to pluripotent cells).

[0060] As used interchangeably in this document, the terms "hematopoietic stem cells and progenitor cells," "hematopoietic stem cells," "hematopoietic progenitor cells," or "hematopoietic precursor cells" generally refer to cells that are classified as hematopoietic lineages but are capable of further hematopoietic differentiation (e.g., differentiation into NK cells), and include pluripotent hematopoietic stem cells (hematopoietic cells), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells and progenitor cells (HSCs) are pluripotent stem cells that produce all types of blood cells, 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, HSCs can be CD34+ hematopoietic cells capable of producing mature myeloid and lymphoid cell types (including T cells, NK cells, and B cells).

[0061] The term "immune cells" generally refers to differentiated hematopoietic cells. Non-limiting examples of immune cells may include NK cells, T cells, monocytes, intrinsic lymphocytes, tumor-infiltrating lymphocytes, macrophages, granulocytes, etc.

[0062] In some cases, such as the term "proGuide" as used generally in this paper, it can refer to a vector (e.g., a plasmid) that encodes an activatable gNA. ProGuide can be an example of a phylum. ProGuide can be an example of a gene regulatory part.

[0063] In some embodiments, the proGuide provided herein can encode an activatable guide nucleic acid molecule, for example, having an inactivating polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). In some cases, a portion of the proGuide encoding the activatable guide nucleic acid molecule may comprise sequentially linked (e.g., from 5' to 3') regions including an upstream stem (e.g., an upstream cleavage site), a polyT unit (or, as used interchangeably herein, "proUnit" or "proGuide Unit"), and a downstream stem (e.g., a downstream cleavage site). The upstream and downstream stems may correspond to "stem region" polynucleotide sequences that are at least partially complementary to each other. Overview

[0064] Biological programming, such as cell programming, allows cells to be engineered to produce desired outcomes. The results of cell programming can include inducing or preventing a wide range of common and / or novel cellular functions; outcomes can also include enhancing or suppressing cellular functions that have already occurred. Cell programming can be accomplished by using genetic circuits. Cell programming can also be accomplished by manipulating biomolecules, such as DNA. For example, due to the versatility and programmability of CRISPR or CRISPR / Cas systems, CRISPR or CRISPR / Cas systems have been adopted for genome editing across many species. Cell programming can affect endogenous or exogenous genes. Cell programming can be implemented to operate in a time-dependent or time-independent manner.

[0065] Genetic circuits used in cell programming can be used to control the cascade of multiple desired expression and / or activity profiles of multiple genes in a cell. To allow for better control over specific cellular outcomes, genetic circuits can be multiplexed to create positive and / or negative feedback systems.

[0066] While the CRISPR / Cas system is widely used for gene editing, Cas is essentially a single-turnover nuclease because it remains bound to the double-strand breaks it generates, and many regions of the genome are resistant to genome editing. Increased understanding of CRISPR / Cas-based genome editing has encouraged the development of cascade regulatory systems to further utilize this technology for engineered cell development. By implementing a series of activatable gRNAs, genome editing can be regulated more temporally from target site to target site, sequential genome editing can be performed to act like a domino effect, and cells can be barcoded. However, this simple barcoding, which typically uses exogenous fluorophores, does not allow for the multivariate regulation of endogenous genes to achieve cell differentiation.

[0067] Therefore, there remains an unmet need for activatable multiplexed CRISPR / Cas systems and their use for editing target polynucleotides (e.g., the genome of cells, particularly eukaryotic cells), utilizing gRNA cascades to form gene circuits including feedback loops in order to independently influence gene regulation and consequently cell fate determination. Given that their multiplexing capabilities are improved through the use of internal positive and / or negative feedback loops, pre-programmed, activatable, and self-regulating gRNA cascaded CRISPR / Cas systems are being applied, for example, in gene therapy, gene circuitry, and / or complex cell fate determination and / or control.

[0068] This disclosure provides systems and methods for engineering a CRISPR / Cas9 system, comprising a Cas endonuclease and an array of homologous single guide RNAs (sgRNAs or gRNAs) having inactivating sequences in non-essential regions and being activatable to allow for regulation and modification of the system using positive and negative feedback loops. This disclosure also provides engineered cells that may contain any of the above-described systems or be capable of performing any of the above-described methods.

[0069] Differentiation of iPSCs into hematopoietic progenitor cells (HPCs) has been hampered by several obstacles stemming from various pathogens. While attempts to generate HPCs using extracellular control mechanisms (culture conditions, growth factors, small molecules, etc.) have achieved limited success on a small scale, they suffer from reproducibility issues and batch-to-batch variability in efficiency and potency. Cells generated by these methods typically exhibit limited lineage potential and fail to contribute to key lymphoid cell lineages. Like other iPSC differentiation procedures based on controlled cellular microenvironment, these methods are also difficult to scale up to the production and manufacturing levels required for therapeutic products.

[0070] Previous work has shown that the activity of TF gene products can drive iPSCs to an HPC state, which is capable of generating lymphoid, myeloid, and erythroid cell lineages. A set of seven TFs was expressed by inserting a cDNA-expressing lentivirus into the genome of human pluripotent stem cells. The resulting cell lines had multiple gene insertions, which pose significant safety concerns and hinder their use in humans. Therefore, although uncontrolled genomic modifications from lentiviral insertions prevent their use in humans, cells with TF transgene insertions exhibited improved HPC stem cell properties and contributions to lymphoid lineages compared to methods that only manipulate extracellular conditions.

[0071] The method described herein uses non-integrating plasmid DNA to deliver genetic instructions to iPSCs. Activation of the TF gene provides the cell with capabilities similar to those of HPCs, capabilities that have not been readily achieved using externally controlled methods. Plasmid DNA does not require genome integration to activate endogenous genes, thus eliminating the safety risks associated with multiple lentiviral transgene insertions. The genetic instructions also have the benefit of being sequentially defined, allowing for improved transitions from one cellular state to the next through timely activation of stage-specific TF gene expression.

[0072] Systems and methods for engineered gene circuits, cells containing them, and methods of using them Various aspects of this disclosure provide systems for inducing desired expression and / or activity levels (or profiles) of one or more target genes in cells. Various aspects of this disclosure also provide methods for inducing desired expression and / or activity levels (or profiles) of one or more target genes in cells.

[0073] In one aspect, this disclosure provides a system for inducing a desired expression and / or activity profile of a target gene in cells. The system may include a heterologous gene circuit comprising multiple gate units. The 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. The 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. Multiple gate units can be different (e.g., containing different polynucleotide sequences).

[0074] The heterologous gene circuits disclosed herein can be operated using multiple gate units in series (e.g., multiple gate units connected sequentially end-to-end to form a single path), multiple gate units in parallel (e.g., multiple gate units cross-connected to each other to form, for example, two or more parallel paths), or combinations thereof.

[0075] As disclosed herein, multiple phylogenetic units can operate synergistically (e.g., as predetermined by the design of heterologous gene circuits) to induce outcomes in cells. These outcomes can include cell function (e.g., motility, reproduction; response to external stimuli, nutrient output, excretion, respiration, growth) and / or cell state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be determined in vitro, ex vivo, and / or in vivo. For example, outcomes disclosed herein can be determined in vitro by: (i) measuring the expression levels of genes of interest by polymerase chain reaction (PCR) or Western blotting; (ii) staining with small molecules or antibodies; (iii) cell sorting based on cell size, morphology, and / or surface protein expression; (iv) measuring phenotypic differentiation and cell function using assays (e.g., cell proliferation assays, metabolic activity assays, cell killing assays); (v) microscopy; and / or (iv) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0076] The cellular outcome may involve the regulation of a target gene. This regulation may involve multiple distinct regulation of the target gene. Multiple phylogenetic units may each induce one of these distinct regulation processes, such that the ensemble of these regulation processes synergistically produces the final expression and / or activity profile of the target gene. At least two of these distinct regulation processes may both increase the expression and / or activity level of the target gene. At least two of these distinct regulation processes may both decrease the expression and / or activity level of the target gene. Alternatively, a first distinct regulation may increase the expression and / or activity level of the target gene, while a second distinct regulation may decrease it. In this case, the first distinct regulation may occur before or after the second distinct regulation. Alternatively, different regulation processes (e.g., the first and / or second regulation) may maintain the expression and / or activity level of the target gene at its pre-regulation level.

[0077] In some cases, as disclosed herein, each of the multiple distinct regulation of a target gene may be necessary to achieve the desired expression and / or activity profile of the target gene, but is insufficient on its own. Therefore, in the absence of any of the multiple distinct regulation of the target gene, cellular outcomes induced by the multiple distinct regulation of the target gene (e.g., enhanced cellular function, induced cellular state, etc.) may be impossible. Alternatively, the extent or measure of cellular outcomes induced by the multiple distinct regulation of the target gene may be greater than the extent or measure of outcomes induced in control cells not by the multiple distinct regulation of the target gene, by one or more but not all of the multiple distinct regulation of the target gene, and / or by all of the multiple distinct regulation of the target gene occurring in different sequences of events.

[0078] The second gate unit can be activated by the first gate unit (e.g., directly or indirectly). For example, the second gate unit can be directly activated by the first gate unit. Alternatively, the second gate unit can be activated by one or more other gate units that are activated by the first gate unit (e.g., directly or indirectly). The one or more other 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. One or more additional gate units may include 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 gate unit. In yet another alternative, the second gate unit may be activated via another portion responsible for activating the first gate unit (e.g., an activation portion, a different gate unit, etc.).

[0079] The second gate unit may be activatable to induce the inactivation of an already activated first gate unit. The terms “inactivation” or “disruption” are used interchangeably herein. Inactivation and, as disclosed herein, can be induced by generating modifications (e.g., cleavage, such as single- or double-strand breaks, and insertion-deletion, etc.) to at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) that is responsible for inducing a first distinct regulation of the target gene.

[0080] Inactivation of the phylum portion and / or gene regulatory portion of the first phylum unit, as disclosed herein, can be achieved using endonuclease-based systems (e.g., CRISPR / Cas systems). Alternatively or additionally, inactivation can be achieved using transcriptional regulator systems (e.g., transcriptional repressors). Endonuclease transcriptional regulator systems (e.g., Cas repressors) can be used to achieve polynucleotide cleavage (e.g., for inactivating the phylum portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or indels. Alternatively or additionally, endonuclease transcriptional regulator systems (e.g., Cas repressors) can be used to regulate target gene expression.

[0081] Alternatively, the second gate unit may be activatable to amplify or enhance the activation of the already activated first gate unit. Amplification or enhancement of the first gate unit can be induced by generating modifications (e.g., cleavage, such as single-strand or double-strand breaks, and insertion-deletion, etc.) to at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) that is responsible for inducing a first distinct regulation of the target gene.

[0082] Multiple gate units can be pre-configured such that one of the gate units is activated upon activation of a heterologous gene circuit to regulate the expression and / or activity levels of additional target genes. These additional target genes may differ from those regulated by the various regulation methods described herein. For example, target genes may include erythroblast transformation-specific (ETS) transcription factors, and additional target genes may include T-box transcription factors (TBX), homeobox proteins, GATA, and / or basic helical-loop-helical transcription factors (bHLH). In some cases, a first gate unit may be configured to be activated to regulate the expression and / or activity levels of additional target genes. For example, the first gate unit may contain multiple activatable gene regulatory portions for regulating the expression and / or activity levels of the target gene and additional target genes, respectively. In some cases, a second gate unit may be configured to be activated to regulate the expression and / or activity levels of additional target genes. For example, the second gate unit may contain multiple activatable gene regulatory portions for regulating the expression and / or activity levels of the target gene and additional target genes, respectively. In some cases, multiple gate units may include at least one additional gate unit configured to be activated to regulate the expression and / or activity levels of additional target genes. For example, a heterologous gene circuit may be configured such that at least one additional gate unit is activated before the activation of the first gate unit, between the activation of the first and second gate units, and / or after the activation of the second gate unit.

[0083] In some cases, the first phylacting unit regulates the first target gene. Alternatively or additionally, the first phylacting unit may also regulate the second phylacting unit. Regulation of the second phylacting unit may occur at least to about 1 millisecond, at least to about 2 milliseconds, at least to about 3 milliseconds, at least to about 4 milliseconds, at least to about 5 milliseconds, at least to about 6 milliseconds, at least to about 7 milliseconds, at least to about 8 milliseconds, at least to about 9 milliseconds, at least to about 10 milliseconds, at least to about 20 milliseconds, at least to about 30 milliseconds, at least to about 40 milliseconds, at least to about 50 milliseconds, at least to about 60 milliseconds, at least to about 70 milliseconds, or at least... About 80 milliseconds, at least or more than about 90 milliseconds, at least or more than about 100 milliseconds, at least or more than about 200 milliseconds, at least or more than about 300 milliseconds, at least or more than about 400 milliseconds, at least or more than about 500 milliseconds, at least or more than about 600 milliseconds, at least or more than about 700 milliseconds, at least or more than about 800 milliseconds, at least or more than about 900 milliseconds, at least or more than about 1 second, at least or more than about 2 seconds, at least or more than about 3 seconds, at least or more than about 4 seconds, at least or more than about 5 seconds, at least or more than about 6 seconds, at least or more than about 7 seconds .... 8 seconds, at least or at most about 9 seconds, at least or at most about 10 seconds, at least or at most about 15 seconds, at least or at most about 20 seconds, at least or at most about 30 seconds, at least or at most about 40 seconds, at least or at most about 50 seconds, at least or at most about 1 minute, at least or at most about 2 minutes, at least or at most about 3 minutes, at least or at most about 4 minutes, at least or at most about 5 minutes, at least or at most about 6 minutes, at least or at most about 7 minutes, at least or at most about 8 minutes, at least or at most about 9 minutes, at least or at most about 10 minutes, at least or at most about 20 minutes, at least or at most about 30 minutes. At least or at most about 40 minutes, at least or at most about 50 minutes, at least or at most about 1 hour, at least or at most about 2 hours, at least or at most about 3 hours, at least or at most about 4 hours, at least or at most about 5 hours, at least or at most about 6 hours, at least or at most about 7 hours, at least or at most about 8 hours, at least or at most about 9 hours, at least or at most about 10 hours, at least or at most about 12 hours, at least or at most about 16 hours, at least or at most about 20 hours, or at least or at most about 24 hours, as determined by rt-qPCR, Western blotting or other methods.

[0084] In some cases, the second gate unit can regulate a second target gene. Regulation of the second target gene can occur at least to about 1 millisecond, at least to about 2 milliseconds, at least to about 3 milliseconds, at least to about 4 milliseconds, at least to about 5 milliseconds, at least to about 6 milliseconds, at least to about 7 milliseconds, at least to about 8 milliseconds, at least to about 9 milliseconds, at least to about 10 milliseconds, at least to about 20 milliseconds, at least to about 30 milliseconds, at least to about 40 milliseconds, at least to about 50 milliseconds, at least to about 60 milliseconds, at least to about 70 milliseconds, or at least... About 80 milliseconds, at least or more than about 90 milliseconds, at least or more than about 100 milliseconds, at least or more than about 200 milliseconds, at least or more than about 300 milliseconds, at least or more than about 400 milliseconds, at least or more than about 500 milliseconds, at least or more than about 600 milliseconds, at least or more than about 700 milliseconds, at least or more than about 800 milliseconds, at least or more than about 900 milliseconds, at least or more than about 1 second, at least or more than about 2 seconds, at least or more than about 3 seconds, at least or more than about 4 seconds, at least or more than about 5 seconds, at least or more than about 6 seconds, at least or more than about 7 seconds, at least or more than about 8 seconds Seconds, at least or more than about 9 seconds, at least or more than about 10 seconds, at least or more than about 15 seconds, at least or more than about 20 seconds, at least or more than about 30 seconds, at least or more than about 40 seconds, at least or more than about 50 seconds, at least or more than about 1 minute, at least or more than about 2 minutes, at least or more than about 3 minutes, at least or more than about 4 minutes, at least or more than about 5 minutes, at least or more than about 6 minutes, at least or more than about 7 minutes, at least or more than about 8 minutes, at least or more than about 9 minutes, at least or more than about 10 minutes, at least or more than about 20 minutes, at least or more than about 30 minutes, at least For as long as about 40 minutes, for at least about 50 minutes, for at least about 1 hour, for at least about 2 hours, for at least about 3 hours, for at least about 4 hours, for at least about 5 hours, for at least about 6 hours, for at least about 7 hours, for at least about 8 hours, for at least about 9 hours, for at least about 10 hours, for at least about 12 hours, for at least about 16 hours, for at least about 20 hours, or for at least about 24 hours or longer, as determined by rt-qPCR, Western blotting or other methods.

[0085] In some cases, modifying a target gene through a phylogenetic unit can inactivate the gene. For example, a modified gene can terminate the expression and / or activity level of the target gene. Alternatively, a modified gene can decrease the expression and / or activity level of the target gene. In some cases, a modified gene can increase the expression and / or activity level of the target gene. Alternatively, a modified gene can maintain the expression and / or activity level of the target gene.

[0086] At least one additional gate unit may include at least or more than about 1 gate unit, at least or more than about 2 gate units, at least or more than about 3 gate units, at least or more than about 4 gate units, at least or more than about 5 gate units, at least or more than about 6 gate units, at least or more than about 7 gate units, at least or more than about 8 gate units, at least or more than about 9 gate units, at least or more than about 10 gate units, at least or more than about 11 gate units, at least or more than about 12 gate units, at least or more than about 13 gate units, at least or more than about 14 gate units, at least or more than about 15 gate units, at least or more than about 20 gate units, at least or more than about 30 gate units, at least or more than about 40 gate units, or at least or more than about 50 gate units. Each of at least one additional phylogenetic unit may contain at least one to about one gene regulatory portion, at least two to about two gene regulatory portions, at least three to about three gene regulatory portions, at least four to about four gene regulatory portions, at least five to about five gene regulatory portions, at least six to about six gene regulatory portions, at least seven to about seven gene regulatory portions, at least eight to about eight gene regulatory portions, at least nine to about nine gene regulatory portions, at least ten to about ten gene regulatory portions, at least fifteen to about fifteen gene regulatory portions, at least twenty to about twenty gene regulatory portions, at least thirty to about thirty gene regulatory portions, at least forty to forty gene regulatory portions, or at least fifty gene regulatory portions. Each of the gene regulatory portions of the phylogenetic unit may be configured to bind to different target genes to regulate the expression and / or activity levels of different target genes, respectively. The heterologous gene circuits provided herein may be configured to regulate the expression and / or activity levels of one or more of the target genes provided herein. In some cases, heterologous gene circuits can be configured to regulate the expression and / or activity levels of some, but not all, of the target genes as described herein. For example, a heterologous gene circuit may be pre-configured to regulate the expression and / or activity levels of a first set of target genes (one or more target genes, such as erythroblast transformation-specific (ETS) genes, such as ETS1, ETV2, LMO2, etc.), but may not be pre-configured to regulate the expression and / or activity of a second set of target genes (one or more target genes, such as TBXT, TBX6, MIXL1, etc.).

