System and method for cell programming

Abstract

Provided herein is a system that regulates the expression of a cargo (e.g., a guide nucleic acid) from a polynucleotide sequence (e.g., a vector).

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,731, filed Jul. 20, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Heterologous proteins and / or nucleic acid molecules can be utilized to elicit a desired response in a cell. Heterologous proteins and / or nucleic acid molecules can regulate a gene of interest (e.g., a transgene and / or an endogenous gene) to program (e.g., differentiate, dedifferentiate) a cell. Optionally, techniques using endonucleases (e.g., clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, i.e., “CRISPR / Cas”) have been employed for manipulation of polynucleotide sequences, their post-formation modification, and / or their expression levels. For example, CRISPR / Cas techniques can be characterized by their versatility and ease of programmability and can be used to facilitate genome editing across various species.

Summary of the Invention

[0003] The present disclosure provides methods and systems for regulating the expression or activity of a target gene. Some aspects of the present disclosure provide methods and systems that utilize a transcription termination sequence (e.g., a polyX sequence) to control an sgRNA-mediated genetic circuit that regulates the expression or activity of a target gene.

[0004] In one aspect, the present disclosure provides a system for regulating the expression or activity of a target gene, the system comprising a polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene for regulating the expression or activity of the target gene, the polynucleotide sequence comprising a domain that (i) corresponds to the tetraloop region of the guide nucleic acid molecule and (ii) contains a polyT sequence, the polyT sequence being sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene.

[0005] In another aspect, the present disclosure provides a system for regulating the expression or activity of a target gene, the system comprising a polynucleotide sequence encoding a guide nucleic acid molecule that (i) exhibits specific affinity for the target gene for regulating the expression or activity of the target gene and (ii) has a size of at least about 12 nucleotides, the polynucleotide sequence comprising a polyX sequence having a length of 5 or more threshold lengths, whereby the polyX sequence is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, and the polyX sequence does not correspond to the terminal domain of the guide nucleic acid molecule.

[0006] In another aspect, the present disclosure provides a method for regulating the expression or activity of a target gene in a cell, the system comprising contacting the cell with a polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene for regulating the expression or activity of the target gene, the polynucleotide sequence comprising a domain that (i) corresponds to the tetraloop region of the guide nucleic acid molecule and (ii) contains a polyT sequence, the polyT sequence being sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene.

[0007] In another aspect, the present disclosure provides a method for regulating the expression or activity of a target gene in a cell, the method comprising the step of providing to the cell a polynucleotide sequence encoding a guide nucleic acid molecule that (i) exhibits specific affinity for the target gene for regulating the expression or activity of the target gene and (ii) has a size of at least about 12 nucleotides, the polynucleotide sequence comprising a polyX sequence having a length of 5 or more threshold lengths, whereby the polyX sequence is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, and the polyX sequence does not correspond to the terminal domain of the guide nucleic acid molecule.

[0008] Further aspects and advantages of the present disclosure will be readily apparent to those of ordinary skill in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present disclosure. As will be appreciated, the present disclosure is capable of other embodiments and different embodiments, and some details thereof are capable of various obvious modifications without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

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

[0010] The novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description, which sets forth exemplary embodiments in which the principles of the invention are utilized, and the appended drawings (also referred to herein as "Figure" and "FIG.").

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[0011] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, modifications, and substitutions may be contemplated by those skilled in the art. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0012] As used herein and in the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. For example, the term "a gate unit" includes a plurality of gate units.

[0013] The term "about" or "approximately" generally means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations in accordance with the conventions in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within fivefold, more preferably within twofold of a value. When a particular value is recited in the present application and claims, unless otherwise specified, the term "about" is assumed to mean within the acceptable error range of the particular value.

[0014] The use of an alternative (e.g., “or”) is to be understood to mean either one, both, or any combination of them. The term “and / or” is to be understood to mean either one or both of the alternatives.

[0015] The terms “guide nucleic acid,” “guide nucleic acid molecule,” and “gNA,” when used interchangeably herein, generally refer to 1) a guide sequence capable of hybridizing to a target sequence, or 2) a scaffold sequence capable of interacting with or forming a complex with a nucleic acid-guided nuclease. The guide nucleic acid can be a single guide nucleic acid (e.g., sgRNA) or a double guide nucleic acid (e.g., dgRNA). The sgRNA can be a single RNA molecule containing both a scaffold tracrRNA and a crRNA that can be complementary to the target sequence. Alternatively, the dgRNA can be a single RNA molecule containing a crRNA annealed to the tracrRNA by direct repeat sequences.

[0016] The terms “genetic circuit,” “biological circuit,” or “circuit,” when used interchangeably herein, generally refer to an assembly of molecular components (e.g., biological materials such as polypeptides and / or polynucleotides, non-biological materials, etc.) operably coupled (e.g., operating simultaneously, sequentially, etc.) according to circuit design. The assembly of molecular components can be capable of providing one or more specific outputs (e.g., regulation of one or more genes) into a cell in response to one or more inputs (e.g., a single input or multiple inputs). Such one or more inputs can be sufficient to induce the molecular components of the genetic circuit to provide one or more specific outputs. For example, the genetic circuit can include one or more molecular switches activatable by one or more inputs (Figure 13).

[0017] A genetic circuit can be a controllable gene expression system that includes an assembly of biological parts that function together (e.g., simultaneously, continuously, etc.) as a logic function. The genetic circuit can include a plurality of gate units, and at least one of the plurality of gate units can be activatable by an activation moiety (e.g., a heterologous input to the cell) that activates other gate units of the plurality of gate units (e.g., simultaneously at once, continuously in a cascade manner, etc.) (FIG. 13). For example, at least one of the plurality of gate units can, as predetermined by the design of the genetic circuit, (i) regulate the expression or activity level of one or more target genes, (ii) activate at least one other gate unit of the plurality of gate units, and / or (ii) inactivate at least one other gate unit of the plurality of gate units, thereby overall regulating the expression and / or activity level of one or more target genes in a desired manner, and can be activatable by another gate unit of the plurality of gate units (e.g., directly or indirectly) (FIG. 13). As used herein, the terms “heterologous genetic circuit,” “HGC,” “cellular algorithm,” or “cellgorithm” may be used interchangeably.

[0018] As used herein, the term “gate unit” generally refers to a part of a genetic circuit that can control gene regulation by functioning in a manner similar to a logic gate, which can control the flow of information and enable the circuit to multiplex decisions at different points. More specifically, the term refers to a nucleic acid that encodes a gene switch, as well as the transcriptional and / or translational regulatory region or series of regions on which the gene switch acts. Inputs to the gate unit can be an activation moiety and / or another gate unit. Outputs from the gate unit can be used to activate another gate unit, inactivate another gate unit, affect a target gene, and / or for any combination of the foregoing. For example, a gate unit can be composed of a plurality of gate parts and / or a plurality of gene regulatory parts (FIG. 13).

[0019] As used herein, the term "activation moiety" generally refers to a moiety that can activate multiple genetic circuits and / or multiple gate units. The activation moiety can be a non-homologous input to the cell. Optionally, the activation moiety can include, but is not limited to, a guide nucleic acid molecule (e.g., gRNA) or other nucleic acid, polypeptide, polynucleotide, small molecule, light, or a combination thereof. For example, the activation moiety can be a guide nucleic acid molecule that forms a complex with an endonuclease (e.g., a Cas protein) and binds to the polynucleotide sequence of an inactivated gate moiety (e.g., a plasmid encoding another guide nucleic acid molecule), activating such a gate moiety that can target one or more gene regulatory moieties (e.g., inducing the expression of a functional form of a further guide nucleic acid molecule).