[0087] The heterologous gene circuits disclosed herein can be operated using multiple gate units in series (e.g., multiple gate units connected sequentially end-to-end to form a single path), multiple gate units in parallel (e.g., multiple gate units cross-connected to each other to form, for example, two or more parallel paths), or combinations thereof. In some embodiments, the multiple gate units in series can operate in a forward cascade order. In some embodiments, the forward order can follow a numerically ascending sequence of steps (e.g., steps 1 to 2 to 3 to 4 to 5, etc.). In some embodiments, the multiple gate units in series can operate in a reverse cascade order. In some embodiments, the reverse cascade can follow a numerically descending sequence of steps (e.g., steps 10 to 9 to 8 to 7 to 6, etc.). In some embodiments, the tandem plurality of 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. In some embodiments, the tandem plurality of 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 plurality of gate units disclosed herein may operate in concert (e.g., as predetermined by the design of a heterologous gene circuit) to induce outcomes in cells. Cellular outcomes can include cell function (e.g., motility, reproduction; response to external stimuli, nutrient output, excretion, respiration, growth) and / or cell state (e.g., cell fate, differentiation, quiescence, programmed cell death). Such outcomes can be determined in vitro, ex vivo, and / or in vivo. For example, outcomes disclosed herein can be determined in vitro by: (i) measuring the expression levels of genes of interest by polymerase chain reaction (PCR) or Western blotting; (ii) staining with small molecules or antibodies; (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 methods such as metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics to screen for molecular and / or genetic differences.

[0088] A heterologous gene circuit may contain multiple gate units that are activated sequentially (e.g., sequentially). These gate units may include functional gate units pre-configured to be activated to regulate (e.g., directly regulate) the expression and / or epigenetic profile of a target gene (e.g., an endogenous target gene). The multiple gate units may further include one or more additional gate units pre-configured to (i) be activated prior to the functional gate unit and (ii) enable subsequent activation of the functional gate unit. In some cases, one or more additional gate units may be pre-configured to be activated to regulate one or more additional target genes. Alternatively, one or more additional gate units may not be pre-configured to regulate any target gene (e.g., any endogenous target gene) upon activation. Such one or more additional gate units may instead be used to delay (e.g., temporally) the activation of the functional gate unit during operation of the heterologous gene circuit, thereby delaying the expression and / or epigenetic profile of the target gene of the functional gate unit, and thus such one or more additional gate units may be referred to as “blank” gate units. The heterologous gene circuit may contain at least or at most about 1 blank gate unit, at least or at most about 2 blank gate units, at least or at most about 3 blank gate units, at least or at most about 4 blank gate units, at least or at most about 5 blank gate units, at least or at most about 6 blank gate units, at least or at most about 7 blank gate units, at least or at most about 8 blank gate units, at least or at most about 9 blank gate units, at least or at most about 10 blank gate units, at least or at most about 11 blank gate units, at least or at most about 12 blank gate units, or at least or at most about 13 blank gate units. At least or at most about 14 blank door units, at least or at most about 15 blank door units, at least or at most about 16 blank door units, at least or at most about 27 blank door units, at least or at most about 18 blank door units, at least or at most about 19 blank door units, at least or at most about 20 blank door units, at least or at most about 25 blank door units, at least or at most about 30 blank door units, at least or at most about 35 blank door units, at least or at most about 40 blank door units, at least or at most about 45 blank door units, at least or at most about 50 blank door units.

[0089] In some cases, using one or more blank gate units can delay the activation of functional gate units (e.g., as determined by measuring the expression / epigenetic profile of the target gene, or as determined by measuring the expression of functional variants or transcripts of the functional gate unit) by at least to about 1 minute, at least to about 5 minutes, at least to about 10 minutes, at least to about 30 minutes, at least to about 1 hour, at least to about 2 hours, at least to about 3 hours, at least to about 4 hours, at least to about 5 hours, at least to about 6 hours, at least to about 7 hours, at least to about 8 hours, or at least to about 9 hours. At least or more to about 10 hours, at least or more to about 11 hours, at least or more to about 12 hours, at least or more to about 13 hours, at least or more to about 14 hours, at least or more to about 15 hours, at least or more to about 16 hours, at least or more to about 17 hours, at least or more to about 18 hours, at least or more to about 19 hours, at least or more to about 20 hours, at least or more to about 21 hours, at least or more to about 22 hours, at least or more to about 23 hours, at least or more to about 24 hours, at least or more to about 2 days, at least or more to about 3 days, at least or more to about 4 days, at least or more to about 5 days, at least or more to about 6 days, or at least or more to about 7 days.

[0090] In some embodiments, the disclosure provided in Appendix A outlines solutions to obstacles in therapeutic HPC production. In some embodiments, Appendix A discloses multi-step cascades for HPC generation. In some embodiments, Appendix A discloses incorporating transcription factor gene activation into the design of a cellular algorithm library. In some embodiments, Appendix A discloses a 10-step cascade for HPC generation. In some embodiments, the 10-step cascade may include blank steps and / or unused steps. In some embodiments, blank steps and / or unused steps may space factor activations, make room for additional genes, maximize the efficiency of gene target induction by the cellular algorithm, and / or regulate the relative timing of each step. Appendix A discloses cellular algorithms for generating a range of HPC scalability and HPC yield.

[0091] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by a second gate unit upon activation can create modifications to at least a portion of the first gate unit. For example, the activated gNA of the second gate unit can generate modifications to a polynucleotide sequence of the first gate unit encoding a gNA (e.g., an activatable gNA) or to a promoter sequence of the first gate unit operatively coupled to such a gNA. Such modifications can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to a target gene). Alternatively, modifications can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0092] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene can be caused by single-strand breaks, where a break exists in one nucleotide chain. Inactivation of a polynucleotide sequence or target gene can be caused by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more single-strand breaks. In some cases, gene inactivation can be caused by up to ten, up to nine, up to eight, up to seven, up to six, up to five, up to four, up to three, up to two, or up to one single-strand break.

[0093] In some cases, modifications to polynucleotide sequences (e.g., as components of phylogenetic units, such as phylogenetic parts) or target genes can be caused by double-strand breaks, where a break exists in both nucleotide chains. In some cases, gene inactivation can be caused by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more double-strand breaks. In some cases, inactivation of polynucleotide sequences or target genes can be caused by up to ten, up to nine, up to eight, up to seven, up to six, up to five, up to four, up to three, up to two, or up to one double-strand break.

[0094] In some cases, the length of 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.

[0095] In some cases, the systems and methods of this disclosure can utilize at least two different endonucleases. A first endonuclease (e.g., a Cas protein not coupled to a transcription regulator) can be conjugated with a guide nucleic acid to induce cleavage of a target polynucleotide sequence in a phylogenetic or gene regulatory plasmid, thereby activating and / or deactivating the phylogenetic or gene regulatory portion, respectively. Furthermore, a second endonuclease (e.g., a Cas protein coupled to a transcription regulator) can be conjugated with another guide nucleic acid to bind to a target gene in the cell (e.g., a target endogenous gene) to regulate the expression and / or activity of the target gene without generating cleavage within the target gene.

[0096] In some cases, the systems and methods of this disclosure can utilize a single endonuclease system (e.g., a Cas repressor) to achieve both (i) polynucleotide cleavage (e.g., for activation / inactivation of gate portions and / or gene regulatory portions) and (ii) regulation of target gene expression. When using a single endonuclease transcription regulator system, unique guide nucleic acid molecules (gNAs) with different spacer region sequence lengths can be used to determine whether the single endonuclease transcription regulator system can (i) hybridize with a polynucleotide sequence to induce Cas-mediated nuclease activity of that 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 through a single heterologous gene circuit. For example, using gNAs with different spacer region sequence lengths bound to different targets can allow a second gate unit, as provided herein, to induce inactivation of an already activated first gate unit and / or induce different regulation of a second target gene.

[0097] As described above, the length of the spacer region sequence of gNA can affect the ability of gNA to mediate Cas nuclease activity. In some cases, gNAs with spacer region sequences of different lengths can be used in the same heterologous gene circuit to influence different types of cleavage, activation, inactivation, and / or regulation of one or more target nucleic acids. In some cases, gNA spacer region sequences shorter than a threshold length (e.g., about 16 nucleotides) can impede the nuclease activity of Cas transcription regulators while still mediating DNA binding for transcriptional regulation of target genes. In some cases, gNA spacer region sequences 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 impede the nuclease activity of Cas proteins while still mediating DNA binding.

[0098] For example, a gNA containing a 20-nucleotide spacer sequence (e.g., a gNA encoded by the phylogenetic part of a plasmid used for targeting gene regulation) may be sufficient to promote the nuclease activity of endonucleases (e.g., Cas or Cas transcription regulator fusion proteins). Alternatively or additionally, a gNA containing a 14-nucleotide spacer sequence (e.g., a gNA encoded by a gene regulation part) may hybridize with DNA, but may not be long enough to mediate nuclease activity—it may only promote endonuclease binding to homologous DNA sequences. Therefore, shorter gNAs can selectively allow transcriptional regulation of target genes without cleaving the target gene by using endonuclease transcription regulator systems (e.g., Cas activator systems, Cas repressor systems).

[0099] In some cases, modifications to polynucleotide sequences (e.g., as components of phylogenetic units, such as phylogenetic parts) or target genes can be caused by insertion-deletion (also known as insertion-deletion mutations). Insertion-deletion mutations can include frameshift or non-frameshift mutations. Insertion-deletion mutations can also include point mutations (also known as base substitutions), where only one base or base pair is modified. The length of an 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 or more bases or base pairs. The length of an insertion-deletion mutation can include up to approximately 2000, up to approximately 1000, up to approximately 900, up to approximately 800, up to approximately 700, up to approximately 600, up to approximately 500, up to approximately 400, up to approximately 300, up to approximately 200, up to approximately 100, up to approximately 90, up to approximately 80, up to approximately 70, up to approximately 60, up to approximately 50, up to approximately 40, up to approximately 30, up to approximately 20, up to approximately 15, up to approximately 10, up to approximately 9, up to approximately 8, up to approximately 7, up to approximately 6, up to approximately 5, up to approximately 4, up to approximately 3, up to approximately 2, or up to approximately 1 base or base pair.

[0100] In some cases, it is possible to modify polynucleotide sequences (e.g., as components of phylogenetic units, such as phylogenetic parts) or target genes without cleaving the polynucleotide sequence or the target gene. For example, gene regulatory parts (e.g., nucleic acid molecules and / or endonucleases, such as complexes containing CRISPR / Cas proteins and guide nucleic acid molecules) can specifically bind to polynucleotide sequences or target genes, thereby modifying the expression and / or activity of those polynucleotide sequences or target genes. Gene regulatory parts may contain transcriptional repressors or transcriptional activators as presented herein.

[0101] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene can inactivate the gene. For example, modifications to a polynucleotide sequence or a target gene can terminate the expression and / or activity level of that polynucleotide sequence or the target gene. Alternatively, modifications to a polynucleotide sequence or a target gene can decrease the expression and / or activity level of the polynucleotide sequence of the target gene. In some cases, modifications to a polynucleotide sequence or a target gene can increase the expression and / or activity level of that polynucleotide sequence or the target gene. Alternatively, modifications to a polynucleotide sequence or a target gene can maintain the expression and / or activity level of that polynucleotide sequence or the target gene.

[0102] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene may comprise a reduction in the expression and / or activity level of the polynucleotide sequence or 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. Modifications to a polynucleotide sequence or target gene may include reductions in expression and / or activity levels of the polynucleotide sequence or 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%, 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.

[0103] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene may comprise an increase in the expression and / or activity level of the polynucleotide sequence or 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%, or at least about 50%, respectively. 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. Modifications to polynucleotide sequences or target genes may include increasing the expression and / or activity levels of the polynucleotide sequence or target gene by up to approximately 1,000,000%, up to approximately 100,000%, up to approximately 9,000%, up to approximately 8,000%, up to approximately 7,000%, up to approximately 6,000%, up to approximately 5,000%, up to approximately 4,000%, up to approximately 3,000%, up to approximately 2,000%, up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, up to approximately 30%. 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.

[0104] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene may comprise a reduction in the expression and / or activity level of that polynucleotide sequence or target gene, compared to a control expression and / or activity level by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, or at least or more than about 3-fold. 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 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 60 times, at least or more than about 70 times, at least or more than about 80 times, at least or more than about 90 times, at least or more than about 100 times, at least or more than about 500 times, at least or more than about 1,000 times, at least or more than about 5,000 times, or at least or more than about 10,000 times. Modifications to a polynucleotide sequence or target gene may involve reducing the expression and / or activity levels of that polynucleotide sequence or 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... Approximately 20 times, up to or less than approximately 10 times, up to or less than approximately 9 times, up to or less than approximately 8 times, up to or less than approximately 7 times, up to or less than approximately 6 times, up to or less than approximately 5 times, up to or less than approximately 4 times, up to or less than approximately 3 times, up to or less than approximately 2 times, up to or less than approximately 1 time, up to or less than approximately 0.9 times, up to or less than approximately 0.8 times, up to or less than approximately 0.7 times, up to or less than approximately 0.6 times, up to or less than approximately 0.5 times, up to or less than approximately 0.4 times, up to or less than approximately 0.3 times, up to or less than approximately 0.2 times, up to or less than approximately 0.1 times.

[0105] In some cases, modifications to a polynucleotide sequence (e.g., as a component of a phylum unit, such as a phylum portion) or a target gene, compared to control expression and / or activity levels, may comprise an increase in the expression and / or activity level of the polynucleotide sequence or target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, or at least or more than about 3-fold. 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 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 60 times, at least or more than about 70 times, at least or more than about 80 times, at least or more than about 90 times, at least or more than about 100 times, at least or more than about 500 times, at least or more than about 1,000 times, at least or more than about 5,000 times, or at least or more than about 10,000 times. Compared to control expression and / or activity levels, modifications to the polynucleotide sequence or target gene may include increasing the expression and / or activity levels 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. Most or less than about 10 times, most or less than about 9 times, most or less than about 8 times, most or less than about 7 times, most or less than about 6 times, most or less than about 5 times, most or less than about 4 times, most or less than about 3 times, most or less than about 2 times, most or less than about 1 time, most or less than about 0.9 times, most or less than about 0.8 times, most or less than about 0.7 times, most or less than about 0.6 times, most or less than about 0.5 times, most or less than about 0.4 times, most or less than about 0.3 times, most or less than about 0.2 times, most or less than about 0.1 times.

[0106] In some cases, (i) changes in the expression and / or activity levels of the target gene (e.g., enhancement or reduction) in a sequential manner at multiple different regulation of the target gene can be greater than any change in the expression and / or activity levels of the target gene at only one or none of the multiple different regulation, to the extent of at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 11%, at least or more than about 12%, at least or more than about 13%, at least or more than about 14%, at least or more than about 15%, at least or more than about 16%, at least or more than about 17%, at least or more than about 18%. At least or more to about 19%, at least or more to about 20%, at least or more to about 25%, at least or more to about 30%, at least or more to about 35%, at least or more to about 40%, at least or more to about 45%, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 95%, at least or more to about 100%, at least or more to about 125%, at least or more to about 150%, at least or more to about 175%, at least or more to about 200%, at least or more to about 225%, at least or more to about 250%, at least or more to about 275%, or at least or more to about 300%.

[0107] In some cases, (i) alterations (e.g., enhancement or reduction) in the expression and / or activity levels of the target gene in a sequential manner at multiple different regulation of the target gene can last for a longer duration than (ii) any alterations in the expression and / or activity levels of the target gene at only one or none of the multiple different regulation, to the extent of at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 11%, at least or more than about 12%, at least or more than about 13%, at least or more than about 14%, at least or more than about 15%, at least or more than about 16%, at least or more than about 17%, at least or more than about 15%, at least or more than about 16%, at least or more than about 17%, at least or more than about 10%. About 18%, at least or more to about 19%, at least or more to about 20%, at least or more to about 25%, at least or more to about 30%, at least or more to about 35%, at least or more to about 40%, at least or more to about 45%, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 95%, at least or more to about 100%, at least or more to about 125%, at least or more to about 150%, at least or more to about 175%, at least or more to about 200%, at least or more to about 225%, at least or more to about 250%, at least or more to about 275%, or at least or more to about 300%.

[0108] In some cases, (i) the expression and / or activity level of the target gene may be less than (ii) the expression and / or activity level of the target gene in a sequential manner (e.g., including order disruption) under multiple different regulation of the target gene, to the extent of at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 11%, at least or more than about 12%, at least or more than about 13%, at least or more than about 14%, at least or more than about 15%, at least or more than about 16%, at least or more than about 17%, at least or more than about 10%. 18%, at least or more to about 19%, at least or more to about 20%, at least or more to about 25%, at least or more to about 30%, at least or more to about 35%, at least or more to about 40%, at least or more to about 45%, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least more to about 91%, at least more to about 92%, at least more to about 93%, at least more to about 94%, at least more to about 95%, at least more to about 96%, at least more to about 97%, at least more to about 98%, at least more to about 99%, or substantially about 100%.

[0109] In some cases, (i) the expression and / or activity levels of the target gene may be shorter in a sequential manner (e.g., including order disruption) under multiple different regulation of the target gene than (ii) the expression and / or activity levels of the target gene may be shorter in a single first different regulation but not a second different regulation, to the extent of at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 11%, at least or more than about 12%, at least or more than about 13%, at least or more than about 14%, at least or more than about 15%, at least or more than about 16%, at least or more than about 17%, at least or more than Up to 18%, at least or more to 19%, at least or more to 20%, at least or more to 25%, at least or more to 30%, at least or more to 35%, at least or more to 40%, at least or more to 45%, at least or more to 50%, at least or more to 55%, at least or more to 60%, at least or more to 65%, at least or more to 70%, at least or more to 75%, at least or more to 80%, at least or more to 85%, at least or more to 90%, at least more than or about 91%, at least more than or about 92%, at least more than or about 93%, at least more than or about 94%, at least more than or about 95%, at least more than or about 96%, at least more than or about 97%, at least more than or about 98%, at least more than or about 99%, or substantially about 100%.

[0110] In some cases, the first and second gate units, as provided herein, can be activated to induce a first distinct regulation and a subsequent second distinct regulation of a common gene (e.g., the expression and / or activity profile of that common gene). The first and second gate units can be activated sequentially (e.g., at different time points) to achieve the first and second distinct regulation of the common gene in a sequential manner. Alternatively, the first and second gate units can be subsequently activated simultaneously, and such gates can be pre-configured to still achieve the first and second distinct regulation of the common gene in this sequential manner.

[0111] In some cases, the activation of multiple gate units may be the result of a single activation of a heterogeneous gene circuit (e.g., through a single activated portion at a single time point). The multiple gate units may include one of a first gate unit and a second gate, which are pre-configured to be activated sequentially 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 activated portion (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by a different activated portion (e.g., a different guide nucleic acid) that is different from the activated portion of the heterogeneous gene circuit. The other activated portion may be a part of the heterogeneous gene circuit that is generated (e.g., expressed) only when the heterogeneous gene circuit is activated. Alternatively or additionally, the first and second gate units may each be activated by a different activated portion that is different from the activated portion of the heterogeneous gene circuit. Such a different activated portion may be a part of the heterogeneous gene circuit that is generated (e.g., expressed) only when the heterogeneous gene circuit is activated.

[0112] In some embodiments of any of the systems disclosed herein, a gate unit may comprise a gate portion (e.g., at least one, two, three, four, five, or more different gate portions) and / or a gene regulatory portion (e.g., at least one, two, three, four, five, six, seven, eight, nine, ten, or more different gene regulatory portions). A gate portion as disclosed herein may comprise a guide nucleic acid molecule (gNA) (e.g., at least one, two, three, four, five, or more gNAs). A gene regulatory portion as disclosed herein may comprise gNAs (e.g., at least one, two, three, four, five, or more gNAs). A guide nucleic acid molecule as disclosed herein may include, but is not limited to, DNA, RNA, any analogues of such, or any combination thereof. In some embodiments of any of the systems disclosed herein, the phylogenetic and / or gene regulatory portions may be activatable to form a complex with an enzyme (e.g., an endonuclease and / or an exonuclease), and this complex may be configured or capable of binding to a target polynucleotide, for example, to regulate the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operatively coupled to the target polynucleotide. For example, the complex may regulate the expression and / or activity level of a gene containing the target polynucleotide.

[0113] In some embodiments of any of the systems disclosed herein, the initial (or first) gate unit of the heterologous gene circuit disclosed herein may be activated by an activation portion (e.g., direct activation). The activation portion may directly bind to at least a portion of the initial gate unit to activate the initial gate unit, e.g., thereby sequentially activating the heterologous gene circuit. Alternatively, the activation portion (e.g., electromagnetic energy) may 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 may comprise at least one gate portion and at least one gene regulatory portion. In some cases, the initial gate unit may comprise at least one gate portion but may not include, and does not need to include, a gene regulatory portion. In some cases, the initial gate unit may comprise at least one gene regulatory portion but may not include, and does not need to include, a gate portion (e.g., the activation portion may be configured to activate the initial gate unit and at least one additional gate unit).

[0114] In some embodiments of any of the systems disclosed herein, the gNA of the phylum and / or gene regulatory portion (e.g., the gNA encoded by the phylum and / or gene regulatory portion) may be an activatable gNA. An activatable gNA may be, but is not limited to, any of the following: ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogues of such, or any combination thereof. In some embodiments, the activatable gNA molecule may be a self-cleaving gNA (e.g., gRNA containing a cis-ribozyme). For example, when the activatable gNA is expressed in a cell, the activatable gNA may be self-cleavable to become nonfunctional (e.g., not configured to bind a target gene) unless the gene encoding the activatable gNA is modified prior to the expression of the activatable gNA. In some embodiments, the activatable gNA molecule contains a non-canonical transcription termination sequence (e.g., a polyX sequence, such as a polyU sequence or a polyT sequence) such that a functional gNA molecule is not expressed until the gene encoding the activatable gNA having the non-canonical transcription termination sequence can be modified (e.g., to remove some or all of the transcription termination sequence). Therefore, nonfunctional variants of gNA (e.g., nonfunctional fragments) can be expressed in the absence of transcription termination sequence modifications. In some embodiments, gNA may be synthetic. In some embodiments, gNA may have an attached fluorescent label.

[0115] In some cases, a non-canonical termination sequence may comprise or consist substantially of the following polynucleotide sequences: the polynucleotide sequence exhibiting at least or more to about 40%, at least or more to about 45%, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 86%, at least or more to about 87%, at least or more to about 88%, at least or more to about 89%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100% sequence identity with a polynucleotide sequence selected from one or more members of SEQ ID NO: 136 and 145 or their complementary sequences.

[0116] In some cases, a polynucleotide sequence containing a non-canonical termination sequence (or its complementary sequence) may have the following structure (II):

[0117] Wherein: (i) T' is a non-canonical termination sequence as provided herein (e.g., polyT); and (ii) M and M' are as described above for structure (II).

[0118] In some cases, in pairs containing M and M' as shown in structure (II) and / or structure (III), the pair can form an isolation region sequence as provided herein. Alternatively, the pair can form a stem sequence as provided herein.

[0119] In some cases, in pairs containing M and M' as shown in structure (II), the polynucleotide sequence of M and the additional polynucleotide sequence of M' can exhibit the same characteristics as those selected from (1) SEQ ID NO: 117 and SEQ ID NO: 154; (2) SEQ ID NO: 118 and SEQ ID NO: 155; (3) SEQ ID NO: 119 and SEQ ID NO: 156; (4) SEQ ID NO: 120 and SEQ ID NO: 157; (5) SEQ ID NO: 121 and SEQ ID NO: 158; (6) SEQ ID NO: 122 and SEQ ID NO: 159; (7) SEQ ID NO: 123 and SEQ ID NO: 160; (8) SEQ ID NO: 124 and SEQ ID NO: 161; (9) SEQ ID NO: 126 and SEQ ID NO: 162; (10) SEQ ID NO: 127 and SEQ ID NO: 163; (11) SEQ ID NO: 128 and SEQ ID NO: 164; (12) SEQ ID NO: 129 and SEQ ID NO: 165; (13) SEQ ID NO: 130 and SEQ ID NO: 166; (14) SEQ ID NO: 131 and SEQ ID NO: 167; (15) SEQ ID NO: 132 and SEQ ID NO: 168; (16) SEQ ID NO: 133 and SEQ ID NO: 169; (17) SEQ ID NO: 134 and SEQ ID NO: 170; and (18) SEQ ID NO: 135 and SEQ ID NO: The corresponding pair of 171 or its complementary sequence pair has at least or more to about 40%, at least or more to about 45%, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 86%, at least or more to about 87%, at least or more to about 88%, at least or more to about 89%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100% sequence identity.

[0120] In some cases, the size of the polyT sequence is greater than or equal to a threshold length, where the threshold length is sufficient to reduce the expression of the guiding nucleic acid molecule from the polynucleotide sequence. Therefore, plasmids (e.g., phylogenetic or gene regulatory portions) can encode inactive gNAs containing polyT sequences greater than or equal to the threshold length, and editing such plasmids to reduce the length of the polyT to below the threshold length can allow gNAs to be expressed in their entirety without premature termination, thereby activating gNAs. In some cases, the polyT sequence contains at least 5 Ts. In some cases, the polyT sequence contains at least 7 Ts. In some cases, the polyT sequence contains at least 8 Ts. In some cases, the polyT sequence contains at least 10 Ts. In some cases, the polyT sequence contains 5 to 15 Ts. In some cases, the polyT sequence contains one or more additional nucleotides that are not Ts.

[0121] In some cases, gene regulatory portions (e.g., guide nucleic acids and / or endonucleases) can be configured to bind to a target polynucleotide sequence operatively coupled to a target gene in the cell. The target gene may contain a coding polynucleotide sequence encoding a target nucleic acid molecule or a target protein. The target polynucleotide sequence may be part of a coding polynucleotide sequence. Alternatively, the target polynucleotide sequence may not be part of a coding polynucleotide sequence. For example, the target polynucleotide sequence may be upstream of the coding polynucleotide sequence (e.g., part of a promoter encoding a polynucleotide sequence, such as a transcription start site (TSS)).

[0122] In some implementations, proGuide may contain a target polynucleotide domain at or near the inactivating polynucleotide sequence (e.g., at or near the 5' and / or 3' ends of the inactivating polynucleotide sequence), which can be targeted (e.g., via a sequence activation mechanism of a heterologous gene circuit as described herein) to modify (e.g., edit, cleave) the inactivating polynucleotide sequence, thereby causing proGuide to express an activated guide nucleic acid molecule. The target polynucleotide domain of proGuide may not exhibit sequence identity with any comparable endogenous polynucleotide sequence in the cell, thereby avoiding unintentional targeting and regulation of endogenous target genes.

[0123] In some implementations, the inactivating polynucleotide sequence of proGuide can be positioned between two target polynucleotide domains, which may or may not be targetable by a common guide nucleic acid sequence. In some cases, the two target polynucleotide domains can be anticomplementary to each other, allowing the inactivating polynucleotide sequence to be modified or cleaved by the same mechanism (e.g., the same spacer region sequence of the guide nucleic acid molecule).

[0124] In some embodiments, proGuide may comprise a polynucleotide sequence exhibiting at least or at most about 50%, at least or at most about 55%, at least or at most about 60%, at least or at most about 65%, at least or at most about 70%, at least or at most about 75%, at least or at most about 80%, at least or at most about 85%, at least or at most about 86%, at least or at most about 87%, at least or at most about 88%, at least or at most about 89%, at least or at most about 90%, at least or at most about 91%, at least or at most about 92%, at least or at most about 93%, at least or at most about 94%, at least or at most about 95%, at least or at most about 96%, at least or at most about 97%, at least or at most about 98%, at least or at most about 99%, or substantially about 100% sequence identity with any of the following: from SEQ ID NO: 190-191 (e.g., targeting BCL11A), SEQ ID NO: 192-194 (e.g., targeting BCL11B), SEQ ID NO: SEQ ID NO: 195-198 (e.g., targeting BCL2), SEQ ID NO: 199-206 (e.g., targeting BCL2L1), SEQ ID NO: 207-210 (e.g., targeting BMI1), SEQ ID NO: 211-214 (e.g., targeting CD34), SEQ ID NO: 215-218 (e.g., targeting DLL4), SEQ ID NO: 219-224 (e.g., targeting DNMT3B), SEQ ID NO: 225-227 (e.g., targeting DTX1), SEQ ID NO: 228-230 (e.g., targeting EBF1), SEQ ID NO: 231-233 (e.g., targeting EOMES), SEQ ID NO: 234-241 (e.g., targeting ERG), SEQ ID NO: 242-245 (e.g., targeting ETS1), SEQ ID NO: 246-248 (e.g., targeting ETV2), SEQ ID NO: SEQ ID NO: 249-252 (e.g., targeting EZH1), SEQ ID NO: 253-256 (e.g., targeting FLT1), SEQ ID NO: 257-260 (e.g., targeting FOS), SEQ ID NO: 261-264 (e.g., targeting FOXO1), SEQ ID NO: 265-267 (e.g., targeting FOXP3), SEQ ID NO: 268-271 (e.g., targeting GATA1), SEQ ID NO: 272-278 (e.g., targeting GATA2), SEQ ID NO: 279-282 (e.g., targeting GATA3), SEQ ID NO: 283-289 (e.g., targeting GFI1), SEQ ID NO: 290-293 (e.g.,Targeting GFI1B), SEQ ID NO: 294-297 (e.g., targeting HES1), SEQ ID NO: 298-301 (e.g., targeting HHEX), SEQ ID NO: 302-304 (e.g., targeting HOXA10), SEQ ID NO: 305-307 (e.g., targeting HOXA5), SEQ ID NO: 308-311 (e.g., targeting HOXA7), SEQ ID NO: 312 (e.g., targeting HOXA9), SEQ ID NO: 313-315 (e.g., targeting HOXB4), SEQ ID NO: 316-322 (e.g., targeting ID2), SEQ ID NO: 323-325 (e.g., targeting ID3), SEQ ID NO: 326-329 (e.g., targeting IKZF1), SEQ ID NO: 330-336 (e.g., targeting IKZF2), SEQ ID SEQ ID NO:337-340 (e.g., targeting IL7R), SEQ ID NO:341-347 (e.g., targeting IRF4), SEQ ID NO:348-351 (e.g., targeting IRF8), SEQ ID NO:352-355 (e.g., targeting KDR), SEQ ID NO:356-363 (e.g., targeting LCOR), SEQ ID NO:364-371 (e.g., targeting LEF1), SEQ ID NO:372-374 (e.g., targeting LMO2), SEQ ID NO:375-378 (e.g., targeting LYL1), SEQ ID NO:379-382 (e.g., targeting MEF2C), SEQ ID NO:383-386 (e.g., targeting MIXL1), SEQ ID NO:387-389 (e.g., targeting MYB), SEQ ID NO:390-393 (e.g., targeting MYC), SEQ ID NO: SEQ ID NO: 394-397 (e.g., targeting NFIL3), SEQ ID NO: 398-401 (e.g., targeting NR4A1), SEQ ID NO: 402-405 (e.g., targeting PAX5), SEQ ID NO: 406-409 (e.g., targeting PGF), SEQ ID NO: 410-416 (e.g., targeting RORC), SEQ ID NO: 417-419 (e.g., targeting RUNX1), SEQ ID NO: 420-426 (e.g., targeting RUNX3), SEQ ID NO: 427-430 (e.g., targeting SATB1), SEQ ID NO: 431-434 (e.g., targeting SNAI1), SEQ ID NO: 435-438 (e.g.,Targeting sox17), SEQ ID NO: 439-446 (e.g., targeting SOX4), SEQ ID NO: 447-448 (e.g., targeting SPI1), SEQ ID NO: 449-452 (e.g., targeting SUV39H1), SEQ ID NO: 453-460 (e.g., targeting TAL1), SEQ ID NO: 461-463 (e.g., targeting TBX21), SEQ ID NO: 464-470 (e.g., targeting TCF12), SEQ ID NO: 471-478 (e.g., targeting TCF3), SEQ ID NO: 479-480 (e.g., targeting TCF7), SEQ ID NO: 481-484 (e.g., targeting TOX), SEQ ID NO: 485-488 (e.g., targeting TOX2), SEQ ID NO: 489-492 (e.g., targeting VEGFA), SEQ ID NO: SEQ ID NOs 493-496 (e.g., targeting VEGFB), SEQ ID NOs 497-500 (e.g., targeting VEGFC), SEQ ID NOs 501-504 (e.g., targeting VEGFD), SEQ ID NOs 505-507 (e.g., targeting ZBTB16), SEQ ID NOs 508-511 (e.g., targeting ZBTB17), SEQ ID NOs 512-514 (e.g., targeting ZBTB7B), SEQ ID NOs 515-518 (e.g., targeting ZSCAN4), or one or more members of their complementary sequences (shown in Table 5).

[0125] The multiple different regulation of the target gene can be different (e.g., different degrees of change in the expression and / or activity levels of the target gene). For example, the different degrees of the first regulation exerted by the first gene unit and the second regulation exerted by the second gene unit can be 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 degree of difference between the first and second regulation can be 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 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%. Alternatively or additionally, the different regulation of the target gene can be substantially the same (e.g., identical).

[0126] Multiple different regulations may be sufficient, individually, to induce the desired alteration in the expression and / or activity levels of a target gene. Alternatively, different regulations may be insufficient, individually, to induce the desired alteration in the expression and / or activity levels of a target gene.

[0127] As disclosed herein, one or more target genes may include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0128] One or more target genes disclosed herein may include cell differentiation regulators, molecular function regulators, binding factors, membrane fusion (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.

[0129] One or more target genes disclosed herein may include cell differentiation regulators, including growth factors, transcription factors, myogenic regulators, immune cell regulators, neuronal regulators, stem cell differentiation factors, chondrogenic regulators, osteogenic regulators, senescence factors, stemness factors (e.g., dedifferentiation factors), etc.

[0130] In some cases, one or more target genes (e.g., one or more immune cell regulatory factors) may include MYCN, PTCRA, BCL11B, HHEX, NOTCH1, TCF3, RAG2, DTX1, RUNX1, HOXA9, HOXA5, HOXB4, RAG1, SPI1, EVT2, SCL, ID2, BCL11A, ID3, TCF7, IKZF1, TCF12, RUNX3, LMO2, LEF1, GFI1, LYL1, AQP3, MEIS1, GATA2, G ATA3, HES1, ST18, NR4A1, C20ORF100, IKAROS, SPIB, RORC, TCF1, LCOR, IRF8, SATB1, BMI1, MYC, AHR, MIXL1, FOXO1, NOTCH1, NOTCH3, IL-2, IL4, IL7, IL15, EBF1, PAX5, TAL1, MYB, ERG, HHEX, E4PB4, GFI1B, PTA, TBXT, TBX6, TBX21, ETS1, ETS2, or MEF2C. For example, one or more target genes for inducing hematopoietic stem cell differentiation can be selected from TBXT, TBX6, MIXL1, ETS1, ETV2, GATA2, SCL, LMO2, HOXA5, HOXA9, ERG, SPIB1, MYB, RUNX1, HOXB4, GFI1, LCOR, IRF8, SATB1, and BMI1. Figure 2 In some cases, one or more target genes (e.g., one or more hematopoietic stem cell differentiation factors) may not include TBXT, TBX6, and / or MIXL1.

[0131] In some cases, one or more target genes (e.g., one or more stem cell differentiation factors) may include HOXB4, NOTCH1, SOX11, SOX17, RUNX1, GATA2, FLI1, ERG, WNT, HNF1, HNF3, HNF4, CDX2, and LIN28A.

[0132] In some cases, one or more target genes (e.g., one or more stemness factors) may include HES1, HES5, CBF1, SOC2, HMGA2, OLIG2, ID2, ID4, HESR1, HESR2, GLI1, GLI2, GLI3, SOXB, and BMI1.

[0133] 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-restrictive examples of TBX transcription factors may include TBX1, TBX2, TBX3, TBX4, TBX5, TBX6, TBX10, TBX15, TBX18, TBX19, TBX20, TBX21, TBX22, and TBXT (Brachyury protein). In some cases, one or more target genes may not include one or more TBX transcription factors (e.g., TBXT or TBX6).

[0134] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of the TBX gene. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of the TBX gene. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of the TBX gene.

[0135] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of TBXT. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of TBXT. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of TBXT. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of TBXT. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of TBXT.

[0136] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of TBX6. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of TBX6. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of TBX6. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of TBX6. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of TBX6.

[0137] In some cases, one or more target genes may include basic helical-loop-helical 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 helical-loop-helical protein structure. Non-restrictive examples of bHLH transcription factors may 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.

[0138] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of the bHLH gene. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of the bHLH gene. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of the bHLH gene. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of the bHLH gene. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of the bHLH gene.

[0139] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of SCL. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of SCL. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of SCL. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of SCL. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of SCL.

[0140] In some cases, one or more target genes may include SRY-associated box transcription factors (SOX genes). SOX transcription factors are involved in developmental regulation. Non-restrictive 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.

[0141] In some cases, one or more target genes may include SOX A group, which includes SRY. In some cases, one or more target genes may include SOX B1 group, which includes SOX1, SOX2, and / or SOX3. In some cases, one or more target genes may include SOX B2 group, which includes SOX14 and / or SOX21. In some cases, one or more target genes may include SOX C group, which includes SOX4, SOX11, and / or SOX12. In some cases, one or more target genes may include SOX D group, which includes SOX5, SOX6, and / or SOX13. In some cases, one or more target genes may include SOX E group, which includes SOX8, SOX9, and / or SOX10. In some cases, one or more target genes may include SOX F group, which includes SOX7, SOX17, and / or SOX18. In some cases, one or more target genes may include SOX G group, which includes SOX15. In some cases, one or more target genes may include SOX H group, which includes SOX30.

[0142] In some cases, one or more target genes may include forkhead boxes (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 cell differentiation. Non-restrictive 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.

[0143] In some cases, one or more target genes may include erythroblast transformation-specific (ETS) genes. ETS are animal-specific transcription factors and are involved in tissue development. Non-restrictive 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).

[0144] In some cases, one or more target genes may include collagen. Collagen is a fibrous protein and a major component of skin, bone, 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, and 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.