[0020] As used herein, the term "gate portion" generally refers to a portion that can affect the function of a gene regulatory portion within a gate unit. The gate portion can activate and / or inactivate the gene regulatory portion. For example, the gate portion can regulate the expression of a gene regulatory portion by editing a nucleic acid sequence, thereby activating or inactivating the gene regulatory portion. For example, the gate portion can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of a gene regulatory portion (e.g., a plasmid encoding another guide nucleic acid molecule), and activate a gene regulatory portion that can target one or more endogenous genes of a cell (e.g., induce the expression of a functional form of another guide nucleic acid molecule), and can be a guide nucleic acid molecule. Alternatively or additionally, the gate portion can activate and / or inactivate another gate unit of a genetic circuit (Figure 13). For example, the gate portion can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of another gate portion to be inactivated (e.g., a plasmid encoding another guide nucleic acid molecule), and activate another gate portion (e.g., induce the expression of a functional form of another guide nucleic acid molecule), and can be a guide nucleic acid molecule. In another example, the gate portion can form a complex with an endonuclease (e.g., a Cas protein) and bind to the polynucleotide sequence of another gate portion to be activated (e.g., a plasmid encoding another guide nucleic acid molecule), and inactivate another gate portion (e.g., reduce the expression of a functional form of another guide nucleic acid molecule), and can be a guide nucleic acid molecule.

[0021] When the terms "gene regulatory element" or "gene editing element" are used interchangeably herein, they generally refer to elements that can regulate the expression and / or activity profile of nucleic acid sequences or proteins, whether exogenous or endogenous to the cell (Figure 13). For example, a gene editing element can regulate gene expression by editing a nucleic acid sequence (e.g., CRISPR-Cas, zinc finger nuclease, TALEN, or siRNA). Optionally, a gene editing element can regulate gene expression by editing genomic DNA sequences. Optionally, a gene editing element can regulate gene expression by editing mRNA templates. Editing of the nucleic acid sequence can optionally modify the template (e.g., a CRISPR-Cas inspired RNA targeting system) underlying gene expression. Alternatively, a gene editing element can suppress the translation of a gene (e.g., Cas13).

[0022] Alternatively or additionally, a gene editing element can specifically bind to a target sequence operably linked to a gene (or a target sequence within a gene) and regulate gene expression or activity by regulating the production of mRNA from DNA such as chromosomal DNA or cDNA. For example, a gene editing element can recruit or contain at least one transcription factor that controls the rate of transfer of genetic information from DNA to mRNA by binding to a specific DNA sequence. A gene editing element can itself bind to DNA and regulate transcription by physical occlusion, for example, preventing proteins such as RNA polymerase and other associated proteins from assembling on the DNA template. A gene editing element can regulate gene expression at the translational level, for example, by regulating the production of proteins from the mRNA template. Optionally, a gene editing element can regulate gene expression by affecting the stability of the mRNA transcript. Optionally, a gene editing element can regulate a gene by epigenetic editing (e.g., Cas12).

[0023] In some cases, the plasmid may encode a non-functional form of the gene editing moiety. The plasmid can be activated (e.g., genetically recombined) to express a functional form of the gene editing moiety, for example, by activation of the functional gate moiety. For example, the plasmid may encode a non-functional form of a guide nucleic acid molecule that can otherwise bind to a target gene of a cell. When a functional gate moiety (e.g., another guide nucleic acid molecule complexed with a Cas protein) binds to the plasmid, the plasmid is edited (e.g., cleaved at one or more sites) to allow expression of a functional form of the gene editing moiety (e.g., a functional form of a guide nucleic acid molecule that specifically binds to the target gene of the cell) and allow regulation of the target gene in the cell, and can then be repaired via endogenous mechanisms (e.g., homologous recombination, non-homologous end joining).

[0024] Optionally, the gene regulatory part may include a nucleic acid molecule (e.g., a guide nucleic acid molecule that forms a complex with an endonuclease such as a Cas protein). Alternatively or additionally, the gene regulatory part may include an endonuclease or be operably linked to an endonuclease. An endonuclease can be an enzyme that cleaves phosphodiester bonds within a polynucleotide chain. The endonuclease can include a restriction endonuclease that cleaves DNA at specific sites without damaging the bases. Restriction endonucleases can include type I, II, III, and IV endonucleases, and can further include their subtypes. Optionally, 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, Csn2. The endonuclease can be a dead endonuclease that exhibits reduced cleavage activity. For example, the endonuclease can be a nuclease-inactivated Cas such as dCas (e.g., dCas9).

[0025] The above-mentioned Cas protein can form a complex with a guide nucleic acid (gNA (e.g., guide RNA (gRNA)) and specifically bind to a target polynucleotide sequence (e.g., target DNA sequence, target RNA sequence) using the gNA. Thus, in some cases, such a Cas protein may be referred to as a "NA-guided nuclease" (e.g., RNA-guided nuclease). As used herein, the term "guide nucleic acid" (gNA) generally can refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid may be RNA. The guide nucleic acid may be DNA. The guide nucleic acid may be programmed to bind site-specifically to a nucleic acid sequence. The nucleic acid to be targeted or the target nucleic acid may contain nucleotides. The guide nucleic acid may contain nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. The strand of the double-stranded target polynucleotide that is complementary to the guide nucleic acid and hybridizes to the guide nucleic acid may also be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand and thus may not be complementary to the guide nucleic acid may also be called the non-complementary strand. The guide nucleic acid may contain a polynucleotide strand and may also be called a "single guide nucleic acid". The guide nucleic acid may contain two polynucleotide strands and may also be called a "double guide nucleic acid". Otherwise, the term "guide nucleic acid" may be inclusive and may refer to both single guide nucleic acids and double guide nucleic acids. The guide nucleic acid may contain a segment that may be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence" or "spacer sequence". The nucleic acid targeting segment may contain a sub-segment that may be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment" or "scaffold sequence".

[0026] The gene regulatory moiety can be a transcriptional modulator system (e.g., a gene repressor complex or a gene activator complex). For example, the gene regulatory moiety can be a gene repressor complex comprising a dCas protein operably bound (e.g., bound or fused) to a transcriptional repressor. Non-limiting examples of transcriptional repressors include KRAB, SID, MBD2, MBD3, DNMT1, DNMT2A, DNMT3A, DNMT3B, DNMT3L, Mecp2, FOG1, ROM2, LSD1, ERD, SRDX repression domain, Pr-SET7 / 8, SUV4-20H1, RIZ1, JMJD2A, JHDM3A, JMJD2B, JMJD2C, GASC1, JMJD2D, JARID1A, RBP2, JARID1B / PLU-1, JARIDIC / SMCX, JARIDID / SMCY, HDACl, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDACl1, M.Hhal, METI, DRM3, ZMET2, CMT1, CMT2, lamin A, and lamin B. Alternatively, the gene regulatory moiety can be a gene activator complex comprising a dCas protein operably bound (e.g., fused) to a transcriptional activator. Non-limiting examples of transcriptional activators 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.

[0027] In some cases, the gene regulatory moiety has enzymatic activity to modify a target gene without cleaving the target gene. Modification of the target gene can cause epigenetic modifications that can, for example, modify gene expression and / or activity levels. Examples of enzymatic activities that can be provided by the gene regulatory moiety include nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3, ZMET2, CMT1, CMT2), demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity and / or resolvase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the GinH106Y hyperactive mutant of Gin invertase, human immunodeficiency virus type 1 integrase (IN), Tn3 resolvase, etc.), transposase activity, recombinase activity such as that provided by a recombinase (e.g., the catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity, but are not limited thereto.