[0145] In some cases, one or more target genes may include homeobox genes. Homeobox genes are genes that regulate, for example, a wide range of anatomical features during early stages of embryonic development. 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-restrictive examples of homeobox genes may 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, HOXD1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA ...A9, HOXA10, HOXA11, HOXC12, HOXC13, HOXD1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA12, HOXC13, HOXA1, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11, HOXA12, HOXC13, HOXA1, HOXA3, HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA OXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD11, HOXD12, HOXD13, CDX1, CDX2, CDX4, GSX1, GSX2, PDX1, EVX1, EVX2, GBX1, GBX2, MEOX 1. MEOX2, MNX1, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, IRX1, IRX2, IRX3, IRX4, IRX5, IRX6, MEIS1, MEIS2, MEIS3, MKX, PBX1, PBX2, P BX3, PBX4, PKNOX1, PKNOX2, TGIF1, TGIF2, TGIF2LX, TGIF2LY, ISL1, ISL2, LHX1, LHX2, LHX3, LHX4, LHX5, LHX6, LHX8, LHX9, LMX1 A. LMX1B, HDX, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1, POU5F1P1, P OU5F1P4, POU5F2, POU6F1, POU6F2, LASS2, LASS3, LASS4, LASS5, LASS6, HMBOX1, HNF1A, HNF1B, SIX1, SIX2, SIX3, SIX4, SIX5, SI X6,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, T PRX1, UNCX, VSX1, VSX2, BARHL1, BARHL2, BARX1, BARX2, BSX, DBX1, DBX2, EMX1, EMX2, EN1, EN2, HHEX, HLX1, LBX1, LBX2, MSX1, MSX2, NANOG, NOTO, TLX1, TLX2, TLX3, TSHZ1, TSHZ2, TSHZ3, VAX1, VAX2, VENTX, NKX2-1, NKX2-4, NKX2-2, NKX2-8, NKX3-1, NKX3-2, NKX2-3, NKX2-5, NKX2-6, HMX1, HMX2, HMX3, NKX6-1, NKX6-2, and NKX6-3. In some cases, one or more target genes may not include homeobox genes (e.g., MIXL1).

[0146] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of the homeobox gene. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of the homeobox gene. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of the homeobox gene. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of the homeobox gene. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of the homeobox gene.

[0147] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of MIXL1. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of MIXL1. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of MIXL1.

[0148] In some cases, one or more target genes may include GATA genes. GATA genes are transcription factors characterized by their ability to bind to the DNA sequence “GATA”. Non-limiting examples of GATA genes may include GATA1, GATA2, GATA3, GATA4, GATA5, and GATA6.

[0149] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of the GATA gene. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of the GATA gene. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of the GATA gene. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of the GATA gene. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of the GATA gene.

[0150] In some cases, multiple gate units are pre-configured such that one of the gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of GATA2. In some cases, a first gate unit can be activated to regulate the expression and / or activity level of GATA2. Alternatively or additionally, a second gate unit can be activated to regulate the expression and / or activity level of GATA2. Alternatively or additionally, a gate unit can be activated before the first gate unit to regulate the expression and / or activity level of GATA2. Alternatively or additionally, a gate unit can be activated between the first and second gate units to regulate the expression and / or activity level of GATA2.

[0151] In some cases, guide nucleic acid (gNA) molecules, as presented herein, can be configured to bind to target genes (e.g., coding or non-coding regions of the target gene) to regulate the expression and / or activity of the target gene. gNA molecules may contain a multinucleotide sequence (e.g., a spacer sequence) that exhibits specific binding to a target polynucleotide sequence of the target gene. The polynucleotide sequence of the gNA molecule may exhibit at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100% sequence identity with the polynucleotide sequences of any of SEQ ID NO. 1-112 (e.g., the polynucleotide sequences of any of SEQ ID NO. 13-16 for targeting ETS1). The polynucleotide sequence of the gNA molecule that binds to the target gene may contain at least or at most about 10 nucleotides, at least or at most about 11 nucleotides, at least or at most about 12 nucleotides, at least or at most about 13 nucleotides, at least or at most about 14 nucleotides, at least or at most about 15 nucleotides, at least or at most about 16 nucleotides, at least or at most about 17 nucleotides, at least or at most about 18 nucleotides, at least or at most about 19 nucleotides, at least or at most about 20 nucleotides, at least or at most about 21 nucleotides, at least or at most about 22 nucleotides, at least or at most about 23 nucleotides, at least or at most about 24 nucleotides, or at least or at most about 35 nucleotides.

[0152] 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, mesogenic progenitor cells, mesodermal stem cells) into hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells), thereby resulting in 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 cells generated using heterologous gene circuits as disclosed herein being hematopoietic lineage cells.

[0153] 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, mesodermal progenitor cells, mesoderm stem cells) into hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells), thereby resulting in 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 cells generated using heterologous gene circuits as disclosed herein being of the target cell type. In some embodiments, target cells can be identified by characterizing one or more cell surface markers (which may include KDR, CD34, CD43, CD45, and CD309).

[0154] In some cases, the use of heterologous gene circuits as disclosed herein can be used, for example, to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, or mesogenic progenitor cells) into hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) in the absence of feeder cells, serum, and one or more exogenous factors (e.g., one or more exogenous growth factors). Otherwise, in some cases, one or more exogenous factors may be required to achieve the differentiation of stem cells into hematopoietic lineage cells in the absence of heterologous gene circuits as provided herein. Non-limiting examples of one or more exogenous factors may include bone morphogenetic protein (BMP), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase receptor ligand (FLTL), and / or interleukin (IL).

[0155] Non-limiting examples of BMPs may include BMP1, BMP1b, BMP2, BMP2A, BMP2B, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A, and BMP8B. In some embodiments, BMPs include BMP4. Non-limiting examples of VEGFs may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF. Non-limiting examples of FGFs may include FGF1, FGF2 (basic FGF or bFGF), FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23. Non-limiting examples of FLTL may include FLTL-1, FLTL-2, FLTL-3, and FLTL-4. Non-limiting examples of ILs may include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-1 8. IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and IL-36.

[0156] Using heterologous gene circuits as disclosed in this article, up to approximately 1 x 10⁻⁶ can be obtained. 6 One, at most about 9x10 5 One, at most about 8x10 5 One, at most about 7x10 5 One, at most about 6x10 5 One, at most about 5x10 5 One, at most about 4x10 5 One, at most about 3x10 5 One, at most about 2x10 5 One, at most about 1x10 5 One, at most about 5x10 4 One, at most about 2x10 4 One, at most about 1x10 4 One or fewer pluripotent stem cells (e.g., human PSCs) generate at least approximately 1 x 102 4 One, at least about 2 x 10 4 One, at least about 5 x 10 4 One, at least about 1x10 5 One, at least about 2 x 105 One, at least about 5 x 10 5 One, at least about 1x10 6 One, at least about 2 x 10 6 One, at least about 5 x 10 6 One, at least about 1x10 7 One, at least about 2 x 10 7 One, at least about 5 x 10 7 One, at least about 1x10 8 One, at least about 2 x 10 8 One, at least about 5 x 10 8 One, at least about 1x10 9 One, at least about 2 x 10 9 One, at least about 5 x 10 9 One, at least about 1x10 10 One, at least about 2 x 10 10 One, at least about 5 x 10 10 One, at least about 1x10 15 One, at least about 2 x 10 15 One, at least about 5 x 10 15 One or more hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells).

[0157] Such generation of hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) using heterologous genetic circuits as disclosed herein can be achieved within a time span of up to about 60 days, up to about 55 days, up to about 50 days, up to about 45 days, up to about 40 days, up to about 35 days, up to about 30 days, up to about 25 days, up to about 20 days, up to about 15 days, up to about 10 days, up to about 9 days, up to about 8 days, up to about 7 days, up to about 6 days, up to about 5 days, up to about 4 days, up to about 3 days, up to about 2 days, or less. When injected into mice, the resulting hematopoietic lineage cells (e.g., hematopoietic stem cells or hematopoietic progenitor cells) generated using heterologous gene circuits as disclosed herein can extend the lifespan of 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 longer.

[0158] In some cases, multiple type 1 cells can transform into multiple type 2 cells within up to approximately 20 days, up to approximately 19 days, up to approximately 18 days, up to approximately 17 days, up to approximately 16 days, up to approximately 15 days, up to approximately 14 days, up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days, up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, or up to approximately 1 day after culturing (e.g., in vitro culture) stem cells. In some cases, the conversion rate from multiple type I cells to multiple type II cells can be at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 25%, at least or more than about 30%, at least or more than about 35%, at least or more than about 40%, at least or more than about 45%. %, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or substantially about 100%.

[0159] In some cases, multiple stem cells can transform into multiple hematopoietic lineage cells within up to approximately 20 days, up to approximately 19 days, up to approximately 18 days, up to approximately 17 days, up to approximately 16 days, up to approximately 15 days, up to approximately 14 days, up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days, up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, or approximately 1 day after culturing (e.g., in vitro culture). In some cases, the conversion rate from multiple stem cells to multiple hematopoietic lineage cells can be at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 25%, at least or more than about 30%, at least or more than about 35%, at least or more than about 40%, at least or more than about 45%. %, at least or more to about 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or about 100%.

[0160] In some cases, multiple stem cells can transform into multiple hematopoietic lineage cells, wherein the hematopoietic lineage cells are CD45+ (e.g., CD34+CD43-CD45+), within up to approximately 20 days, up to approximately 19 days, up to approximately 18 days, up to approximately 17 days, up to approximately 16 days, up to approximately 15 days, up to approximately 14 days, up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days, up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, or approximately 1 day after culturing (e.g., in vitro culture) stem cells. In some cases, the conversion rate from multiple stem cells to multiple hematopoietic lineage cells can be at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 25%, at least or more than about 30%, at least or more than about 35%, at least or more than about 40%, at least or more than about 45%, at least or more than about 50%, at least or more than about 1%. 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or about 100%, wherein the hematopoietic lineage cells are CD45+ (e.g., CD34+CD43-CD45+).

[0161] In some cases, multiple stem cells can transform into multiple hematopoietic lineage cells, which are CD34+, within up to approximately 20 days, up to approximately 19 days, up to approximately 18 days, up to approximately 17 days, up to approximately 16 days, up to approximately 15 days, up to approximately 14 days, up to approximately 13 days, up to approximately 12 days, up to approximately 11 days, up to approximately 10 days, up to approximately 9 days, up to approximately 8 days, up to approximately 7 days, up to approximately 6 days, up to approximately 5 days, up to approximately 4 days, up to approximately 3 days, up to approximately 2 days, or approximately 1 day after culturing (e.g., in vitro culture) stem cells. In some cases, the conversion rate from multiple stem cells to multiple hematopoietic lineage cells can be at least or more than about 1%, at least or more than about 2%, at least or more than about 3%, at least or more than about 4%, at least or more than about 5%, at least or more than about 6%, at least or more than about 7%, at least or more than about 8%, at least or more than about 9%, at least or more than about 10%, at least or more than about 15%, at least or more than about 20%, at least or more than about 25%, at least or more than about 30%, at least or more than about 35%, at least or more than about 40%, at least or more than about 45 ... 50%, at least or more to about 55%, at least or more to about 60%, at least or more to about 65%, at least or more to about 70%, at least or more to about 75%, at least or more to about 80%, at least or more to about 85%, at least or more to about 90%, at least or more to about 91%, at least or more to about 92%, at least or more to about 93%, at least or more to about 94%, at least or more to about 95%, at least or more to about 96%, at least or more to about 97%, at least or more to about 98%, at least or more to about 99%, or about 100%, wherein the hematopoietic lineage cells are CD34+.

[0162] In some cases, the use of heterologous gene circuits in stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, intermediate progenitor cells) can induce stem cell differentiation into hematopoietic progenitor 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 progenitor (EMP) cells, intermediate progenitor cells) can induce stem cell differentiation into hematopoietic progenitor 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 progenitor (EMP) cells, intermediate progenitor cells) can induce stem cell differentiation into hematopoietic progenitor cells in the absence of one or more exogenous factors (e.g., one or more exogenous growth factors). In some cases, the use of heterologous gene circuits as disclosed herein can be used, for example, to differentiate stem cells (e.g., pluripotent stem cells (PSCs), erythroid myeloid progenitor (EMP) cells, intermediate progenitor cells) into hematopoietic progenitor cells in the absence of one or both of embryoids and serum. The hematopoietic progenitor cells generated by using the heterologous gene circuits disclosed herein constitute 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 cell population.

[0163] In some cases, at least two distinct regulation, including a first regulation and a second regulation, can be applied to a target gene. The timing of the first and second regulation can be controlled (e.g., predetermined by the design of a heterologous gene circuit). For example, the initiation of the second regulation (e.g., by at least a portion of a second gate unit, such as a second gene regulatory portion) can occur at least approximately 1 second, at least approximately 2 seconds, at least approximately 3 seconds, at least approximately 4 seconds, at least approximately 5 seconds, at least approximately 6 seconds, at least approximately 7 seconds, at least approximately 8 seconds, at least approximately 9 seconds, at least approximately 10 seconds, at least approximately 20 seconds, at least approximately 30 seconds, at least approximately 40 seconds, at least approximately 50 seconds, at least approximately 1 minute, at least approximately 2 minutes, at least approximately 3 minutes, at least approximately 4 minutes, at least approximately 5 minutes, at least approximately 6 minutes, or at least approximately 6 seconds after the initiation of the first regulation (e.g., by at least a portion of a first gate unit, such as a first gene regulatory portion). Minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 20 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days. The initiation of the second regulation (e.g., by at least a portion of the second gate unit, such as the second gene regulatory portion) may occur at most approximately 10 days, at most approximately 9 days, at most approximately 8 days, at most approximately 7 days, at most approximately 6 days, at most approximately 5 days, at most approximately 4 days, at most approximately 3 days, at most approximately 2 days, at most approximately 1 day, at most approximately 20 hours, at most approximately 10 hours, at most approximately 9 hours, at most approximately 8 hours, at most approximately 7 hours, at most approximately 6 hours, at most approximately 5 hours, at most approximately 4 hours, at most approximately 3 hours, at most approximately 3 hours, or at most approximately 10 days after the initiation of the first regulation (e.g., by at least a portion of the first gate unit, such as the first gene regulatory portion). It may occur in 2 hours, up to about 1 hour, up to about 50 minutes, up to about 40 minutes, up to about 30 minutes, up to about 20 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6 minutes, up to about 5 minutes, up to about 4 minutes, up to about 3 minutes, up to about 2 minutes, up to about 1 minute, up to about 50 seconds, up to about 40 seconds, up to about 30 seconds, up to about 20 seconds, up to about 10 seconds, up to about 9 seconds, up to about 8 seconds, up to about 7 seconds, up to about 6 seconds, up to about 5 seconds, up to about 4 seconds, up to about 3 seconds, up to about 2 seconds, or up to about 1 second.

[0164] In some cases, the number of gate units that need to be activated (e.g., sequentially activated) between the activation of the first regulation by the first gate unit and the subsequent activation of the second regulation by the second gate unit can be at least partially determined (e.g., substantially determined) of the timing between the first and second regulation. After the first regulation of the target gene is activated by the 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 the second gate unit for inducing the second regulation. After the first regulation of the target gene is activated by the first gate unit, 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 1 additional gate unit may need to be activated (e.g., sequentially activated) to activate the second gate unit for inducing the second regulation.

[0165] Cellular results can include regulation of multiple target genes. For example, results can include 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. Results can include regulation of 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 target gene. Each gene disclosed herein may be subject to 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 regulation. Each gene disclosed herein may be subject to 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 regulation. If one or more regulation of a target gene (e.g., an endogenous gene) induced by the heterologous gene circuit of this disclosure can be artificial regulation (or heterologous regulation), such artificial regulation would not occur in the cell in the absence of (i) the heterologous gene circuit and / or (ii) the activated portion of the heterologous gene circuit.

[0166] Multiple gate units can operate sequentially (e.g., each of the multiple gate units is activated sequentially). For example, a gate unit in the multiple gate units is activated to activate a subsequent gate unit in the multiple gate units. The sequential operation of the gate units can be linear. Alternatively, the sequential operation of the gate units can return to each other as inputs to form a loop. For example, the multiple gate units can cause feedback loops, such as positive feedback loops or negative feedback loops.

[0167] In some embodiments of any of the systems disclosed herein, the first gate unit may include a first gene regulatory portion, which may be activated to exhibit specific binding to a target gene to induce a first distinct regulation. Alternatively or additionally, the first gate unit may include a first gene regulatory portion, which may be activated to exhibit non-specific binding to a target gene to induce a first distinct regulation.

[0168] The first different regulation can induce changes (e.g., 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 different regulation can induce changes (e.g., increases or decreases) in the expression and / or activity levels of 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%, 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.

[0169] Compared to control expression and / or activity levels, the first distinct regulation (e.g., induced by a first phylum unit) disclosed herein can induce a change (e.g., increase or decrease) in the expression and / or activity levels of the target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than Approximately 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 20 times, at least or more than about 30 times, at least or more than about 40 times, at least or more than about 50 times, at least or more than about 60 times, at least or more than about 70 times, at least or more than about 80 times, at least or more than about 90 times, at least or more than about 100 times, at least or more than about 500 times, at least or more than about 1,000 times, at least or more than about 5,000 times, or at least or more than about 10,000 times. Compared to control expression and / or activity levels, the first distinct regulation can induce changes (e.g., increases or decreases) in the expression and / or activity levels 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, or at most or less than about 20-fold. Most or less than about 10 times, most or less than about 9 times, most or less than about 8 times, most or less than about 7 times, most or less than about 6 times, most or less than about 5 times, most or less than about 4 times, most or less than about 3 times, most or less than about 2 times, most or less than about 1 time, most or less than about 0.9 times, most or less than about 0.8 times, most or less than about 0.7 times, most or less than about 0.6 times, most or less than about 0.5 times, most or less than about 0.4 times, most or less than about 0.3 times, most or less than about 0.2 times, most or less than about 0.1 times.

[0170] Subsequently, a second distinct regulation as disclosed herein (e.g., induced by a second phylum unit) can induce additional alterations (e.g., increases, decreases, or selective attenuations) in the expression and / or activity levels 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%, and 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 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 regulation can induce additional alterations (e.g., increases or decreases) in the expression and / or activity levels of the target gene by up to approximately 1,000,000%, up to approximately 100,000%, up to approximately 9,000%, up to approximately 8,000%, up to approximately 7,000%, up to approximately 6,000%, up to approximately 5,000%, up to approximately 4,000%, up to approximately 3,000%, up to approximately 2,000%, up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, up to approximately 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 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%.

[0171] Compared to control expression and / or activity levels, additional alterations via a second distinct regulation can induce additional alterations (e.g., increases or decreases) in the expression and / or activity levels of the target gene by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than 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. Compared to control expression and / or activity levels, a second distinct regulation can induce additional alterations (e.g., increases or decreases) in the expression and / or activity levels of the target gene by at least or less than about 10,000-fold, at least or less than about 5,000-fold, at least or less than about 1,000-fold, at least or less than about 500-fold, at least or less than about 100-fold, at least or less than about 90-fold, at least or less than about 80-fold, at least or less than about 70-fold, at least or less than about 60-fold, at least or less than about 50-fold, at least or less than about 40-fold, at least or less than about 30-fold, at least or less than about 20-fold. The values ​​are: 10 times, up to or less than about 10 times, up to or less than about 9 times, up to or less than about 8 times, up to or less than about 7 times, up to or less than about 6 times, up to or less than about 5 times, up to or less than about 4 times, up to or less than about 3 times, up to or less than about 2 times, up to or less than about 1 time, up to or less than about 0.9 times, up to or less than about 0.8 times, up to or less than about 0.7 times, up to or less than about 0.6 times, up to or less than about 0.5 times, up to or less than about 0.4 times, up to or less than about 0.3 times, up to or less than about 0.2 times, up to or less than about 0.1 times.

[0172] When the expression and / or activity levels of the target gene reach the target level through the action of a first different regulation (e.g., through the design of a heterologous gene circuit), further changes through a second different regulation can occur.

[0173] Compared to control expression and / or activity levels, when the expression and / or activity levels of the target gene are altered (e.g., increased or decreased) by at least or more than about 0.1-fold, at least or more than about 0.2-fold, at least or more than about 0.3-fold, at least or more than about 0.4-fold, at least or more than about 0.5-fold, at least or more than about 0.6-fold, at least or more than about 0.7-fold, at least or more than about 0.8-fold, at least or more than about 0.9-fold, at least or more than about 1-fold, at least or more than about 2-fold, at least or more than about 3-fold, at least or more than about 4-fold, at least or more than about 5-fold, at least or more than Additional changes via a second different adjustment may occur when the value is approximately 6 times, at least or more than approximately 7 times, at least or more than approximately 8 times, at least or more than approximately 9 times, at least or more than approximately 10 times, at least or more than approximately 20 times, at least or more than approximately 30 times, at least or more than approximately 40 times, at least or more than approximately 50 times, at least or more than approximately 60 times, at least or more than approximately 70 times, at least or more than approximately 80 times, at least or more than approximately 90 times, at least or more than approximately 100 times, at least or more than approximately 500 times, at least or more than approximately 1,000 times, at least or more than approximately 5,000 times, or at least or more than approximately 10,000 times. Compared to control expression and / or activity levels, when the expression and / or activity levels of the target gene are altered (e.g., increased or decreased) by a first different regulatory action, 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 1 When the difference is 0 times, up to or less than about 9 times, up to or less than about 8 times, up to or less than about 7 times, up to or less than about 6 times, up to or less than about 5 times, up to or less than about 4 times, up to or less than about 3 times, up to or less than about 2 times, up to or less than about 1 time, up to or less than about 0.9 times, up to or less than about 0.8 times, up to or less than about 0.7 times, up to or less than about 0.6 times, up to or less than about 0.5 times, up to or less than about 0.4 times, up to or less than about 0.3 times, up to or less than about 0.2 times, or up to or less than about 0.1 times, further changes may occur through a second different adjustment.

[0174] Alternatively or additionally, a second distinct regulation (e.g., induced by a second phylum unit) as disclosed herein may induce changes (e.g., increases or decreases) in the expression and / or activity levels of additional target genes 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%, 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 distinct regulation can induce changes (e.g., increases or decreases) in the expression and / or activity levels of additional target genes by up to approximately 1,000,000%, up to approximately 100,000%, up to approximately 9,000%, up to approximately 8,000%, up to approximately 7,000%, up to approximately 6,000%, up to approximately 5,000%, up to approximately 4,000%, up to approximately 3,000%, up to approximately 2,000%, up to approximately 1,000%, up to approximately 900%, up to approximately 800%, up to approximately 700%, up to approximately 600%, up to approximately 500%, up to approximately 400%, up to approximately 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 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%. Cells may include prokaryotic cells, eukaryotic cells, or artificial cells.

[0175] In some embodiments, the second plurality of cells comprises KDR- and CD34+ hematopoietic endothelial cells, and further optionally, wherein the conversion rate from the first plurality of cells to the second plurality of cells is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% within about 14 days or about 7 days after exposure. In some embodiments, the conversion rate from the first plurality of cells to the second plurality of cells via a heterologous gene circuit is at least or at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times greater than the conversion rate in the absence of a heterologous gene circuit.

[0176] In some embodiments, the second plurality of cells comprises hematopoietic cells (HPCs) that are CD34+ and CD43+, and further optionally, wherein the conversion rate from the first plurality of cells to the second plurality of cells is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% within about 14 days or about 7 days after exposure. In some embodiments, the conversion rate from the first plurality of cells to the second plurality of cells via a heterologous gene circuit is at least or at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times greater than the conversion rate in the absence of a heterologous gene circuit.

[0177] In some embodiments, the second plurality of cells comprises hematopoietic cells (HPCs) that are CD34+ and CD45+, and further optionally, the conversion rate from the first plurality of cells to the second plurality of cells is at least or at most about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% within about 14 days or about 7 days after exposure. In some embodiments, the conversion rate from the first plurality of cells to the second plurality of cells via a heterologous gene circuit is at least or at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times greater than the conversion rate in the absence of a heterologous gene circuit.

[0178] Cells (e.g., initial cells to be modified into engineered cells as disclosed herein, final cell products generated from engineered cells as disclosed herein) may include muscle cells, immune cells, neurons, osteoblasts, endothelial cells, mesenchymal cells, epithelial cells, stem cells, secretory cells, blood cells, germ cells, nurturing cells, storage cells, enteroendocrine cells, pituitary cells, neurosecretory cells, ductal cells, odontoblasts, cementoblasts, glial cells, mesenchymal cells, mesodermal stem cells, endoderm stem cells, ectoderm stem cells, hematopoietic progenitor cells, mesodermal progenitor cells, mesenchymal stem cells (MSCs) or erythroid myeloid progenitor (EMP) cells.

[0179] 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, for example, US20080241194); myeloid cells such as granulocytes (basophils, eosinophils, neutrophils / segmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, coagulating cells / 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); and cells from the nervous system, including glial cells (astrocytes, microglia), large neurosecreting cells, astrocytes, and Boettcher cells.Cells of the respiratory system, including lung cells (type I and type II lung cells), 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 and peripheral cells), goblet cells, Panette cells, G cells, D cells, ECL cells, I cells, K cells, and S cells; enteroendocrine cells, including enterochromaffin cells, APUD cells, hepatocytes (hepatocytes and Kupffer cells), and cartilage / bone / muscle cells; bone cells, including osteoblasts, osteocytes, osteoclasts, and tooth cells (cementogenic cells and ameloblasts); chondrocytes, including chondrogenic cells and chondrocytes; skin cells, including hair cells, keratinocytes, and melanocytes (nevus cells); muscle cells, including myocytes; and cells of the urinary system, including podocytes, juxtaglobinocytes, and intraglobinic / extraglobinic mesangial cells. Cells, including brush border cells of the proximal renal tubules and dense macula cells; reproductive system cells, including sperm, Sartori cells, testicular interstitial cells, and oocytes; and other cells, including adipocytes, fibroblasts, tendon cells, epidermal keratinocytes (differentiated epidermal cells), epidermal basal cells (stem cells), keratinocytes of the nails and toenails, nail bed basal cells (stem cells), medullary hair stem cells, cortical hair stem cells, keratinized hair stem cells, keratinized root sheath cells, root sheath cells of Huxley's layer, and root sheath cells of Henle's layer. Cells, outer root sheath cells, hair matrix cells (stem cells), moist stratified barrier epithelial cells, surface epithelial cells of stratified squamous epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cells (stem cells) of epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urinary epithelial cells (lining the urethra, bladder and ureter), exocrine epithelial cells, salivary gland mucin cells (rich in polysaccharide secretions), salivary gland serous cells (rich in glycoprotein proteases), von Ebner cells in the tongueEbner's gland cells (washing taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), ceruminous gland cells in the ear (wax secretion), dark cells of eccrine sweat glands (glycoprotein secretion), light cells of eccrine sweat glands (small molecule secretion), apocrine sweat gland cells (odor secretion, sex hormone sensitive), eyelid eyelash gland cells (dedicated sweat glands), sebaceous gland cells (lipid-rich sebum secretion), Baumann's gland cells in the nose (washing olfactory epithelium), Brenner's gland cells in the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components, including fructose from motile sperm), prostate cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin's gland cells (vaginal lubricant secretion), Litrex's gland cells (mucus secretion), endometrial cells (carbohydrate secretion), separated goblet cells of the respiratory and digestive tracts (mucus secretion), gastric lining mucus cells (mucus secretion), gastric gland zymogen cells (pepsinogen secretion), gastric gland acid-secreting cells (salt secretion). Acid secretion), pancreatic acinar cells (bicarbonate and digestive enzyme secretion), Panthen cells of the small intestine (lysozyme secretion), type II lung cells of the lung (surfactant secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, somatic cells, prolactin cells, thyroid-stimulating hormone cells, gonadotropin cells, adrenocorticotropic hormone cells, intermediate pituitary cells, large neurosecreting cells, intestinal and respiratory tract cells, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, parathyroid chief cells, eosinophilic cells, adrenal cells, chromaffin cells, testicular interstitial cells, follicular endometrial cells, luteal cells of ruptured follicles, granulosa luteal cells, membranous luteal cells, juxtaglomerular cells (renin secretion), dendritic cells of the kidney, metabolic and storage cells, barrier function cells (lung, intestine, exocrine glands and urogenital tract), kidney cells, type I lung cells (lining air space of the lung) Air space), pancreatic duct cells (vacuole cells), streaked duct cells (of sweat glands, salivary glands, mammary glands, etc.), duct cells (of seminal vesicles, prostate glands, etc.), epithelial cells lining closed internal body cavities, propulsive ciliated cells, extracellular matrix secretory cells, contractile cells; skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, erythrocytes (red blood cells)Cells, megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, 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 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. Transducer cells, autonomic neurons, sensory organ and peripheral neurons, supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oocytes (Oogonium / Oocyte), sperm cells, spermatocytes, spermatogonia (sperm stem cells of spermatocytes), sperm, fertilizing cells, follicular cells, settler cells (in the testes), thymic epithelial cells, interstitial cells and interstitial kidney cells.

[0180] Stem cells can include induced pluripotent stem cells (iPSCs), pluripotent stem cells (PSCs), embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), erythroid myeloid progenitor cells (EMPs), intermediate progenitor cells, hematopoietic stem cells (HSCs), hematopoietic progenitor cells, muscle stem cells, neural stem cells, epithelial stem cells, epidermal stem cells, breast stem cells, intestinal stem cells, neural crest stem cells, or testicular stem cells.

[0181] Stem cells (e.g., pluripotent stem cells) as described herein can be engineered to disrupt the expression of T cell receptors (TCRs) (e.g., TCRα, TCRβ, CD3γ, CD3δ, CD3ε, and / or CD247) and / or major histocompatibility complexes (MHCs) (e.g., MHC class I, MHC class II, and / or MHC class III, where human MHCs may be referred to as human leukocyte antigens (HLA)). For example, genes encoding at least a portion of the TCR or MHC can be edited (e.g., via non-homologous end joining (NHEJ) or homologous recombination (HR) mutations) to disrupt expression. Disruption of TCR and / or MHC expression can reduce or eliminate (i) graft-versus-host disease (GvHD) and / or (ii) host-versus-graft disease (HvGD) when administered to subjects in need of differentiated cells as described herein. For example, disrupting TCR and / or MHC expression can reduce or eliminate the effects of administering differentiated cells as allogeneic cells (e.g., allogeneic T cells) to HLA mismatched (or partially mismatched) individuals. Alternatively, differentiated cells can be administered as allogeneic cells to HLA-matched individuals (e.g., without disrupting TCR and / or MHC expression).

[0182] Disruption of TCR and / or MHC expression can occur before, concurrently with, or after the transformation of multiple stem cells (e.g., pluripotent stem cells) into hematopoietic lineage cells (e.g., hematopoietic stem cells). Disruption of TCR and / or MHC expression can also occur before, concurrently with, or after the introduction (e.g., expression) of heterologous gene circuits into multiple stem cells. Disruption of TCR and / or MHC expression can occur before, concurrently with, or after the activation of heterologous gene circuits in multiple stem cells. In some cases, disruption of TCR and / or MHC expression can occur after the generation of hematopoietic lineage cells.

[0183] Different aspects of this disclosure provide engineered cells programmed to induce desired levels (or profiles) of expression of one or more target genes in cells.

[0184] In some embodiments, the engineered cells of this disclosure (e.g., engineered hematopoietic progenitor 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., phylogenetic units, phylogenetic portions, activation portions, etc.) can be introduced during any stage (or cell state) between and including: (a) isolated stem cells and (b) their differentiated hematopoietic progenitor cell state.

[0185] In some cases, engineered hematopoietic progenitor cells can be derived from PSCs, and heterologous gene circuits and / or components thereof (e.g., heterologous gate units, heterologous activation portions, heterologous gate portions, etc.) can be introduced into the cells in (A) a PSC state, (B) a mesodermal stem cell state, (C) a hematopoietic progenitor cell state, or (D) any other intermediate cell state. During one of (A), (B), (C), or (D), the heterologous gene circuit and / or components thereof (e.g., heterologous gate units, heterologous activation portions, heterologous gate portions, etc.) can be introduced into the cells once. Alternatively, during two, three, or all of (A), (B), (C), or (D), the heterologous gene circuit and / or components thereof (e.g., heterologous gate units, heterologous activation portions, heterologous gate portions, etc.) can be introduced into the cells multiple times.

[0186] The engineered cells disclosed herein (e.g., engineered hematopoietic progenitor cells) can be used (e.g., administered) to treat subjects in need. Subjects may have or 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 the subject, and such cells can be cultured in vitro and genetically modified to generate any subject-engineered cells (e.g., any immune cells) disclosed herein. Subsequently, the engineered immune cells can be administered to the subject for adaptive immunotherapy. Thus, the engineered cells for the subject in need can be autologous. Alternatively, the engineered cells for the subject can be allogeneic (e.g., allogeneic stem cell transplantation, allogeneic adoptive immunotherapy, etc.).

[0187] The cells or constructs disclosed herein can be used (e.g., administered) in pharmaceutical formulations. Pharmaceutical formulations may further include additional therapeutic agents. These additional therapeutic agents may include chemotherapeutic agents, immunosuppressants, and / or antibiotics.

[0188] Chemotherapy agents, also known as antitumor agents, are a type of cancer treatment used to directly or indirectly inhibit the growth and proliferation of cancer cells. Chemotherapy agents can be alkylating agents (e.g., oxazaphosphorine, nitrogen mustard, imidazotetrazine, nitrosourea, alkyl sulfonates, hydrazine, or platinum-based reagents), antimetabolites (e.g., antifolate agents, pyrimidine antagonists, purine antagonists, or ribonuclease reductase inhibitors), topoisomerase inhibitors (e.g., topoisomerase I 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.

[0189] Immunosuppressants are agents that reduce the immune response. Non-limiting examples of immunosuppressants may 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, biologics targeting B cell growth factors, T cell, cytokine, or JAK inhibitors.

[0190] Antibiotics are agents that destroy or inhibit the growth of microorganisms. Non-limiting examples of antibiotics may include tetracycline, oxytetracycline, methacycline, doxycycline, minocycline, erythromycin, lincomycin, penicillin G, clindamycin, kanamycin, chloramphenicol, neomycin, streptomycin, norfloxacin, ciprofloxacin, ofloxacin, gapfloxacin, levofloxacin, sparfloxacin, ampicillin, carbenicillin, methicillin, cephalosporins, vancomycin, bacitracin, gentamicin, fusidic acid, ciprofloxacin and other quinolones, erythromycin, gentamicin, sulfonamides, trimethoprim, dapsone, isoniazid, teicoplanin, avopacin, synercidin, and vitamin. Virginiamycin, piperacillin, ticarcillin, cefepime, cefpirome, rifampin, pyrazinamide, enrofloxacin, amikacin, netilmicin, imipenem, meropenem, linezolid, cefuroxime, ceftriaxone, cefadroxil, cefazolin, ceftazidime, cefotaxime, roxithromycin, cefaclor, cefalexin, cefoxitin, amoxicillin, co-amoxiclavic acid, mupirocin, cloxacillin, and co-trimoxazole.

[0191] Pharmaceutical formulations may further include excipients. Excipients may be buffers, carriers, stabilizers, solubilizers, fillers, preservatives, diluents, mediators, detergents, salts, peptides, surfactants, oligosaccharides, amino acids, adjuvants, carbohydrates, and / or swelling agents.

[0192] As disclosed herein, engineered cells can be administered to a subject before, simultaneously with, or after the activation of heterologous genetic circuits in engineered stem cells. For example, engineered cells can be activated after being administered to a subject, for instance, by administering an activator of a heterologous genetic circuit to the subject.

[0193] Subjects may be treated (e.g., administered) with the engineered cell (e.g., engineered hematopoietic progenitor cell) population disclosed herein for at least or more than about 1 dose, at least or more than about 2 doses, at least or more than about 3 doses, at least or more than about 4 doses, at least or more than about 5 doses, at least or more than about 6 doses, at least or more than about 7 doses, at least or more than about 8 doses, at least or more than about 9 doses, or at least or more than about 10 doses. Alternatively or additionally, subjects may be treated (e.g., administered) with the engineered cell (e.g., engineered hematopoietic progenitor cell) populations disclosed herein 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.

[0194] The target cells, target tissues, target conditions, or target diseases of the subject can be treated using any of the methods disclosed herein.

[0195] Non-limiting examples of target tissues may include cells that can be obtained from the object, such as hematopoietic progenitor cells. Non-limiting examples of objects include humans, dogs, cats, mice, rats, and their transgenic species. Examples of samples from objects from which cells can be derived include, but are not limited to, skin, heart, lungs, kidneys, bone marrow, mammary glands, pancreas, liver, muscle, smooth muscle, bladder, gallbladder, colon, intestine, brain, prostate, esophagus, thyroid gland, serum, saliva, urine, gastric juice and digestive juices, tears, feces, semen, vaginal fluid, interstitial fluid derived from tumor tissue, eye discharge, sweat, mucus, earwax, oil, glandular secretions, cerebrospinal fluid, hair, nails, plasma, nasal swabs or nasopharyngeal washes, cerebrospinal fluid, cerebrospinal fluid, tissue, pharyngeal swabs, biopsy, amniotic fluid, amniotic fluid, umbilical cord blood, emphatic fluids, cavity fluid, sputum, pus, microbiota, meconium, breast milk, and / or other excretions or body tissues.