[0028] Unless otherwise specified or clear from the context, when the terms "polynucleotide", "oligonucleotide", or "nucleic acid" are used interchangeably herein, they generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof, in single-stranded, double-stranded, or multi-stranded form. Polynucleotides can be exogenous or endogenous to a cell. Polynucleotides can exist in a cell-free environment. Polynucleotides can be a gene or a fragment thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can assume any three-dimensional structure and can perform any known or unknown function. Polynucleotides can contain one or more analogs (e.g., modified backbone, sugar, or nucleotide). When present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophore (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotide, biotin-linked nucleotide, fluorescent base analog, CpG island, methyl-7-guanosine, methylated nucleotide, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, 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 probe, and primer.The nucleotide sequence can be interfered with by non-nucleotide components.

[0029] The term "gene" generally refers to nucleic acids (e.g., DNA such as genomic DNA or cDNA) involved in encoding an RNA transcript and its corresponding nucleotide sequence. As used herein with respect to genomic DNA, this term includes regulatory regions, as well as the 5' and 3' ends, in addition to intervening non-coding regions. In some applications, this term encompasses transcribed sequences that include 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region contains an "open reading frame" that encodes a polypeptide. In some applications of this term, "gene" includes only the coding sequences necessary to encode a polypeptide (e.g., an "open reading frame" or "coding region"). Optionally, a gene does not encode a polypeptide, such as a ribosomal RNA gene (rRNA) and a transfer RNA (tRNA) gene. Optionally, the term "gene" includes not only the transcribed sequence, but also non-transcribed regions that include upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene," i.e., a natural gene in its natural position in an organism's genome. A gene can refer to an "exogenous gene" or a non-natural gene. A non-natural gene can refer to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A non-natural gene can also refer to a gene that is not in its natural position in an organism's genome. A non-natural gene can also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-natural sequence) that includes mutations, insertions, and / or deletions.

[0030] "Sequence identity" generally refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid match between two polynucleotide or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity". The percent identity between two sequences, whether nucleic acid or amino acid sequence, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the longer sequence and multiplying by 100. Percent identity can be determined, for example, by comparing sequence information using sophisticated BLAST computer programs available from the National Institutes of Health, including version 2.2.9. The BLAST programs are based on the alignment methods of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, Vol. 87: pp. 2264-2268 (1990), Altschul et al., J. Mol. Biol. 215: pp. 403-410 (1990), Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: pp. 5873-5877 (1993), and Altschul et al., Nucleic Acids Res. 25: pp. 3389-3402 (1997). The program can be used to determine percent identity over the entire length of the proteins being compared. For example, default parameters are provided to optimize searches with short query sequences by the blastp program. The program also allows the use of a SEG filter to mask-off segments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: pp. 149-163 (1993).The range of sequence identity to the desired degree is approximately 50% to 100%, and integer values in between. Generally, the present disclosure encompasses sequences having 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 of the sequences provided herein.

[0031] The term "expression" generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is later translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product". When the polynucleotide is derived from genomic DNA, expression may include the splicing of mRNA in eukaryotic cells. "Upregulated" with respect to expression generally refers to an increase in the expression level of a polynucleotide (such as an RNA, e.g., mRNA) and / or polypeptide sequence compared to the expression level in the wild-type state, while "downregulated" generally refers to a decrease in the expression level of a polynucleotide (such as an RNA, e.g., mRNA) and / or polypeptide sequence compared to the expression in the wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression", the transfected gene is not transferred to daughter cells during cell division. Since its expression is limited to the transfected cells, the expression of the gene decreases over time. During transient expression, episomal DNA can be transferred to daughter cells, but since episomal DNA is not replicated, it is not inherited permanently and is diluted over time. In contrast, stable expression of a transfected gene can occur when a gene is co-transfected with another gene that confers a selective advantage to the transfected cells. During stable expression, the plasmid may have DNA replication elements that allow the plasmid to be inherited or integrated into the genome. Such a selective advantage may be resistance to a particular toxin presented to the cell.

[0032] When the terms "peptide", "polypeptide", or "protein" are used interchangeably herein, they generally refer to a polymer of at least two amino acid residues linked by peptide bonds. This term does not imply a polymer of a specific length, nor is it intended to suggest or distinguish whether the peptide is produced using recombinant techniques, chemical synthesis, or enzymatic synthesis, or whether it exists naturally. This term applies to amino acid polymers that contain at least one modified amino acid in addition to naturally occurring amino acid polymers. Optionally, the polymer can be interfered with by non-amino acids. This term includes amino acid chains of any length, including full-length proteins, as well as proteins that do or do not have secondary and / or tertiary structures (e.g., domains). This term also encompasses amino acid polymers modified by any other operations, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and conjugation with labeling components. The terms "amino acid" and "amino acids" as used herein 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 natural and non-natural amino acids that have been chemically modified to contain groups or chemical moieties that do not naturally occur 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.

[0033] When the terms "derivative", "variant", or "fragment" are used interchangeably herein with respect to a polypeptide, they generally refer to a polypeptide related to a wild-type polypeptide by, for example, any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Derivatives, variants, and fragments of a polypeptide can include one or more amino acid modifications (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to the wild-type polypeptide.

[0034] As used herein, the terms "engineered," "chimeric," or "recombinant," when referring to a polypeptide molecule (e.g., a protein), generally refer to a polypeptide molecule having a non-homologous amino acid sequence or a modified amino acid sequence, as a result of the application of genetic engineering techniques to the nucleic acid encoding the polypeptide molecule, as well as to a cell or organism expressing the polypeptide molecule. The terms "engineered" or "recombinant," when used herein with respect to a polynucleotide molecule (e.g., a DNA molecule or an RNA molecule), generally refer to a polynucleotide molecule having a non-homologous nucleic acid sequence or a modified nucleic acid sequence as a result of applying 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. Optionally, an engineered polynucleotide or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.

[0035] Unless otherwise defined or clear from the context, the term "nucleotide," as used herein, generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be the monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term "nucleotide" can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates, such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term "nucleotide," as used herein, can refer to dideoxyribonucleoside triphosphates (ddNTP) and their derivatives. Exemplary examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides may not be labeled or may be detectably labeled by well-known techniques. Labeling can also be performed using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides can include, but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2’7’-dimethoxy-4’5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N’,N’-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4’ dimethylaminophenylazo)benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides 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, available from Perkin Elmer, Foster City, CA; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, IL; fluorescein-15-dATP, fluorescein-12-dUTP, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, IN; and chromosome-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, available from Molecular Probes, Eugene, OR. Nucleotides can also be labeled or marked by chemical modification.The chemically modified single nucleotide can be a biotin-dNTP. Some non-limiting examples of biotinylated dNTPs 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).

[0036] The term "cell" generally refers to a biological cell. A cell can be the basic structural, functional, and / or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotes, protozoan cells, cells derived from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, sorghum, potatoes, cotton, cannabis, tobacco, flowering plants, coniferous plants, gymnosperms, ferns, clubmosses, horsetails, mosses, liverworts), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardlii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast cells, cells derived from mushrooms), animal cells, cells derived from invertebrates (e.g., flies, cnidarians, echinoderms, nematodes, etc.), cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells derived from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.). Sometimes, a cell does not originate from a natural organism (e.g., a cell can be produced synthetically and can sometimes be referred to as an artificial cell).

[0037] Summary

[0038] Biological programming, such as cell programming, enables the manipulation of cells to bring about desired results. The results of cell programming can include inducing or preventing a wide range of common and / or new cell functions. The results can also include enhancing or suppressing cell functions that have already occurred. Cell programming can be achieved by the use of genetic circuits. Cell programming can be achieved by the manipulation of biomolecules (e.g., DNA). For example, the CRISPR or CRISPR / Cas system has been adopted for genome editing across many species due to its versatility and easy programmability. Cell programming can affect endogenous or exogenous genes. Cell programming can be implemented to function in a time-dependent or non-time-dependent manner.