[0196] The target disease of the subject can be cancer or tumor. Non-limiting examples of cancer may include cancerous cells, including acanthoma, acinar carcinoma, acoustic neuroma, acral lentigines-like melanoma, acral hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryocytic leukemia, acute monocytic leukemia, acute myeloid leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, ameloma, adenocarcinoma, adenoid cystic carcinoma, adenoma, odontogenic adenoma-like tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, acinar soft tissue sarcoma, etc. Ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, undifferentiated thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdomyosarcoma, basal cell carcinoma, basoid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini ductal carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner's tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary origin, carcinoid tumor, carcinoma, carcinoma in situ, penile carcinoma, cancer of unknown primary origin, carcinosarcoma, Castleman's disease Diseases, embryonic tumors of the central nervous system, cerebellar astrocytoma, brain astrocytoma, cervical cancer, chondroma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease.Disease), dermatofibrosarcoma protuberans, dermoid cyst, connective tissue proliferative small round cell tumor, diffuse large B-cell lymphoma, dysembryonic neuroepithelial tumor, embryonal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, olfactory neuroblastoma, Ewing's family tumor, Ewing's family sarcoma, Ewing's sarcoma, extracranial germ cell tumor, gonadal extragerminoma, extrahepatic bile duct carcinoma, extramammary Paget's disease, fallopian tube cancer, fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid carcinoma, gallbladder cancer, gallbladder cancer Ganglioglioma, ganglioma, 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, cerebral glioma, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast cancer and ovarian cancer syndrome, Hodgkin lymphoma. 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, renal cancer, Klatskin tumor, Krukenberg tumor, laryngeal cancer, laryngeal cancer, malignant lentigines melanoma, leukemia, lip and oral cancer, liposarcoma, lung cancer, corpus luteum tumor, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, bone malignant fibrous histiocytoma, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyosarcoma, malignant salamander tumor Tumor), MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck cancer (Metastatic Squamous Neck Cancer with OccultPrimary, metastatic urothelial carcinoma, mixed Müllerian tumor, monocytic leukemia, oral cancer, myxoma, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorders, myxoma, nasal cavity carcinoma, nasopharyngeal carcinoma, nasopharyngeal carcinoma, vegetation, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular tumors. Oncology), oligodendroastrocytoma, oligodendroglioma, eosinophilic cytoma, optic nerve sheath meningioma, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low-grade potential ovarian tumor, Paget's disease of the breast, superior sulcus tumor of the lung, pancreatic cancer, papillary thyroid carcinoma, papilloma, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor (Pineal Parenchymal Tumor of Intermediate) Differentiation), pineal blastoma, pituitary adenoma, pituitary tumor, plasma cell tumor, pleural pulmonary blastoma, polyembryoma, precursor T-cell lymphoma, primary central nervous system lymphoma, primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, respiratory tract cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma, Richter's transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannoma, sebaceous gland carcinoma, secondary vegetations, 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, sootoma (SootSpinal cord tumors, spinal tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial diffuse melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphoblastic 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 neoplasm, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, visual pathway glioma, vulvar cancer, Waldenstrom's macroglobulinemia. Macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. 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 hematopoietic lineage cancer, such as lymphoma. The antigen may be a tumor-associated antigen.

[0197] This disclosure also provides compositions comprising engineered gene circuits as disclosed herein. The composition may further comprise an actuator for the heterologous gene circuit. This disclosure also provides kits comprising the compositions. The kits may further comprise activators of the heterologous gene circuits. The activators may be in the same composition as the engineered cells. Alternatively or additionally, the activators may be in a different and separate composition from the engineered cells. Example

[0198] Example 1: Differentiation of hematopoietic progenitor cells In this embodiment, multiple heterologous gene circuits (HGCs) are used to differentiate induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells.

[0199] iPSC maintains Free iPSCs (Gibco, CatA18945) were maintained in fully Essential 8™ Flex medium (Gibco, Catalog # A2858501) or StemFlex™ medium (Gibco, Catalog # A3349401) on flasks coated with fibronectin (Gibco, Catalog # A31804), and passaged with velene (Gibco, Catalog # 15040066) to maintain 50%-90% confluence to enhance cell growth while maintaining cell clumps.

[0200] nuclear transfection On day 0 of the experiment, iPSC cells were collected as single-cell suspensions using ACCUTASE™ (Stemcell Technologies, catalog #07920). Cells were resuspended at 1.2e9 cells / ml in P3 primary Nucleofector cells. TM In solution, and using optimized pulse codes, nuclear transfection was performed in 96-well nucleocuvettes (Lonza, catalog # V4SP-3096) with 8e5 cells per well on a 96-well shuttle system (catalog #AAF-1003B / S) on a Lonza 4D Nucleofector.

[0201] Cells were nuclear transfected with plasmids encoding Cas9-VPR, a core cascade, and gene-targeting spacers. Cas9-VPR is a Cas transcription regulator system that, depending on the specific gNA used, can be used for both cleavage (e.g., phylogenetic plasmids, gene-regulatory plasmids) and non-cleavage gene regulation (e.g., CRISPR activation of target endogenous genes, CRISPR repression of target endogenous genes). The activatable gNA used is simply a polyT continuous tract with a constant core cascade that performs four sequential steps. Nucleotide gNA spacers were used to target the genome according to the sequences in Table 1. Figure 2 The gene targets used in each heterologous gene circuit cascade are described. Steps refer to the sequence in which the expected gene is expressed as a result of Cas9-VPR-mediated targeting and activation.

[0202] Table 1: Spacer sequence

[0203] Cell culture Five nuclear-transfected cells per well were cultured in 24-well tissue culture plates treated with anti-adhesion washing solution (Stemcell Technologies, catalog #07010) and rotated at 100 RPM in a humidified 5% CO2 tissue culture incubator under standard normoxic conditions, 500 μL 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), 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).

[0204] On day 1, the culture was treated with StemPro-34 (Gibco, catalog # 10639011), 1X penicillin-streptomycin (Gibco catalog # 15140122), 2 mL Glutamax (Gibco, catalog # 35050061), 50 μg / mL ascorbic acid (Santa Cruz Biotechnology, catalog # sc-39430), 4 mM monothioglycerol (Sigma-Aldrich, catalog # M1753), 1X 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) Underwent a complete culture medium change. On day 2, 6 μM SB431542 (Selleckchem, catalog # 101762-616) was added to the culture. On day 4, the culture was treated with StemPro-34 (Gibco, catalog # 10639011), 1X penicillin-streptomycin (Gibco catalog # 15140122), 2 mL Glutamax (Gibco, catalog # 35050061), 50 μg / mL ascorbic acid (Santa Cruz Biotechnology, catalog # sc-39430), 4 mM 1-thioglycerol (Sigma-Aldrich, catalog # M1753), 1X 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) underwent changes in culture medium.

[0205] Flow cytometry Cultures were analyzed by flow cytometry on a Penteon (Agilent, catalog #2010284AA) on days 3, 5, and 9. Embryoids were harvested into 96-well plates (Nest, catalog #503162) and depolymerized at 37°C in 500 ml TrypLE™ Express enzyme (Gibco, catalog #12605028) for 20–30 min, followed by mechanical disruption by pipetting. The TrypLE™ Express enzyme was quenched using DMEM-F12 (Stemcell Technologies, catalog #36254). Cells were stained with Zombie Aqua™ Fixable (Biolegend, catalog # 423102, 1:2000) for 20 min at room temperature, washed with 1XDPBS, 2% FBS, and 0.02% NaN3, and blocked with human IgG (Lee Biosolutions catalog # 340-21) on ice for 10 min. Cells were stained on ice for 30 minutes with PDGFR-BV421 (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), and CD45-AF700 (Biolegend, catalog # 304024, 1:200). Cells were washed with 1X DPBS, 2% FBS, and 0.02% NaN3. Cells were resuspended in 1X DPBS, 2% FBS, and 0.02% NaN3 for analysis. Data were analyzed using FlowJo™ v10.8 software (BD Life Sciences). Dead cells and duplexes were excluded from the analysis using Zombie Aqua and the duplex identification gate.

[0206] Aggregate flow cytometry data to form Figures 4A-4D It depicts the relative frequency of cells among appropriately sized, viable, singlet cells on day 5 of culture. Scatter plots were created at different time points to represent the magnitude of the increase in hematopoietic gene expression induced by HGC relative to the control. Figure 3 Visualization.

[0207] Exemplary results from flow cytometry analysis are shown in Figures 5A-5B The diagram illustrates the frequency of cells expressing the indicated surface markers in live, single-peak cells on day 5 of culture, compared to DNA-free controls (e.g., undergoing nuclear transfection in the absence of exogenous DNA) for HSCs (Cell Algorithm) #7 and #12. Summary charts are available in... Figures 5C-5D As shown in the image.

[0208] Example 2: Multi-step cascading The systems and methods provided herein (e.g., based on polynucleotide sequences encoding activatable sgRNAs containing one or more polyT sequences) can be used to induce sequentially delineated multi-step cascade effects, whereby the expression of endogenous gene products can be activated at any step of the cascade.

[0209] For example, a multi-step cascade effect can be a 10-step cascade effect, such as a 10-step forward cascade or a 10-step reverse cascade.

[0210] Experimental details Overall, the experiments involved preparing a mixture of plasmid DNAs encoding components of the proGuide cascade, then introducing those DNAs into cells (e.g., HEK 293 cells) via nuclear transfection, and finally using flow cytometry to detect cell surface gene products (e.g., CXCR4) to assess the effect of activation of the target gene products at different time points.

[0211] The basic components of the cell algorithm (e.g., Cas9-VPR expression plasmid, proGuide encoding plasmid) are mixed, and a GFP expression plasmid is included to identify transfected cells. To construct plasmid combinations that activate endogenous genes at different steps in the proGuide cascade, the mixture of cascade plasmid DNA uses the components described in Tables 2 and 3. The core cascade plasmid is progressively included in the transfection mixture as follows to add additional steps in the cascade. For example, the first step (e.g., step 1) condition includes no proGuide and an sgRNA with a spacer sequence that targets the 5' and 3' cleavage sites within the second step (e.g., step 2) proGuide plasmid. The second step (e.g., step 2) condition includes all plasmids from the first step (e.g., step 1) condition plus the proGuide plasmid described for the second step (e.g., step 2). The third step (e.g., step 3) condition includes all plasmids from the second step (e.g., step 2) condition plus the proGuide plasmid described for the third step (e.g., step 3), and so on. To maintain a constant quality of each proGuide plasmid DNA for all transfections and to maintain a constant total DNA quality, genetically inert plasmid DNA (e.g., pUC19) is used as a "filler" for conditions with fewer proGuide plasmids.

[0212] To activate the expression of an endogenous gene product (e.g., CXCR4), a 14-nt spacer sequence is used to target the Cas9-VPR to the promoter region of the gene (e.g., CXCR4). For activation at the first step (e.g., step 1), gene (e.g., CXCR4) activation is stimulated by sgRNA containing the gene's associated spacer (e.g., a 14-nt CXCR4 spacer). For subsequent steps, a proGuide plasmid containing the gene's associated spacer (e.g., a 14-nt CXCR4 spacer) is added to the plasmid DNA mixture. By matching the 5' and 3' cleavage sites of a specific step in the cascade with the 5' and 3' cleavage sites in the gene (e.g., CXCR4) activation proGuide, gene (e.g., CXCR4) activation is effectively programmed to occur at a specific step in the cascade for each condition / plasmid DNA mixture.

[0213] A mixture of plasmid DNA was introduced into cells (e.g., hematopoietic cells) using a nuclear transfection system (e.g., Lonza 4D) following standard procedures. The transfected cells were plated (e.g., in multi-well tissue culture plates) and maintained using standard mammalian tissue culture methods. At specified time points following nuclear transfection (e.g., 12, 24, 36, 48, and 72 hours), the cells were treated for flow cytometry and detection of cell surface expression of the gene product (e.g., CXCR4). For each condition, independent replicaseries (e.g., n = 4) (nuclear transfection) were examined by flow cytometry.

[0214] result The aforementioned multi-step cascade method is partially used to generate hematopoietic progenitor cells (HPCs). The multi-step cascade produces a series of HPC scalability and HPC yields. HPCs derived from the multi-step cascade possess the ability to remain in the HPC state and acquire lymphoid cell potential. Some cell algorithms promote greater loss of CD34 expression, maintain CD45 expression, and lack an increase in CD7. Some cell algorithms enable HPCs to exit the progenitor state and promote an increase in CD7+ cell yield. The CD34+CD45+ count, CD34-CD45+ count, total CD45 count, and / or CD7 count after the multi-step cascade can be... Figures 13A-13D The frequency of triple-positive cells obtained in vitro was 9.18% for CD34+ cells and 8.77% for CD43+ cells.

[0215] Compared to control conditions, the degree to which the cell algorithm (described in Table 4) induced iPSC differentiation into early stages of the cell lineage was comparable, as indicated by the increase in CD309+ (KDR) cells lacking compared to control conditions. Figures 8A-8D In contrast, some (but not all) cells processed by the cell algorithm exhibited the hematopoietic endothelial stage (KDR-CD34+). Figures 9A-9D , Figures 10A-10D ) and HPC phase (CD34+ CD43+; Figures 11A-11D ; and CD34+ CD45+; Figures 12A-12D A significant increase in ) was observed. CGO178 induced the greatest increase in HPC stage cells.

[0216] The control conditions described in this article include “directed differentiation” (e.g., “directed differentiation,” “directed differentiation 1,” “directed differentiation 2”), in which cells undergo treatment and DNA addition, but are not treated with electric field pulses from the nuclear transfection instrument (e.g., “no electrical stimulation”). The control condition “DNA-free” described in this article means that cells undergo treatment and electrical pulses from the nuclear transfection instrument, but plasmid DNA is not added to the cells during nuclear transfection. The control condition “GFP + core cascade” described in this article means that cells are nuclear transfected with a GFP expression plasmid, Cas9-VPR, and a proGuide that runs through all steps of the process, but does not include a gene-targeting proGuide. For the control condition “Cas-VPR control” described in this article, cells are engineered with a control cell algorithmic gene loop designed to target one or more genes (e.g., CXCR4 and CD105) that are different from any of the genes listed in Table 4.

[0217] The identity and activation order of the TFs activated by the cell algorithm are crucial for HPC differentiation. A comparison between CGO178 and CGO181 shows that CGO178 includes the five TFs activated in step 6 (Table 4). The addition of these TFs increases the percentage of hematopoietic endothelial cells and HPCs in the culture. Figure 10D , Figure 11D , Figure 12D A comparison of CGO176, CGO181, and CGO192 reveals a difference in their sequential activation of the same TF set (CGO176 and CGO181) compared to the one-time total activation of TFs by CGO192 (Table 4). The sequential activation of TFs by CGO176 and CGO181 resulted in a higher percentage of HPC cell types compared to the one-time total activation by CGO192. Figure 11D , Figure 12D For the production of hematopoietic cells (including lymphoid marker cells), similar improvements were observed from cell algorithms that sequentially activate TF compared to a one-time total activation of TF and a control with no activation. Figures 13A-13D ).

[0218] Furthermore, CGO178 was compared with a baseline scheme as described in Sugimura et al. (Haematopoietic stem and progenitor cells from human pluripotent stem cells. Nature 545, 432–438 (2017).). Compared to the baseline, the method disclosed in this paper yielded a higher and faster conversion rate. Figures 14A-14B ).

[0219] Table 2: Examples of heterologous gene circuits used to test multi-step cascades (e.g., 10-step forward cascades).

[0220]

[0221] Table 3: Examples of additional heterologous gene circuits used to test multi-step cascades (e.g., 10-step reverse cascades based on reversing the order of downstream / upstream cleavage site pairs from the heterologous gene circuits in Table 2).

[0222]

[0223] Table 4: Gene Activation Procedure for HPC Cell Algorithm

[0224] Table 5: Spacer sequences used for target gene activation Implementation Plan

[0225] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[0226] Implementation Scheme 1. A method for guiding the differentiation of multiple stem cells, the method comprising: The plurality of stem cells are contacted with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially induce a plurality of different regulation of target genes in the plurality of stem cells, each of the plurality of different regulation being necessary for the induction of differentiation of the plurality of stem cells, but insufficient individually to achieve the induction, wherein the plurality of gate units include: (i) a first gate unit, which, upon activation of the heterologous gene circuit, induces a first distinct regulation among the plurality of distinct regulations; and (ii) A second gate unit, which is activated upon activation of the heterologous gene circuit, to induce a second distinct regulation among the plurality of distinct regulation, wherein the second distinct regulation is induced after the first distinct regulation, such that both the first distinct regulation and the second distinct regulation increase or decrease the expression and / or activity level of the target gene in the cell. The target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and Upon contact, the plurality of gate units operate in concert to guide the differentiation of the plurality of stem cells. Optional, wherein: (1) The plurality of stem cells include pluripotent stem cells; and / or (2) After the contact, the plurality of gate units operate in concert to achieve the differentiation of the plurality of stem cells into tissue-specific progenitor cells. Further, optionally, wherein: i. The tissue-specific progenitor cells include hematopoietic lineage cells; Further, optionally, wherein: 1. Upon contact, the plurality of gate units operate in concert to guide the plurality of stem cells into hematopoietic stem cells; and / or 2. The hematopoietic lineage cells are characterized as CD45+; and / or 3. The hematopoietic lineage cells are characterized as CD34+CD43-CD45+; and / or ii. The activation of the heterologous gene circuit, wherein at least about 10%, at least about 15%, at least about 20%, at least about 30%, or at least about 40% of the plurality of stem cells differentiate into the tissue-specific progenitor cells; and / or iii. The differentiation is observed in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 6 days; and / or (3) The ETS transcription factors include ETS1; and / or (4) The ETS transcription factors include ETV2; and / or (5) The ETS transcription factors include LMO2; and / or (6) The plurality of gate units are pre-configured such that one of the plurality of gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of an additional target gene, wherein the additional target gene includes a T-box transcription factor (TBX) or a homeobox protein. Further, optionally, wherein: i. The regulation of the expression and / or activity levels of the additional target genes is induced prior to at least one of the plurality of different regulations of the target genes encoding the ETS transcription factor; and / or ii. The TBX includes at least two different types of TBX; and / or iii. The TBX includes TBXT and / or TBX6; and / or iv. The homeobox proteins include PRD-type homeobox proteins; and / or v. The homeobox protein includes MIXL1; and / or (7) The plurality of gate units are not pre-configured to regulate the expression and / or activity levels of one or more members selected from TBXT, TBX6, and MIXL1 prior to the plurality of different regulation of the target gene encoding the ETS transcription factor; and / or (8) The plurality of gate units are pre-configured such that one of the plurality of gate units is activated upon activation of the heterologous gene circuit to regulate the expression and / or activity levels of different target genes, wherein the different target genes include GATA transcription factors or basic helical-loop-helical transcription factors (bHLH); and / or Further, optionally, wherein: i. The GATA transcription factor includes GATA2; and / or ii. The bHLH includes SCL; and / or (9) The contact occurs in a culture medium that is substantially free of exogenous thrombopoietin (TPO) and / or exogenous FLT-3 ligand (FLT3L); and / or (10) The contact occurs in a culture medium that is substantially free of exogenous interleukins (ILs). Further, optionally, wherein: i. The exogenous IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (11) The plurality of gate units includes a third gate unit, which is activatable upon activation of the heterologous gene circuit to induce a third distinct regulation among the plurality of distinct regulation, wherein the third distinct regulation is induced after the first distinct regulation and the second distinct regulation, such that the first distinct regulation, the second distinct regulation, and the third distinct regulation all increase or decrease the expression and / or activity level of the target gene in the plurality of stem cells; and / or (12) The plurality of gate units are pre-configured such that the expression and / or activity level of the target gene is sequentially regulated by no more than three different gate units from the plurality of gate units; and / or (13) No more than three different gate units among the plurality of gate units are pre-configured to sequentially regulate the expression and / or activity levels of the target gene; and / or (14) Both the first different regulation and the second different regulation increase the expression and / or activity level of the target gene in the cell; and / or (15) Both the first different regulation and the second different regulation reduce the expression and / or activity level of the target gene in the cell; and / or (16)(i) The first gate unit includes a first gene regulatory portion, which is activated upon activation of the first gate unit to induce the first different regulation via the specific binding of the first gene regulatory portion to the target gene, and (ii) The second gate unit includes a second gene regulatory portion, which is activated upon activation of the second gate unit to induce the second distinct regulation via the specific binding of the first gene regulatory portion to the target gene. Further, optionally, wherein: i. The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a polynucleotide sequence substantially identical to that of the target gene; and / or ii. The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene; and / or iii. The second gate unit further includes a second gate portion, which is activated upon the 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. Further, optionally, wherein: 1. The first gate unit further includes a first gate portion, which is activated after the activation of the first gate unit, to: (a) Activation of the first gene regulatory region is induced by the specific binding of the first phylum portion to the first gene regulatory region, and (b) Activation of the second gate portion is induced via specific binding of the first gate portion to the second gate portion; and / or 2. The activation portion is capable of: (a) Activation of the first gene regulatory portion is induced via the specific binding of the activation portion to the first gene regulatory portion, and (b) Inducing the activation of the second gate portion; and / or (17) 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 contain activatable gNA. Specifically, after gNA activation, gNA forms a complex with a nuclease. Further, optionally, wherein: i. The activatable gNA contains a non-canonical termination sequence; and / or ii. The endonuclease comprises a CRISPR / Cas protein, further optionally, wherein: 1. The CRISPR / Cas protein has essentially no cleavage activity; and / or 2. The CRISPR / Cas protein is coupled to a transcription activator; and / or (18) The target genes include multiple types of ETS transcription factors, wherein the first different regulation via the first gate unit is the regulation of a first type of ETS transcription factor, and wherein the second different regulation via the second gate unit is the regulation of a second type of ETS transcription factor. Further optionally, wherein (i) the first type of ETS transcription factor includes one or more members selected from ETS1, ETV2, LMO2 and combinations thereof, and (ii) the second type of ETS transcription factor includes ERG.