[0039] The genetic circuits used in cell programming can be used to control cascades of multiple desired expression and / or activity profiles of multiple genes in a cell. To enable better control of the fate of specific cells, genetic circuits can be multiplexed, and positive feedback systems and / or negative feedback systems can be created.

[0040] Although the CRISPR / Cas system is widely used in gene editing, Cas can be a single-turnover nuclease since it remains bound to the double-strand breaks it generates, and many regions of the genome are resistant to genome editing. With the increasing understanding of CRISPR / Cas-based genome editing, the development of cascade regulatory systems for further leveraging this technology for use in the development of engineered cells is encouraged. By implementing a series of activatable gRNAs, genome editing can be more temporally regulated from target site to target site, sequential genome editing can be carried out to function like a domino effect, and cells can be barcoded. However, this barcoding does not enable epigenetic gene regulation that can be used for cell differentiation.

[0041] Therefore, there remains an unmet need for an activatable multiplexed CRISPR / Cas system and its use for editing a target polynucleotide (e.g., the genome of a cell, specifically a eukaryotic cell) using a cascade of gRNAs to form a genetic circuit that can independently affect gene regulation and thus cell fate determination, including feedback loops. Considering the improved multiplexing ability through the use of internal positive and / or negative feedback loops, CRISPR / Cas systems with pre-programmed, activatable, self-regulating gRNA cascades have found utility, for example, in gene therapy, genetic circuits, and / or complex cell fate determination and / or control.

[0042] Accordingly, the present disclosure provides systems, compositions, and methods for controlling a gene regulatory moiety (e.g., a guide nucleic acid molecule of a CRISPR / Cas system) such that the activity of the gene regulatory moiety for achieving regulation of one or more target genes (e.g., in a cell) can be controlled. In some embodiments, the control of the gene regulatory moiety can include control of the expression or activity level of the gene regulatory moiety. In some embodiments, the present disclosure provides systems, compositions, and methods for manipulating the activity of a CRISPR / Cas9 system comprising (i) an array of cognate single guide RNAs (sgRNAs or gRNAs) having a deactivation sequence in a non-essential region and (ii) being activatable, to enable regulation and modification of the Cas endonuclease and its system.

[0043] Systems and methods for activation and deactivation of guide nucleic acids

[0044] Various aspects of the present disclosure provide systems and methods for controlling the expression of a molecule of interest (e.g., a polynucleotide molecule) from a polynucleotide sequence encoding the molecule of interest. In some embodiments, the polynucleotide sequence can be a vector or expression cassette encoding a polynucleotide sequence encoding the molecule of interest. For example, the polynucleotide sequence can be a DNA sequence, and the expression can be the transcription of at least a portion of the DNA sequence into an RNA sequence. As provided herein, the molecule of interest, once expressed, can be utilized as a therapeutic molecule. Optionally, a variant expressed from the molecule of interest can exhibit specific binding to a target gene for the regulation (or modulation) of the expression or epigenetic profile of the target gene. For example, the molecule of interest can be at least a portion of a shRNA or guide nucleic acid molecule (e.g., a partial or complete) to form a complex with an endonuclease (e.g., a Cas protein).

[0045] A domain of a polynucleotide sequence encoding (or corresponding to) a molecule of interest can include a polyX sequence. The polyX sequence can be sufficient to reduce the expression of the molecule of interest (e.g., a guide nucleic acid molecule) from the polynucleotide sequence. For example, the polyX sequence can be placed within the domain encoding the molecule of interest (e.g., neither at the 5' end nor the 3' end of such domain) such that the expression of the molecule of interest (e.g., the transcription of the RNA molecule of interest) is disrupted (e.g., terminated) during expression.

[0046] Accordingly, the polyX sequence (e.g., in a polynucleotide sequence encoding a molecule of interest) can be referred to as a termination sequence (e.g., its sequence and / or a non-standard termination sequence at its position), a disruption sequence (e.g., for disrupting the complete expression of the molecule of interest), an inactivation sequence (e.g., for inactivating the function of the polynucleotide sequence or the molecule of interest).

[0047] As provided herein, a molecule of interest can be a guide nucleic acid molecule that, when expressed in an active or functional state, includes a spacer region (e.g., for binding to a target gene) and a scaffold region (e.g., for forming a complex with a Cas protein). In a domain of a polynucleotide sequence encoding a guide nucleic acid molecule of interest, polyX can be located within the sequence encoding the spacer region, between the sequence encoding the spacer region and the sequence encoding the scaffold, and / or within the sequence encoding the scaffold. Optionally, the scaffold region can include one or more loops (e.g., formed by two polynucleotide segments that are partially or wholly complementary to each other), such as a tetraloop and one or more stem-loops. Optionally, polyX can be part of, adjacent to, or located within a polynucleotide sequence encoding one or more loops.

[0048] Optionally, the polynucleotide sequence can be described as having a polyX sequence.

[0049] Optionally, a molecule of interest encoded by a polynucleotide sequence can be described as having a polyX sequence. In some examples, the description of a molecule of interest (e.g., a guide nucleic acid molecule) having a polyX sequence may refer to the molecule of interest in its expressed (e.g., transcribed) form. Alternatively or additionally, the description of a molecule of interest having a polyX sequence may refer to the polynucleotide sequence encoding such molecule of interest.

[0050] Accordingly, a further aspect of the disclosure provides systems and methods for activating a polynucleotide sequence (e.g., by modifying such polyX sequence within the polynucleotide sequence, such as by mutating it, removing part or all of it) to express a molecule of interest in an active state / functional state, or for activating a molecule of interest (e.g., to express in such active state / functional state).

[0051] In some cases, the tetraloop domain can be a polyX sequence. The polyX sequence can be a polyA sequence, a polyG sequence, a polyC sequence, a polyT sequence, or a polyU sequence. In some cases, the polyX sequence can be a polyT sequence. The polyX sequence can cause premature termination. In some cases, the polyT sequence can cause premature termination. In eukaryotes, RNA polymerase III (Pol III) is a protein that can transcribe DNA to synthesize small non-coding ribosomal nucleic acids. Termination of Pol III-regulated transcription can occur at a stretch of polyT sequences at the end of a gene.

[0052] In some cases, the polyX sequence can be located within a polynucleotide sequence, such as a DNA sequence or an RNA sequence (e.g., not at the end). In some cases, the polyX sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 3’ end of the polynucleotide sequence. In some cases, the polyX sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 5’ end of the polynucleotide sequence. In some cases, the polyX sequence can be located at the end of the nucleic acid sequence.

[0053] Optionally, the polyT sequence or polyU sequence can be located within a polynucleotide sequence such as a DNA sequence or an RNA sequence (e.g., not at the ends). Optionally, the polyT sequence or polyU sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 3' end of the polynucleotide sequence. Optionally, the polyT sequence or polyU sequence can be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 5' end of the polynucleotide sequence. Optionally, the polyT sequence or polyU sequence can be located at the end of the nucleic acid sequence. Optionally, an RNA containing a PolyU sequence can also be represented by a DNA containing a PolyT sequence.

[0054] A polyX sequence (e.g., a polyT sequence or a polyU sequence) can contain 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50X, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 bases. The polyX sequence can contain up to about 100, up to about 90, up to about 80, up to about 70, up to about 60, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, 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, or up to about 2 X bases. The polyX sequence can be represented by a complementary polyX sequence in the corresponding complementary DNA strand (e.g., polyT disclosed herein as a DNA sequence can also be referred to as polyA in the complementary DNA strand). The disclosed polyX sequence can contain multiple X bases. The multiple X bases can be disclosed sequentially adjacent to each other (e.g., TT, TTT, TTTT, TTTTT, etc.). Alternatively or additionally, the multiple X bases can be separated by one or more additional nucleotides that are not X. The one or more additional nucleotides can contain a single type of nucleotide or different types of nucleotides.