[0227] Implementation Scheme 2. A method for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), the method comprising: The first plurality of cells are cultured in vitro in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL). Within approximately 14 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+. Optionally, the culture includes contacting a target gene in the first plurality of cells with a heterologous gene regulatory portion to modulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor.

[0228] Implementation Scheme 3. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The target gene in the first plurality of cells is brought into contact with the regulatory portion of a heterologous gene to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor. The contact enables the transformation from the first plurality of cells to the second plurality of cells. Optional, wherein: (1) Within approximately 14 days following the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+; and / or (2) Within approximately 14 days following the contact, an additional conversion rate of at least approximately 5% from the first plurality of cells to the second plurality of cells, wherein the second plurality of cells are CD34+; and / or (3) The contact in vitro occurs in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L) and interleukin (IL).

[0229] Implementation Scheme 4. The method as described in any one of Implementation Scheme 1, Implementation Scheme 2 and Implementation Scheme 3, further optionally, wherein: (1) The culture medium is substantially free of the TPO; and / or (2) The culture medium is substantially free of the FLT3L; and / or (3) The culture medium is substantially free of the IL. Further optionally, the IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (4) The second plurality of cells are CD34+CD43-CD45+; and / or (5) Observe the conversion rate approximately 10 days, 7 days, or 6 days after the culture or contact; and / or (6) The conversion rate is at least about 10% or at least about 15%; and / or (7) Observe the additional conversion rate over approximately 6 days or approximately 5 days; and / or (8) The additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%; and / or (9) Within approximately 7 days following the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+); and / or (10) Within approximately 7 days following the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+); and / or (11) Within approximately 7 days following the culture, the additional conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%, wherein the second plurality of cells are CD34+; and / or (12) Within approximately 7 days following the culture, the additional conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 10%, wherein the second plurality of cells are CD34+; and / or (13) The ETS transcription factors include one or more members selected from ETS1, ETV2 and / or LMO2; and / or (14) The heterologous gene regulatory portion increases the expression and / or activity level of the target gene; and / or (15) The heterologous gene regulatory portion reduces the expression and / or activity level of the target gene; and / or (16) The target gene is endogenous for the first plurality of cells; and / or (17) The method further includes contacting an additional target gene in the first plurality of cells with an additional heterologous gene regulatory portion to regulate the expression and / or activity level of the additional target gene, thereby achieving the conversion, wherein the additional target gene includes one or more members selected from T-box transcription factors (TBX), homeobox proteins, GATA transcription factors, and basic helical-loop-helical transcription factors (bHLH). Further, optionally, wherein: a. The additional heterologous gene regulatory portion enhances the expression and / or activity level of the additional target gene; and / or b. The additional heterologous gene regulatory portion reduces the expression and / or activity level of the additional target gene; and / or c. The additional target genes are endogenous to the first plurality of cells; and / or (18) The heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to the target gene; and / or (19) The additional heterologous gene regulatory portion comprises (i) nucleic acid molecules and / or (ii) polypeptide molecules that exhibit specific binding to the additional target gene; and / or (20) The polypeptide molecule includes a nuclease, and the nuclease molecule includes a guide nucleic acid (gNA) that forms a complex with the nuclease. Further, optionally, the endonuclease includes a CRISPR / Cas protein. Further, optionally, wherein: i. The CRISPR / Cas protein has essentially no cleavage activity; and / or ii. The CRISPR / Cas protein is coupled to a transcription activator; and / or (21) The method further includes guiding the plurality of hematopoietic lineage cells to transform into lymphoid cells, Further optionally, the lymphoid cells are selected from T cells, NK cells, and B cells.

[0230] Implementation Scheme 5. A system for guiding the differentiation of multiple stem cells, the system comprising: A heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units cooperate to sequentially induce multiple distinct regulation of target genes in multiple stem cells, each of the multiple distinct regulation being necessary for the induction of differentiation of the multiple stem cells, but insufficient individually to achieve the induction, wherein the multiple gate units include: (i) a first gate unit, which, upon activation of the heterologous gene circuit, induces a first distinct regulation among the plurality of distinct regulations; and (ii) A second gate unit, which is activated upon activation of the heterologous gene circuit, to induce a second distinct regulation among the plurality of distinct regulation, wherein the second distinct regulation is induced after the first distinct regulation, such that both the first distinct regulation and the second distinct regulation increase or decrease the expression and / or activity level of the target gene in the cell. The target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and Specifically, after the plurality of stem cells are brought into contact with the heterologous gene circuit, the plurality of gate units operate in concert to guide the differentiation of the plurality of stem cells. Optional, wherein: (1) The plurality of stem cells include pluripotent stem cells; and / or (2) After the contact, the plurality of gate units operate in concert to achieve the differentiation of the plurality of stem cells into tissue-specific progenitor cells. Further, optionally, wherein: i. The tissue-specific progenitor cells include hematopoietic lineage cells, further optionally, wherein: 1. Upon contact, the plurality of gate units operate in concert to guide the plurality of stem cells into hematopoietic stem cells; and / or 2. The hematopoietic lineage cells are characterized as CD45+; and / or 3. The hematopoietic lineage cells are characterized as CD34+CD43-CD45+; and / or ii. The activation of the heterologous gene circuit, wherein at least about 10%, at least about 15%, at least about 20%, at least about 30%, or at least about 40% of the plurality of stem cells differentiate into the tissue-specific progenitor cells; and / or iii. The differentiation is observed in less than about 14 days, less than about 10 days, less than about 7 days, or less than about 6 days; and / or (3) The ETS transcription factors include ETS1; and / or (4) The ETS transcription factors include ETV2; and / or (5) The ETS transcription factors include LMO2; and / or (6) The plurality of gate units are pre-configured such that one of the plurality of gate units can be activated upon activation of the heterologous gene circuit to regulate the expression and / or activity level of an additional target gene, wherein the additional target gene includes a T-box transcription factor (TBX) or a homeobox protein. Further, optionally, wherein: i. The regulation of the expression and / or activity levels of the additional target genes is induced prior to at least one of the plurality of different regulations of the target genes encoding the ETS transcription factor; and / or ii. The TBX includes at least two different types of TBX; and / or iii. The TBX includes TBXT and / or TBX6; and / or iv. The homeobox proteins include PRD-type homeobox proteins; and / or v. The homeobox protein includes MIXL1; and / or (7) The plurality of gate units are not pre-configured to regulate the expression and / or activity levels of one or more members selected from TBXT, TBX6, and MIXL1 prior to the plurality of different regulation of the target gene encoding the ETS transcription factor; and / or (8) The plurality of gate units are pre-configured such that one of the plurality of gate units is activated upon activation of the heterologous gene circuit to regulate the expression and / or activity levels of different target genes, wherein the different target genes include GATA transcription factors or basic helical-loop-helical transcription factors (bHLH); and / or Further, optionally, wherein: i. The GATA transcription factor includes GATA2; and / or ii. The bHLH includes SCL; and / or (9) The contact occurs in a culture medium that is substantially free of exogenous thrombopoietin (TPO) and / or exogenous FLT-3 ligand (FLT3L); and / or (10) The contact occurs in a culture medium that is substantially free of exogenous interleukins (ILs). Further, optionally, wherein: i. The exogenous IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (11) The plurality of gate units includes a third gate unit, which is activatable upon activation of the heterologous gene circuit to induce a third distinct regulation among the plurality of distinct regulation, wherein the third distinct regulation is induced after the first distinct regulation and the second distinct regulation, such that the first distinct regulation, the second distinct regulation, and the third distinct regulation all increase or decrease the expression and / or activity level of the target gene in the plurality of stem cells; and / or (12) The plurality of gate units are pre-configured such that the expression and / or activity level of the target gene is sequentially regulated by no more than three different gate units from the plurality of gate units; and / or (13) No more than three different gate units among the plurality of gate units are pre-configured to sequentially regulate the expression and / or activity levels of the target gene; and / or (14) Both the first different regulation and the second different regulation increase the expression and / or activity level of the target gene in the cell; and / or (15) Both the first different regulation and the second different regulation reduce the expression and / or activity level of the target gene in the cell; and / or (16)(i) The first gate unit includes a first gene regulatory portion, which is activated upon activation of the first gate unit to induce the first different regulation via the specific binding of the first gene regulatory portion to the target gene, and (ii) The second gate unit includes a second gene regulatory portion, which is activated upon activation of the second gate unit to induce the second distinct regulation via the specific binding of the first gene regulatory portion to the target gene. Further, optionally, wherein: i. The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with a polynucleotide sequence substantially identical to that of the target gene; and / or ii. The first gene regulatory portion and the second gene regulatory portion exhibit complementarity with different polynucleotide sequences of the target gene; and / or iii. The second gate unit further includes a second gate portion, which is activated upon the 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, further optionally, wherein: 1. The first gate unit further includes a first gate portion, which is activated after the activation of the first gate unit, to: (a) Activation of the first gene regulatory region is induced by the specific binding of the first phylum portion to the first gene regulatory region, and (b) Activation of the second gate portion is induced via specific binding of the first gate portion to the second gate portion; and / or 2. The activation portion is capable of: (a) Activation of the first gene regulatory portion is induced via the specific binding of the activation portion to the first gene regulatory portion, and (b) Inducing the activation of the second gate portion; and / or (17) 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 contain activatable gNA. Specifically, after gNA activation, gNA forms a complex with a nuclease. Further, optionally, wherein: i. The activatable gNA contains a non-canonical termination sequence; and / or ii. The endonuclease comprises a CRISPR / Cas protein, further optionally, wherein: 1. The CRISPR / Cas protein has essentially no cleavage activity; and / or 2. The CRISPR / Cas protein is coupled to a transcription activator; and / or (18) The target genes include multiple types of ETS transcription factors, wherein the first different regulation via the first gate unit is the regulation of a first type of ETS transcription factor, and wherein the second different regulation via the second gate unit is the regulation of a second type of ETS transcription factor. Further optionally, wherein (i) the first type of ETS transcription factor includes one or more members selected from ETS1, ETV2, LMO2 and combinations thereof, and (ii) the second type of ETS transcription factor includes ERG.

[0231] Implementation Scheme 6. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), said system comprising: The culture medium used for in vitro culture of the first plurality of cells, wherein the culture medium is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL). Within approximately 14 days after the first plurality of cells are cultured in vitro in the culture medium, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+. Optionally, the in vitro culture includes contacting a target gene in the first plurality of cells with a heterologous gene regulatory portion to modulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor.

[0232] Implementation Scheme 7. A system for converting multiple stem cells (a first plurality of cells) into multiple hematopoietic lineage cells (a second plurality of cells), said system comprising: The heterologous gene regulation component exhibits specific binding to target genes in the first plurality of cells to regulate the expression and / or activity levels of the target genes, wherein the target genes encode erythrocyte transformation-specific (ETS) transcription factors. The contact between the target gene and the regulatory portion of the heterologous gene enables the transformation from the first plurality of cells to the second plurality of cells. Optional, wherein: (1) Within approximately 14 days following the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+; and / or (2) Within approximately 14 days following the contact, an additional conversion rate of at least approximately 5% from the first plurality of cells to the second plurality of cells, wherein the second plurality of cells are CD34+; and / or (3) The contact in vitro occurs in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L) and interleukin (IL).

[0233] Implementation Scheme 8. The system as described in any one of Implementation Schemes 5, 6 and 7, further optionally, wherein: (1) The culture medium is substantially free of the TPO; and / or (2) The culture medium is substantially free of the FLT3L; and / or (3) The culture medium is substantially free of the IL, and further optionally, wherein: a. The IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15, and IL-21; and / or (4) The second plurality of cells are CD34+CD43-CD45+; and / or (5) Observe the conversion rate approximately 10 days, 7 days, or 6 days after the culture or contact; and / or (6) The conversion rate is at least about 10% or at least about 15%; and / or (7) Observe the additional conversion rate over approximately 6 days or approximately 5 days; and / or (8) The additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%; and / or (9) Within approximately 7 days following the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+); and / or (10) Within approximately 7 days following the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+); and / or (11) Within approximately 7 days following the culture, the additional conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%, wherein the second plurality of cells are CD34+; and / or (12) Within approximately 7 days following the culture, the additional conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 10%, wherein the second plurality of cells are CD34+; and / or (13) The ETS transcription factors include one or more members selected from ETS1, ETV2, GATA2, SCL and / or LMO2; and / or (14) The ETS transcription factors include one or more members selected from ETS1, ETV2 and / or LMO2; and / or (15) The heterologous gene regulatory portion increases the expression and / or activity level of the target gene; and / or (16) The heterologous gene regulatory portion reduces the expression and / or activity level of the target gene; and / or (17) The target gene is endogenous for the first plurality of cells; and / or (18) The system further includes an additional heterogeneous gene regulation portion configured to bind to additional target genes in the first plurality of cells to regulate the expression and / or activity levels of the additional target genes, thereby achieving the conversion, wherein the additional target genes include one or more members selected from T-box transcription factors (TBX), homeobox proteins, GATA transcription factors, and basic helical-loop-helical transcription factors (bHLH), further optionally, wherein: a. The additional heterologous gene regulatory portion enhances the expression and / or activity level of the additional target gene; and / or b. The additional heterologous gene regulatory portion reduces the expression and / or activity level of the additional target gene; and / or c. The additional target genes are endogenous to the first plurality of cells; and / or (19) The heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule that exhibits specific binding to the target gene; and / or (20) The additional heterologous gene regulatory portion comprises (i) nucleic acid molecules and / or (ii) polypeptide molecules that exhibit specific binding to the additional target gene; and / or (21) The polypeptide molecule includes a nuclease, and the nuclease molecule includes a guide nucleic acid (gNA) that forms a complex with the nuclease. Further, optionally, the endonuclease includes a CRISPR / Cas protein. Further, optionally, wherein: i. The CRISPR / Cas protein has essentially no cleavage activity; and / or ii. The CRISPR / Cas protein is coupled to a transcription activator.

[0234] Implementation Scheme 9. An engineered cell comprising the system as described in any one of Implementation Scheme 5, Implementation Scheme 6, Implementation Scheme 7 and Implementation Scheme 8.

[0235] Implementation Scheme 10. A composition comprising the system as described in any one of Implementation Schemes 5, 6, 7 and 8, or engineered cells as described in Implementation Scheme 9.

[0236] Implementation Scheme 11. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The first plurality of cells are brought into contact with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially regulate the expression levels of a plurality of different target genes, wherein the plurality of gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The heterologous gene circuit is programmed such that: (A) The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and / or (B) The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (C) The target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (D) The plurality of gate units includes a third gate unit that is activated upon activation of the second gate unit to regulate the expression of another target gene that is different from the target gene and the other target gene, such that regulation of the expression of the other target gene is induced after regulation of the expression of the other target gene, wherein the target gene includes one or more members selected from TBXT, TBX6, MIXL1 and combinations thereof; the other target gene includes one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2 and combinations thereof; and the other target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof. Upon contact, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells. Optional, wherein: (1) The heterologous gene circuit is characterized by (A); and / or (2) The heterologous gene circuit is characterized by (B); and / or (3) The heterologous gene circuit is characterized by (C); and / or (4) The heterologous gene circuit is characterized by (D); and / or (5) The plurality of gate units includes an additional gate unit that is activated upon activation of the first gate unit, wherein activation of the additional gate unit is necessary to induce activation of the second gate unit, thereby inducing a time delay between the regulation of the target gene by the first gate unit and the regulation of the additional target gene by the second gate unit; and / or (6) The second plurality of cells includes KDR- and CD34+ hematopoietic endothelial cells, further optionally, wherein: a. Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%; and / or b. Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 10%; and / or (7) The second plurality of cells includes (i) CD34+ and CD43+ or (ii) CD34+ and CD45+ hematopoietic cells (HPCs), further optionally, wherein: a. Within approximately 14 days or approximately 7 days following the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%; and / or b. Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%; and / or (8) The conversion rate from the first plurality of cells to the second plurality of cells via the heterologous gene circuit is at least about 1 times greater than the conversion rate in the absence of the heterologous gene circuit; and / or (9) The conversion rate from the first plurality of cells to the second plurality of cells via the heterologous gene circuit is at least about 3 times greater than the conversion rate in the absence of the heterologous gene circuit; and / or (10) The contact in vitro occurs in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof; and / or (11) Each of (i) the regulation of the expression of the target gene, (ii) the regulation of the expression of the additional target gene, and / or (ii) the regulation of the expression of the other target gene is necessary to achieve the transition from the first plurality of cells to the second plurality of cells, but is insufficient alone to achieve the transition; and / or (12) The gate unit in the plurality of gate units includes a gene regulatory portion, which is activated upon activation of the gate unit to induce regulation of the corresponding target gene via the specific binding of the gene regulatory portion to the corresponding target gene; and / or (13) The gene regulatory portion comprises a nucleic acid sequence exhibiting complementarity to at least a portion of the corresponding target gene; and / or (14) The gene regulatory portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with a nuclease, wherein the complex exhibits specific binding to the corresponding target gene; and / or (15) The endonuclease comprises a CRISPR / Cas protein, further optionally wherein (i) the CRISPR / Cas protein has substantially no cleavage activity and / or (ii) the CRISPR / Cas protein is coupled to a transcription activator; and / or (16) The plurality of different target genes are endogenous to the plurality of cells.