[0055] Optionally, the polyX sequence (e.g., polyT sequence) can include a continuous sequence of the same X nucleobase (e.g., the same T nucleobase). Such a continuous sequence can include at least, or up to about 2, at least, or up to about 3, at least, or up to about 4, at least, or up to about 5, at least, or up to about 6, at least, or up to about 7, at least, or up to about 8, at least, or up to about 9, at least, or up to about 10, at least, or up to about 11, at least, or up to about 12, at least, or up to about 13, at least, or up to about 14, at least, or up to about 15, at least, or up to about 16, at least, or up to about 17, at least, or up to about 18, at least, or up to about 19, at least, or up to about 20, at least, or up to about 21, at least, or up to about 22, at least, or up to about 23, at least, or up to about 24, at least, or up to about 25, at least, or up to about 26, at least, or up to about 27, at least, or up to about 28, at least, or up to about 29, at least, or up to about 30, at least, or up to about 35, at least, or up to about 40, at least, or up to about 45, or at least, or up to about 50 of the same X nucleobase (e.g., such a number of consecutive T bases, such a number of consecutive T bases, etc.).

[0056] Optionally, one or more additional nucleotides that are not X can be adjacent to (or disposed between) (i) one or more 5’X bases and (ii) one or more 3’X bases. Optionally, the region where the 5’X base and the 3’X base are adjacent can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 bases in length. Optionally, the region where the 5’X base and the 3’X base are adjacent can be at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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 base in length. See, for example, Structure (I) described below.

[0057] Optionally, one or more X sequences may be adjacent to either the 5' end and / or the 3' end of one or more additional nucleotides that are not X. Optionally, 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 X sequences may be at the 5' of one or more additional nucleotides that are not X. Optionally, 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 X sequences may be at the 3' of one or more additional nucleotides that are not X. Optionally, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, 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 X sequence may be at the 5' of one or more additional nucleotides that are not X. Optionally, up to about 50, up to about 40, up to about 30, up to about 20, up to about 15, up to about 14, up to about 13, up to about 12, up to about 11, 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 X sequence may be at the 3' of one or more additional nucleotides that are not X.

[0058] Optionally, there may be additional nucleotides other than X by a number greater than the number of X nucleotides (e.g., within a tetraloop domain containing a polyX sequence). For example, there may be additional nucleotides other than U by a number greater than the number of U nucleotides within a tetraloop domain of an RNA containing a polyU sequence. Optionally, there may be additional nucleotides other than X that are 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 more than the number of X nucleotides. Optionally, there may be additional nucleotides other than X that are the same number as the number of X nucleotides. Optionally, there may be additional nucleotides other than X by a number less than the number of X nucleotides. Optionally, there may be additional nucleotides other than X that are 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 less than the number of X nucleotides.

[0059] The polyX sequence can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50X, at least 60, at least 70, at least 80, at least 90, or at least 100 bases in length. The polyX sequence can be at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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, or at most about 2 bases in length. The polyX sequence can be represented by the corresponding polyX sequence in the corresponding RNA. For example, the PolyT sequence can be represented by the corresponding PolyU sequence in the corresponding RNA. The polyX sequence can be about 4 - 8, about 4 - 10, about 5 - 7, about 5 - 8, about 5 - 10, about 5 - 15, about 6 - 8, about 6 - 10, about 6 - 15, or about 7 - 15 bases in length.

[0060] The polyT sequence can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, at least 50X, at least 60, at least 70, at least 80, at least 90, or at least 100 T bases in length. The polyT sequence can be at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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, or at most about 2 T bases in length. The PolyT sequence can be represented by a PolyU sequence in the corresponding RNA. The polyT sequence can be about 4 - 8, about 4 - 10, about 5 - 7, about 5 - 8, about 5 - 10, about 5 - 15, about 6 - 8, about 6 - 10, about 6 - 15, or about 7 - 15 T bases in length.

[0061] Optionally, the threshold length of the polyX sequence may be required to achieve premature termination. The threshold length of the polyX sequence can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. Optionally, the polyX sequence may be sufficient to reduce the expression of the gNA molecule compared to a control without the polyX sequence. Optionally, the polyX sequence may be sufficient to reduce the expression of the gNA molecule compared to a control having a polyX sequence of a length shorter than the threshold polyX sequence.

[0062] In some cases, the threshold length of the polyT array may be necessary to achieve early termination. The threshold length of the polyT array can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 Ts. In some cases, the polyT array may be sufficient to reduce the expression of the gNA molecule as compared to a control that does not have the polyT array. In some cases, the polyT array may be sufficient to reduce the expression of the gNA molecule as compared to a control that has a polyT array of a length shorter than the threshold polyT array.

[0063] As provided herein, polyX arrays can be utilized to control the activation / inactivation of guide nucleic acid molecules. Accordingly, various aspects of the present disclosure provide systems for efficiently inactivating and / or activating a guide nucleic acid (e.g., sgRNA) to enable control over engineered CRISPR / Cas systems designed to regulate the expression or activity of a target gene. Various aspects of the present disclosure provide methods for efficiently inactivating and / or activating a guide nucleic acid (e.g., sgRNA) to enable control over engineered CRISPR / Cas systems designed to regulate the expression or activity of a target gene.

[0064] In one aspect, the present disclosure provides a system for inducing a desired expression and / or activity profile of a target gene in a cell. The system can include a heterologous genetic circuit that includes a plurality of gate units. The plurality of gate units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The plurality of gate units can 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 can be different (e.g., include different polynucleotide sequences).

[0065] The heterologous genetic circuits disclosed herein can operate with a plurality of gate units that are in series (e.g., a plurality of gate units are continuously connected in an end-to-end manner forming a single pathway), in parallel (e.g., a plurality of gate units are cross-connected to each other, forming, for example, two or more parallel pathways), or a combination thereof. In some embodiments, a plurality of gate units in series can operate in a forward cascade. In some embodiments, the forward mode can follow a numerically increasing step order (e.g., from step 1 to step 2, step 3, step 4, step 5, etc.). In some embodiments, a plurality of gate units in series can operate in a reverse cascade. In some embodiments, the reverse cascade can follow a numerically decreasing step order (e.g., from step 10 to step 9, step 8, step 7, step 6, etc.). In some embodiments, a plurality of gate units in series can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. In some embodiments, a plurality of gate units in series can 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 can cooperate (e.g., as predetermined by the design of the heterologous genetic circuit) to induce a cellular outcome. Cellular outcomes can include cellular functions (e.g., responses to movement, regeneration, external stimuli, nutrient production, excretion, respiration, growth) and / or cellular states (e.g., cell fate, differentiation, dormancy, programmed cell death). Such outcomes can be confirmed in vitro, ex vivo, and / or in vivo.For example, the outcomes disclosed herein can be confirmed in vitro by: (i) measuring the expression level of a gene 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) use of assays to measure phenotypic differentiation and cell function (e.g., cell proliferation assays, metabolic activity assays, cell death assays); (v) microscopy; and / or (vi) screening for molecular and / or genetic differences using, for example, metabolomics, genomics, proteomics, lipidomics, epigenomics, and / or transcriptomics.

[0066] Non-identical genetic circuits can include a plurality of gate units that are sequentially activated, for example, activated one after another in series. The plurality of gate units can include functional gate units that are preconfigured 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 plurality of gate units can further include one or more additional gate units that are (i) activated before the functional gate unit and (ii) subsequently preconfigured to achieve the activation of the functional gate unit. Optionally, the one or more additional gate units can be preconfigured to be activated to regulate one or more additional target genes. Alternatively, the one or more additional gate units may not be preconfigured to regulate any target gene (e.g., any endogenous target gene) upon activation. Such one or more additional gate units can instead function to shift (e.g., temporally) the activation of the functional gate unit during the operation of the non-identical genetic circuit, thereby shifting the expression and / or epigenetic profile of the target gene of the functional gate unit backward, and for this reason, the one or more additional gate units may be referred to as "blank" gate units.Non-identical genetic circuits can include at least, or at most, 1 blank gate unit, at least, or at most, 2 blank gate units, at least, or at most, 3 blank gate units, at least, or at most, 4 blank gate units, at least, or at most, 5 blank gate units, at least, or at most, 6 blank gate units, at least, or at most, 7 blank gate units, at least, or at most, 8 blank gate units, at least, or at most, 9 blank gate units, at least, or at most, 10 blank gate units, at least, or at most, 11 blank gate units, at least, or at most, 12 blank gate units, at least, or at most, 13 blank gate units, at least, or at most, 14 blank gate units, at least, or at most, 15 blank gate units, at least, or at most, 16 blank gate units, at least, or at most, 27 blank gate units, at least, or at most, 18 blank gate units, at least, or at most, 19 blank gate units, at least, or at most, 20 blank gate units, at least, or at most, 25 blank gate units, at least, or at most, 30 blank gate units, at least, or at most, 35 blank gate units, at least, or at most, 40 blank gate units, at least, or at most, 45 blank gate units, or at least, or at most, 50 blank gate units.

[0067] In some cases, the use of one or more blank gate units can shift the activation of the functional gate unit by at least, or up to 1 minute, at least, or up to 5 minutes, at least, or up to 10 minutes, at least, or up to 30 minutes, at least, or up to 1 hour, at least, or up to 2 hours, at least, or up to 3 hours, at least, or up to 4 hours, at least, or up to 5 hours, at least, or up to 6 hours, at least, or up to 7 hours, at least, or up to 8 hours, at least, or up to 9 hours, at least, or up to 10 hours, at least, or up to 11 hours, at least, or up to 12 hours, at least, or up to 13 hours, at least, or up to 14 hours, at least, or up to 15 hours, at least, or up to 16 hours, at least, or up to 17 hours, at least, or up to 18 hours, at least, or up to 19 hours, at least, or up to 20 hours, at least, or up to 21 hours, at least, or up to 22 hours, at least, or up to 23 hours, at least, or up to 24 hours, at least, or up to 2 days, at least, or up to 3 days, at least, or up to 4 days, at least, or up to 5 days, at least, or up to 6 days, or at least, or up to 7 days later.

[0068] Intracellular fates can include the regulation of target genes. The regulation of target genes can include multiple individual regulations of the target gene. The plurality of gate units can each induce one of the multiple individual regulations of the target gene such that the collection of individual regulations cooperate to bring about the final expression and / or activity profile of the target gene. At least two of the multiple individual regulations can increase both the expression and / or activity level of the target gene. At least two of the multiple individual regulations can decrease both the expression and / or activity level of the target gene. Alternatively, a first one of the multiple individual regulations can increase the expression and / or activity level of the target gene, and a second one of the multiple individual regulations can decrease the expression and / or activity level of the target gene. In such a case, the first individual regulation can occur before the second individual regulation, or vice versa. Alternatively, one of the multiple individual regulations (e.g., the first and / or second regulation) can maintain the expression and / or activity level of the target gene at the expression and / or activity level prior to regulation.

[0069] Optionally, each individual regulation of the multiple individual regulations of the target gene disclosed herein may be necessary to bring about the desired expression and / or activity profile of the target gene, but may be insufficient individually. For this reason, the intracellular fates induced by the multiple individual regulations of the target gene (e.g., enhancement of cell function, induction of cell state, etc.) may not be possible in the absence of any one of the multiple individual regulations of the target gene. Alternatively, the degree or scale of the intracellular fates induced by the multiple individual regulations of the target gene may not be induced by any, or may not be induced by all, but may be induced by one or more of the multiple individual regulations of the target gene, and / or may be higher than the degree or scale of the intracellular fates induced by all of the multiple individual regulations of the target gene occurring through a different sequential order of events in control cells.

[0070] 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 additional gate units that are activated by the first gate unit (e.g., directly or indirectly). The one or more additional gate units can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more gate units. The one or more additional gate units can be 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 can be activated via another part involved in the activation of the first gate unit (e.g., an activation part, a different gate unit, etc.).

[0071] The second gate unit can be activatable to induce inactivation of the activated first gate unit. The terms "inactivation" or "disruption" may be used interchangeably herein. The inactivation disclosed herein can be induced by generating a modification (e.g., a cleavage such as a single-stranded or double-stranded break, and an indel, etc.) in at least a portion of the first gate unit (e.g., the gate portion and / or the gene regulation portion of the first gate unit) involved in the induction of the first individual regulation of the target gene.

[0072] Inactivation by the gate portion and / or gene regulatory portion of the first gate unit disclosed herein can be achieved by an endonuclease-based system (e.g., the CRISPR / Cas system). Alternatively or additionally, inactivation can be achieved by the use of a transcriptional regulator system (e.g., a transcriptional repressor). An endonuclease transcriptional regulator system (e.g., a Cas repressor) can be used to achieve polynucleotide cleavage (e.g., to inactivate a gate portion and / or gene regulatory portion). Polynucleotide cleavage can create nucleic acid modifications such as single-strand breaks, double-strand breaks, insertions, deletions, or insertions-deletions (indels). Alternatively or additionally, an endonuclease transcriptional regulator system (e.g., a Cas repressor) can be used to regulate target gene expression.

[0073] Alternatively, the second gate unit can be activatable to amplify or enhance the activation of the activated first gate unit. Amplification or enhancement of the first gate unit can be induced by creating modifications (e.g., cleavage such as single-strand or double-strand breaks, and indels, etc.) in at least a portion of the first gate unit (e.g., the gate portion and / or gene regulatory portion of the first gate unit) involved in inducing the first individual regulation of the target gene.

[0074] In some cases, the first gate unit regulates the first target gene. Alternatively or additionally, the first gate unit may also regulate the second gate unit. The regulation of the second gate unit, if ascertained by rt-qPCR, Western blotting, or other methods, is at least, or up to approximately 1 millisecond, at least, or up to approximately 2 milliseconds, at least, or up to approximately 3 milliseconds, at least, or up to approximately 4 milliseconds, at least, or up to approximately 5 milliseconds, at least, or up to approximately 6 milliseconds, at least, or up to approximately 7 milliseconds, at least, or up to approximately 8 milliseconds, at least, or up to approximately 9 milliseconds, at least, or up to approximately 10 milliseconds, at least, or up to approximately 20 milliseconds, at least, or up to approximately 30 milliseconds, at least, or up to approximately 40 milliseconds, at least, or up to approximately 50 milliseconds, at least, or up to approximately 60 milliseconds, at least, or up to approximately 70 milliseconds, at least, or up to approximately 80 milliseconds, at least, or up to approximately 90 milliseconds, at least, or up to approximately 100 milliseconds, at least, or up to approximately 200 milliseconds, at least, or up to approximately 300 milliseconds, at least, or up to approximately 400 milliseconds, at least, or up to approximately 500 milliseconds, at least, or up to approximately 600 milliseconds, at least, or up to approximately 700 milliseconds, at least, or up to approximately 800 milliseconds, at least, or up to approximately 900 milliseconds, at least, or up to approximately 1 second, at least, or up to approximately 2 seconds, at least, or up to approximately 3 seconds, at least, or up to approximately 4 seconds, at least, or up to approximately 5 seconds, at least, or up to approximately 6 seconds, at least, or up to approximately 7 seconds, at least, or up to approximately 8 seconds, at least, or up to approximately 9 seconds, at least, or up to approximately 10 seconds, at least, or up to approximately 15 seconds, at least, or up to approximately 20 seconds, at least, or up to approximately 30 seconds, at least, or up to approximately 40 seconds, at least, or up to approximately 50 seconds, at least, or up to approximately 1 minute, at least, or up to approximately 2 minutes, at least, or up to approximately 3 minutes, at least, or up to approximately 4 minutes, at least, or up to approximately 5 minutes, at least, or up to approximately 6 minutes, at least, or up to approximately 7 minutes,Can occur at least, or up to approximately 8 minutes, at least, or up to approximately 9 minutes, at least, or up to approximately 10 minutes, at least, or up to approximately 20 minutes, at least, or up to approximately 30 minutes, at least, or up to approximately 40 minutes, at least, or up to approximately 50 minutes, at least, or up to approximately 1 hour, at least, or up to approximately 2 hours, at least, or up to approximately 3 hours, at least, or up to approximately 4 hours, at least, or up to approximately 5 hours, at least, or up to approximately 6 hours, at least, or up to approximately 7 hours, at least, or up to approximately 8 hours, at least, or up to approximately 9 hours, at least, or up to approximately 10 hours, at least, or up to approximately 12 hours, at least, or up to approximately 16 hours, at least, or up to approximately 20 hours, or at least, or up to approximately 24 hours, or can occur after adjustment of the first gate unit.,