[0237] Implementation Scheme 12. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), said system comprising: A heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units are configured to cooperate in order to sequentially regulate the expression levels of multiple different target genes, wherein the multiple gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The heterologous gene circuit is programmed such that: (A) The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and / or (B) The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (C) The target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (D) The plurality of gate units includes a third gate unit that is activated upon activation of the second gate unit to regulate the expression of another target gene that is different from the target gene and the other target gene, such that regulation of the expression of the other target gene is induced after regulation of the expression of the other target gene, wherein the target gene includes one or more members selected from TBXT, TBX6, MIXL1 and combinations thereof; the other target gene includes one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2 and combinations thereof; and the other target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1 and combinations thereof. Specifically, after the first plurality of cells come into contact with the heterologous gene circuit, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells. Optional, wherein: (1) The heterologous gene circuit is characterized by (A); and / or (2) The heterologous gene circuit is characterized by (B); and / or (3) The heterologous gene circuit is characterized by (C); and / or (4) The heterologous gene circuit is characterized by (D); and / or (5) The plurality of gate units includes an additional gate unit that is activated upon activation of the first gate unit, wherein activation of the additional gate unit is necessary to induce activation of the second gate unit, thereby inducing a time delay between the regulation of the target gene by the first gate unit and the regulation of the additional target gene by the second gate unit; and / or (6) The second plurality of cells includes KDR- and CD34+ hematopoietic endothelial cells, further optionally, wherein: a. The plurality of gate units are configured to operate cooperatively to achieve a conversion rate of at least about 5% from the first plurality of cells to the second plurality of cells within about 14 days or about 7 days after the contact; and / or b. The plurality of gate units are configured to operate cooperatively to achieve a conversion rate of at least about 10% from the first plurality of cells to the second plurality of cells within about 14 days or about 7 days after the contact; and / or (7) The second plurality of cells includes (i) CD34+ and CD43+ or (ii) CD34+ and CD45+ hematopoietic cells (HPCs), further optionally, wherein: a. The plurality of gate units are configured to operate cooperatively to achieve a conversion rate of at least about 3% from the first plurality of cells to the second plurality of cells within about 14 days or about 7 days after the contact; and / or b. The plurality of gate units are configured to operate cooperatively to achieve a conversion rate of at least about 6% from the first plurality of cells to the second plurality of cells within about 14 days or about 7 days after the contact; and / or (8) The conversion rate from the first plurality of cells to the second plurality of cells via the heterologous gene circuit is at least about 1 times greater than the conversion rate in the absence of the heterologous gene circuit; and / or (9) The conversion rate from the first plurality of cells to the second plurality of cells via the heterologous gene circuit is at least about 3 times greater than the conversion rate in the absence of the heterologous gene circuit; and / or (10) The plurality of gate units are configured to cooperate in operation to achieve the transformation in vitro in a culture medium substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof; and / or (11) Each of (i) the regulation of the expression of the target gene, (ii) the regulation of the expression of the additional target gene, and / or (ii) the regulation of the expression of the other target gene is necessary to achieve the transition from the first plurality of cells to the second plurality of cells, but is insufficient alone to achieve the transition; and / or (12) The gate unit in the plurality of gate units includes a gene regulatory portion, which is activated upon activation of the gate unit to induce regulation of the corresponding target gene via the specific binding of the gene regulatory portion to the corresponding target gene; and / or (13) The gene regulatory portion comprises a nucleic acid sequence exhibiting complementarity to at least a portion of the corresponding target gene; and / or (14) The gene regulatory portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with a nuclease, wherein the complex exhibits specific binding to the corresponding target gene; and / or (15) The endonuclease comprises a CRISPR / Cas protein, further optionally wherein (i) the CRISPR / Cas protein has substantially no cleavage activity and / or (ii) the CRISPR / Cas protein is coupled to a transcription activator; and / or (16) The plurality of different target genes are endogenous to the plurality of cells.

[0238] Implementation Scheme 13. An engineered cell comprising the system described in Implementation Scheme 12.

[0239] Implementation Scheme 14. A composition comprising the system as described in Implementation Scheme 12 or engineered cells as described in Implementation Scheme 13.

[0240] The systems and methods disclosed herein may be combined with or modified by other systems and methods, such as those described in International Patent Application Nos. PCT / US2018 / 052211, PCT / US2018 / 052211, PCT / US2023 / 028169, PCT / US2023 / 028255 and PCT / US2023 / 028033, each of which is incorporated herein by reference in its entirety.

[0241] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The invention is not intended to be limited to the specific embodiments provided herein. While the invention has been described with reference to the foregoing detailed description, the description and illustration of embodiments herein are not intended to be construed as limiting. Various modifications, alterations, and substitutions will now be conceived by those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, and depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. Therefore, it is contemplated that the invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The first plurality of cells are cultured in vitro in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL). Within approximately 14 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+.

2. The method as described in any of the preceding claims, wherein the culture medium is substantially free of the TPO.

3. The method as described in any of the preceding claims, wherein the culture medium is substantially free of the FLT3L.

4. The method as described in any of the preceding claims, wherein the culture medium is substantially free of the IL.

5. The method of claim 4, wherein the IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15 and IL-21.

6. The method as described in any of the preceding claims, wherein the second plurality of cells are CD34+CD43-CD45+.

7. The method as described in any of the preceding claims, wherein, Within approximately 7 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+).

8. The method as described in any of the preceding claims, wherein, Within approximately 7 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+).

9. The method as claimed in any of the preceding claims, wherein the additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%.

10. The method of any of the preceding claims, wherein the culture comprises contacting a target gene in the first plurality of cells with a heterologous gene regulatory portion to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor.

11. The method of claim 10, wherein the ETS transcription factor comprises one or more members selected from ETS1, ETV2, GATA2, SCL and / or LMO2.

12. The method of claim 10, wherein the ETS transcription factor comprises one or more members selected from ETS1, ETV2 and / or LMO2.

13. The method of claim 10, wherein the heterologous gene regulation portion enhances the expression and / or activity level of the target gene.

14. The method of claim 10, wherein the target gene is endogenous for the first plurality of cells.

15. The method of any of the preceding claims, further comprising contacting an additional target gene in the first plurality of cells with an additional heterologous gene regulatory portion to modulate the expression and / or activity level of the additional target gene thereby achieving the conversion, wherein the additional target gene comprises one or more members selected from T-box transcription factors (TBX), homeobox proteins, GATA transcription factors, and basic helical-loop-helical transcription factors (bHLH).

16. The method of claim 15, wherein the additional heterologous gene regulatory portion enhances the expression and / or activity level of the additional target gene.

17. The method of claim 15, wherein the additional target gene is endogenous to the first plurality of cells.

18. The method of any of the preceding claims, wherein each heterologous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule exhibiting specific binding to the target gene.

19. The method of claim 18, wherein the polypeptide molecule comprises a nuclease, and wherein the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the nuclease.

20. The method of claim 19, wherein the endonuclease comprises a CRISPR / Cas protein.

21. The method of claim 20, wherein the CRISPR / Cas protein has substantially no cleavage activity.

22. The method of claim 20, wherein the CRISPR / Cas protein is coupled to a transcription activator.

23. The method as described in any of the preceding claims, further comprising guiding the plurality of hematopoietic lineage cells to transform into lymphoid cells.

24. The method of claim 23, wherein the lymphoid cells are selected from T cells, NK cells, and B cells.

25. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The first plurality of cells are brought into contact with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially regulate the expression levels of a plurality of different target genes, wherein the plurality of gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof. Upon contact, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells.

26. The method of any of the preceding claims, wherein the plurality of gate units includes a third gate unit that is activated upon activation of the second gate unit to regulate the expression of another target gene that is different from the target gene and the other target gene, such that regulation of the expression of the other target gene is induced after regulation of the expression of the other target gene, wherein the other target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof.

27. The method of any of the preceding claims, wherein the plurality of gate units includes an additional gate unit that is activatable upon activation of the first gate unit, wherein activation of the additional gate unit is necessary to induce activation of the second gate unit, thereby inducing a time delay between the regulation of the target gene by the first gate unit and the regulation of the additional target gene by the second gate unit.

28. The method of any of the preceding claims, wherein the second plurality of cells comprises KDR- and CD34+ hematopoietic endothelial cells.

29. The method of claim 28, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%.

30. The method of claim 28, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 10%.

31. The method of any of the preceding claims, wherein the second plurality of cells comprises (i) CD34+ and CD43+ or (ii) CD34+ and CD45+ hematopoietic cells (HPCs).

32. The method of claim 31, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%.

33. The method of claim 31, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%.

34. The method of any of the preceding claims, wherein the contact occurs in vitro in a culture medium that is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof.

35. The method of any of the preceding claims, wherein one of the plurality of gate units comprises a gene regulation portion, the gene regulation portion being activated upon activation of the gate unit to induce regulation of the corresponding target gene via specific binding of the gene regulation portion to the corresponding target gene.

36. The method of any of the preceding claims, wherein the gene regulation portion comprises a nucleic acid sequence exhibiting complementarity to at least a portion of the corresponding target gene.

37. The method of any of the preceding claims, wherein the gene regulation portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with a nuclease, wherein the complex exhibits specific binding to the corresponding target gene.

38. The method of any of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein, and further optionally, wherein (i) the CRISPR / Cas protein is substantially non-cleaving and / or (ii) the CRISPR / Cas protein is coupled to a transcription activator.

39. The method of any of the preceding claims, wherein the plurality of different target genes are endogenous to the plurality of cells.

40. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The first plurality of cells are brought into contact with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially regulate the expression levels of a plurality of different target genes, wherein the plurality of gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The heterologous gene circuit is programmed such that: (x) The target gene includes one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (y) The target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof. Upon contact, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells.

41. A method for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the method comprising: The target gene in the first plurality of cells is brought into contact with the regulatory portion of a heterologous gene to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor. The contact therein enables the transformation from the first plurality of cells to the second plurality of cells.

42. A method for guiding the differentiation of multiple stem cells, the method comprising: The plurality of stem cells are contacted with a heterologous gene circuit comprising a plurality of gate units, wherein, upon activation of the heterologous gene circuit, the plurality of gate units cooperate to sequentially induce a plurality of different regulation of target genes in the plurality of stem cells, each of the plurality of different regulation being necessary for the induction of differentiation of the plurality of stem cells, but insufficient individually to achieve the induction, wherein the plurality of gate units include: (i) a first gate unit, which, upon activation of the heterologous gene circuit, induces a first distinct regulation among the plurality of distinct regulations; and (ii) A second gate unit, which is activated upon activation of the heterologous gene circuit, to induce a second distinct regulation among the plurality of distinct regulation, wherein the second distinct regulation is induced after the first distinct regulation, such that both the first distinct regulation and the second distinct regulation increase or decrease the expression and / or activity level of the target gene in the cell. The target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and Upon contact, the plurality of gate units operate in concert to guide the differentiation of the plurality of stem cells.

43. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the system comprising: The culture medium used for in vitro culture of the first plurality of cells, wherein the culture medium is substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), and interleukin (IL). Wherein, within approximately 14 days after the first plurality of cells are cultured in vitro in the culture medium, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+.

44. The system as claimed in any of the preceding claims, wherein the culture medium is substantially free of the TPO.

45. The system as claimed in any of the preceding claims, wherein the culture medium is substantially free of the FLT3L.

46. ​​The system as claimed in any of the preceding claims, wherein the culture medium is substantially free of the IL.

47. The system of claim 46, wherein the IL includes one or more members selected from IL-2, IL-3, IL-7, IL-15 and IL-21.

48. The system as claimed in any of the preceding claims, wherein the second plurality of cells are CD34+CD43-CD45+.

49. The system as claimed in any of the preceding claims, wherein, Within approximately 7 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+).

50. The system as claimed in any one of the preceding claims, wherein, Within approximately 7 days after the culture, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%, wherein the second plurality of cells are CD45+ (or CD34+ and CD45+).

51. The system as claimed in any of the preceding claims, wherein the additional conversion rate is at least about 20%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%.

52. The system of any of the preceding claims, wherein the in vitro culture comprises contacting a target gene in the first plurality of cells with a heterologous gene regulatory portion to regulate the expression and / or activity level of the target gene, wherein the target gene encodes an erythrocyte transformation-specific (ETS) transcription factor.

53. The system of claim 52, wherein the ETS transcription factor comprises one or more members selected from ETS1, ETV2, GATA2, SCL and / or LMO2.

54. The system of claim 52, wherein the ETS transcription factor comprises one or more members selected from ETS1, ETV2 and / or LMO2.

55. The system of claim 52, wherein the heterologous gene regulation portion enhances the expression and / or activity level of the target gene.

56. The system of claim 52, wherein the target gene is endogenous for the first plurality of cells.

57. The system of any one of the preceding claims further comprises an additional heterogeneous gene regulation portion configured to bind to an additional target gene in the first plurality of cells to regulate the expression and / or activity level of the additional target gene to achieve the conversion, wherein the additional target gene comprises one or more members selected from T-box transcription factor (TBX), homeobox protein, GATA transcription factor, and basic helical-loop-helical transcription factor (bHLH).

58. The system of claim 57, wherein the additional heterologous gene regulatory portion enhances the expression and / or activity level of the additional target gene.

59. The system of claim 57, wherein the additional target gene is endogenous to the first plurality of cells.

60. The system as claimed in any of the preceding claims, wherein each heterogeneous gene regulatory portion comprises (i) a nucleic acid molecule and / or (ii) a polypeptide molecule exhibiting specific binding to the target gene.

61. The system of claim 60, wherein the polypeptide molecule comprises a nuclease, and wherein the nucleic acid molecule comprises a guide nucleic acid (gNA) that forms a complex with the nuclease.

62. The system of claim 61, wherein the endonuclease comprises a CRISPR / Cas protein.

63. The system of claim 62, wherein the CRISPR / Cas protein has substantially no cleavage activity.

64. The system of claim 62, wherein the CRISPR / Cas protein is coupled to a transcription activator.

65. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the system comprising: A heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units are configured to cooperate in order to sequentially regulate the expression levels of multiple different target genes, wherein the multiple gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The target genes include one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof. Wherein, after the first plurality of cells come into contact with the heterologous gene circuit, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells.

66. The system of any of the preceding claims, wherein the plurality of gate units includes a third gate unit that is activated upon activation of the second gate unit to regulate the expression of another target gene that is different from the target gene and the other target gene, such that regulation of the expression of the other target gene is induced after regulation of the expression of the other target gene, wherein the other target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof.

67. The system of any of the preceding claims, wherein the plurality of gate units includes an additional gate unit that is activatable upon activation of the first gate unit, wherein activation of the additional gate unit is necessary to induce activation of the second gate unit, thereby inducing a time delay between the regulation of the target gene by the first gate unit and the regulation of the additional target gene by the second gate unit.

68. The system as claimed in any of the preceding claims, wherein the second plurality of cells comprises KDR- and CD34+ hematopoietic endothelial cells.

69. The system of claim 68, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 5%.

70. The system of claim 68, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 10%.

71. The system as claimed in any of the preceding claims, wherein the second plurality of cells comprises (i) CD34+ and CD43+ or (ii) CD34+ and CD45+ hematopoietic cells (HPCs).

72. The system of claim 71, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 3%.

73. The system of claim 71, wherein, Within approximately 14 days or approximately 7 days after the contact, the conversion rate from the first plurality of cells to the second plurality of cells is at least approximately 6%.

74. The system as claimed in any of the preceding claims, wherein the plurality of gate units are configured to cooperate to achieve the transformation in vitro in a culture medium substantially free of one or more exogenous factors selected from thrombopoietin (TPO), exogenous FLT-3 ligand (FLT3L), interleukin (IL), and combinations thereof.

75. The system of any of the preceding claims, wherein one of the plurality of gate units comprises a gene regulation portion, the gene regulation portion being activated upon activation of the gate unit to induce regulation of the corresponding target gene via specific binding of the gene regulation portion to the corresponding target gene.

76. The system of any of the preceding claims, wherein the gene regulatory portion comprises a nucleic acid sequence exhibiting complementarity to at least a portion of the corresponding target gene.

77. The system of any of the preceding claims, wherein the gene regulation portion comprises a guide nucleic acid (gNA) molecule capable of forming a complex with a nuclease, wherein the complex exhibits specific binding to the corresponding target gene.

78. The system of any one of the preceding claims, wherein the endonuclease comprises a CRISPR / Cas protein, further optionally wherein (i) the CRISPR / Cas protein is substantially non-cleaving and / or (ii) the CRISPR / Cas protein is coupled to a transcription activator.

79. The system as claimed in any of the preceding claims, wherein the plurality of different target genes are endogenous to the plurality of cells.

80. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the system comprising: A heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units are configured to cooperate in order to sequentially regulate the expression levels of multiple different target genes, wherein the multiple gate units include: The first gate unit, which can be activated upon activation of the heterologous gene circuit, regulates the expression of the target gene; and A second gate unit, which is activated upon activation of the first gate unit, regulates the expression of an additional target gene different from the target gene, such that regulation of the expression of the additional target gene is induced following regulation of the expression of the target gene. The heterologous gene circuit is programmed such that: (x) The target gene includes one or more members selected from TBXT, TBX6, MIXL1, and combinations thereof; and the additional target gene includes one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof; and / or (y) The target genes include one or more members selected from ETS1, ETV2, GATA2, SCL, LMO2, and combinations thereof; and the additional target genes include one or more members selected from HOXA5, HOXA9, ERG, LCOR, RUNX1, and combinations thereof. Wherein, after the first plurality of cells come into contact with the heterologous gene circuit, the plurality of gate units operate in concert to achieve the transformation from the first plurality of cells to the second plurality of cells.

81. A system for converting a plurality of stem cells (a first plurality of cells) into a plurality of hematopoietic lineage cells (a second plurality of cells), the system comprising: The heterologous gene regulation component exhibits specific binding to target genes in the first plurality of cells to regulate the expression and / or activity levels of the target genes, wherein the target genes encode erythrocyte transformation-specific (ETS) transcription factors. The contact between the target gene and the regulatory portion of the heterologous gene enables the transformation from the first plurality of cells to the second plurality of cells.

82. A system for guiding the differentiation of multiple stem cells, the system comprising: A heterologous gene circuit comprising multiple gate units, wherein, upon activation of the heterologous gene circuit, the multiple gate units cooperate to sequentially induce multiple distinct regulation of target genes in multiple stem cells, each of the multiple distinct regulation being necessary for the induction of differentiation of the multiple stem cells, but insufficient individually to achieve the induction, wherein the multiple gate units include: (i) a first gate unit, which, upon activation of the heterologous gene circuit, induces a first distinct regulation among the plurality of distinct regulations; and (ii) A second gate unit, which is activated upon activation of the heterologous gene circuit, to induce a second distinct regulation among the plurality of distinct regulation, wherein the second distinct regulation is induced after the first distinct regulation, such that both the first distinct regulation and the second distinct regulation increase or decrease the expression and / or activity level of the target gene in the cell. The target gene encodes an erythrocyte transformation-specific (ETS) transcription factor, and In this process, after the plurality of stem cells are brought into contact with the heterologous gene circuit, the plurality of gate units operate in concert to guide the differentiation of the plurality of stem cells.

83. An engineered cell comprising the system as described in any one of the preceding claims.

84. A composition comprising the system or engineered cells as described in any of the preceding claims.

Images