[0075] In some cases, the second gate unit may regulate the second target gene. When the regulation of the second target gene is grasped by rt-qPCR, Western blotting, or other methods, it is at least, or at most about 1 millisecond, at least, or at most about 2 milliseconds, at least, or at most about 3 milliseconds, at least, or at most about 4 milliseconds, at least, or at most about 5 milliseconds, at least, or at most about 6 milliseconds, at least, or at most about 7 milliseconds, at least, or at most about 8 milliseconds, at least, or at most about 9 milliseconds, at least, or at most about 10 milliseconds, at least, or at most about 20 milliseconds, at least, or at most about 30 milliseconds, at least, or at most about 40 milliseconds, at least, or at most about 50 milliseconds, at least, or at most about 60 milliseconds, at least, or at most about 70 milliseconds, at least, or at most about 80 milliseconds, at least, or at most about 90 milliseconds, at least, or at most about 100 milliseconds, at least, or at most about 200 milliseconds, at least, or at most about 300 milliseconds, at least, or at most about 400 milliseconds, at least, or at most about 500 milliseconds, at least, or at most about 600 milliseconds, at least, or at most about 700 milliseconds, at least, or at most about 800 milliseconds, at least, or at most about 900 milliseconds, at least, or at most about 1 second, at least, or at most about 2 seconds, at least, or at most about 3 seconds, at least, or at most about 4 seconds, at least, or at most about 5 seconds, at least, or at most about 6 seconds, at least, or at most about 7 seconds, at least, or at most about 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 it can occur for 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 or more, or it can occur after the regulation of the first target gene.,

[0076] Optionally, the modification of the target gene by the gate unit can inactivate the gene. For example, the modification of the gene can stop the expression and / or activity level of the target gene. Alternatively, the modification of the gene can lower the expression and / or activity level of the target gene. Optionally, the modification of the gene can increase the expression and / or activity level of the target gene. Alternatively, the modification of the gene can maintain the expression and / or activity level of the target gene.

[0077] The expression and / or activity profile of a gene of interest (e.g., a differentiation marker) can be compared to that of a control gene (e.g., a housekeeping gene such as GAPDH), the relative expression levels of two or more genes of interest (e.g., the ratio of the expression or activity levels between a stem cell marker and a differentiation marker), the relative average expression level of the gene of interest compared to the average expression level of the same gene of interest in the cell type of interest, etc.

[0078] In some cases, the activation of multiple gate units may be the result of a single activation of a heterologous genetic circuit (e.g., by a single activation moiety at a single point in time). The multiple gate units may include one of a first gate unit and a second gate unit preconfigured to be continuously activated upon activation of the heterologous genetic circuit by a single activation. In some cases, one of the first and second gate units may be activated by a single activation moiety (e.g., a guide nucleic acid), while the other of the first and second gate units may be activated by a further activation moiety different from the activation moiety of the heterologous genetic circuit (e.g., a different guide nucleic acid). The further activation moiety may be a part of the heterologous genetic circuit that is generated (e.g., expressed) only upon activation of the heterologous genetic circuit. Alternatively or additionally, the first and second gate units may each be activated by different activation moieties that are not the same as the activation moiety of the heterologous genetic circuit. Such different activation moieties may be parts of the heterologous genetic circuit that are generated (e.g., expressed) only upon activation of the heterologous genetic circuit.

[0079] In some embodiments of any one of the systems disclosed herein, the gate unit may include a gate portion (e.g., at least, or up to about 1 gate portion, at least, or up to about 2 gate portions, at least, or up to about 3 gate portions, at least, or up to about 4 gate portions, at least, or up to about 5 gate portions, etc.) and / or a gene regulatory portion (e.g., at least, or about 1 gene regulatory portion, at least, or up to about 2 gene regulatory portions, at least, or up to about 3 gene regulatory portions, at least, or up to about 4 gene regulatory portions, at least, or up to about 5 gene regulatory portions, at least, or up to about 6 gene regulatory portions, at least, or up to about 7 gene regulatory portions, at least, or up to about 8 gene regulatory portions, at least, or up to about 9 gene regulatory portions, at least, or up to about 10 gene regulatory portions, etc.). The gate portion disclosed herein may include a guide nucleic acid molecule (gNA) (e.g., at least, or up to about 1 gNA molecule, at least, or up to about 2 gNA molecules, at least, or up to about 3 gNA molecules, at least, or up to about 4 gNA molecules, at least, or up to about 5 gNA molecules, etc.). The gene regulatory portion disclosed herein may include a gNA (e.g., at least, or up to about 1 gNA molecule, at least, or up to about 2 gNA molecules, at least, or up to about 3 gNA molecules, at least, or up to about 4 gNA molecules, at least, or up to about 5 gNA molecules, etc.). The guide nucleic acid molecule disclosed herein may include, but is not limited to, DNA, RNA, any analogs thereof, or any combinations thereof.In some embodiments of any one of the systems disclosed herein, the gate portion and / or gene regulatory portion may be activatable to form a complex with an enzyme (e.g., an endonuclease and / or exonuclease), and this complex may be configured to bind to or be capable of binding to a target polynucleotide to regulate, for example, the expression and / or activity level of the target polynucleotide or another polynucleotide sequence operably linked to the target polynucleotide. For example, the complex may regulate the expression and / or activity level of a gene containing the target polynucleotide.

[0080] In some embodiments of any one of the systems disclosed herein, the first (or primary) gate unit of the non-homologous genetic circuit disclosed herein may be activated (e.g., directly activated) by an activation moiety. The activation moiety may directly bind to at least a portion of the first gate unit to activate the first gate unit, thereby, for example, continuously activating the non-homologous genetic circuit. Alternatively, the activation moiety (e.g., electromagnetic energy) may activate the first gate unit without directly binding to at least a portion of the first gate unit. Optionally, the first gate unit may include at least one gate portion and at least one gene regulatory portion. Optionally, the first gate unit may include at least one gate portion, but does not include or need not include a gene regulatory portion. Optionally, the first gate unit may include at least one gene regulatory portion, but does not include or need not include a gate portion (e.g., the activation moiety may be configured to activate the first gate unit and at least one additional gate unit).

[0081] In some embodiments of any one of the systems disclosed herein, the gNA of the gate portion and / or the gene regulatory portion (e.g., the gNA encoded by the gate portion and / or the gene regulatory portion) can be an activatable gNA. The activatable gNA can be any one of, but not limited to, ribonucleotides (e.g., gRNA), deoxyribonucleotides, any analogs thereof, or any combination thereof. In some embodiments, the vector (or expression cassette) encoding the activatable gNA can include an inactivating polynucleotide sequence to inactivate the gNA until it is activated (e.g., until the inactivating polynucleotide sequence is modified or removed from the vector). For example, the inactivating polynucleotide sequence can encode a self-cleaving polynucleotide molecule (e.g., ribozyme). Alternatively or additionally, the inactivating polynucleotide sequence can encode a non-standard transcription termination sequence as described below. The inactivating polynucleotide sequence can be part of or adjacent to a region of the vector that encodes (i) the spacer sequence of the gNA, (ii) the scaffold sequence of the gNA, and / or (ii) any linker sequence between the spacer sequence and the scaffold sequence. The vector can include at least, or at most about 1, at least, or at most about 2, at least, or at most about 3, at least, or at most about 4, at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, or at least, or at most about 10 inactivating polynucleotide sequences.

[0082] In some embodiments, the activatable gNA molecule can be a self-cleaving gNA (e.g., the gRNA contains a ribozyme). For example, when the activatable gNA is expressed intracellularly, the activatable gNA may be self-cleavable such that it becomes non-functional (e.g., not configured to bind to a target gene) unless the gene encoding the activatable gNA is modified prior to the expression of the activatable gNA. In some embodiments, the gNA can be synthetic. In some embodiments, the gNA can have a bound fluorescent label.

[0083] In some embodiments, the guide nucleic acid molecule encoded by the polynucleotide sequences disclosed herein may include an enzymatic polynucleotide domain (e.g., a ribozyme). Alternatively, the guide nucleic acid molecule encoded by the polynucleotide sequences disclosed herein may be capable of exhibiting enzymatic activity by itself.

[0084] In some embodiments, the guide nucleic acid molecule encoded by the polynucleotide sequences disclosed herein may not include an enzymatic polynucleotide domain (e.g., a ribozyme). Alternatively, the guide nucleic acid molecule encoded by the polynucleotide sequences disclosed herein may not be capable of exhibiting enzymatic activity by itself.

[0085] Optionally, the term "proGuide" as used herein may generally refer to such a polynucleotide sequence (e.g., a vector, expression cassette, plasmid, etc.) encoding an activatable gNA. The proGuide can be an example of a gate portion. The proGuide can be an example of a gene regulatory portion. Optionally, the term "matureGuide" as used herein may generally refer to the functional form of the gNA expressed (e.g., transcribed) from the proGuide when an inactivating polynucleotide sequence (e.g., including a polyT sequence) is modified and removed from the proGuide.

[0086] In some cases, a non-homologous genetic circuit can be activated by a guide nucleic acid molecule (gNA) (e.g., a functional gNA). Alternatively or additionally, the gNA may be used to exhibit specific affinity for a target gene in order to regulate the expression or activity of the target gene. In some cases, the gNA can be at least about 10, at least about 12, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, 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 bases in length. In some cases, the gNA can be at most about 500, at most about 400, at most about 300, at most about 200, at most about 150, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 55, at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 15, at most about 14, at most about 12, or at most about 10 bases in length. In some cases, the gNA can be at least about 14 nucleotides in length. In some cases, the gNA can be at most about 300 nucleotides in length. In some cases, the gNA can be introduced into the system exogenously. Alternatively, the gNA can be produced endogenously by the system (e.g., expressed by a gate unit).

[0087] The gNA can be activatable. The gNA can include a domain corresponding to the tetraloop region of the guide nucleic acid molecule. The tetraloop can include a 4-base hairpin loop motif that can cap the double-stranded portion of the nucleic acid in the RNA secondary structure. The tetraloop can play an important role in the structural stability and biological function of the RNA. Also, the tetraloop can include a first hairpin in the gRNA.

[0088] In some embodiments, the proGuide provided herein can encode an activatable guide nucleic acid molecule having, for example, an inactivated polynucleotide sequence (e.g., one or more polyX sequences, such as one or more polyT sequences). Optionally, a portion of the proGuide encoding the activatable guide nucleic acid molecule can include various regions (e.g., in the 5' to 3' direction) that are sequentially linked, including an upstream stem (e.g., an upstream cleavage site), a polyT unit (i.e., a "proUnit" used interchangeably herein), and a downstream stem (e.g., a downstream cleavage site), as shown in Tables 1 and 2. The upstream stem and the downstream stem may correspond to "stem region" polynucleotide sequences that are at least partially complementary to each other, as schematically shown in the shape of the encoded guide nucleic acid molecule structure in FIG. 8. Optionally, the portion of the proGuide encoding the activatable guide nucleic acid molecule can include various regions (e.g., in the 5' to 3' direction) that are sequentially linked, including a spacer sequence, an extra sequence (e.g., a linker sequence, an insulator sequence, or a sequence corresponding to another portion of the scaffold sequence of the guide nucleic acid molecule), an upstream stem, a polyT unit, and a downstream stem. These various regions can be linked in the order shown in FIGS. 22A and 22B, e.g., in the 5' to 3' direction in sequence.

[0089] Optionally, the upstream and / or downstream regions may be, or may include, an endonuclease recognition site provided herein (e.g., targetable by a Cas / guide nucleic acid complex) for modifying or removing the polyT unit.

[0090] In some cases, by modification or removal of the polyT unit, a guide nucleic acid molecule can be expressed, and at least a portion of the upstream stem and at least a portion of the downstream stem can form part of the scaffold sequence of the functional guide nucleic acid molecule. Alternatively or additionally, at least a portion of the upstream stem and at least a portion of the downstream stem may be bound to the scaffold sequence of a functional guide nucleic acid molecule that does not inhibit the activity of the scaffold sequence that forms a complex with the corresponding endonuclease (e.g., Cas protein, dCas protein, etc.), but may not be the actual or active portion of the scaffold sequence. Thus, the upstream stem and / or the downstream stem can be characterized by (1) having a length sufficient to be specifically targetable by a targeting moiety (e.g., a CRISPR / Cas / gRNA complex) for cleavage of adjacent polyT sequences, (2) exhibiting minimal or substantially no sequence identity to any other polynucleotide sequence of equivalent length in the genome of the cell in order to minimize off-target modification (e.g., cleavage) or endogenous gene disruption, and / or (3) not having a secondary structure that can inhibit the ability of the scaffold sequence to form a complex with the corresponding endonuclease. Based at least on (2), the terms "polyX", "polyT", "polyU", "polyT unit", "inactivated polynucleotide sequence", "non-standard sequence", "non-standard termination sequence", and "non-standard disruption sequence" may be used interchangeably throughout this disclosure.

[0091] A set of proGuides for a common non-homologous genetic circuit can have the same (or substantially the same) or different extra sequences disposed between the spacer sequence and the upstream stem.

[0092] In some cases, in proGuide, the distance between (i) the end (e.g., the 3' end) of the region encoding or corresponding to the spacer sequence of the guide nucleic acid molecule and (ii) the end (e.g., the 5' end) of a further region corresponding to the inactivating polynucleotide sequence (e.g., a polyT sequence) is at least, or at most, about 5 nucleic acid bases, at least, or at most, about 10 nucleic acid bases, at least, or at most, about 11 nucleic acid bases, at least, or at most, about 12 nucleic acid bases, at least, or at most, about 13 nucleic acid bases, at least, or at most, about 14 nucleic acid bases, at least, or at most, about 15 nucleic acid bases, at least, or at most, about 16 nucleic acid bases, at least, or at most, about 17 nucleic acid bases, at least, or at most, about 18 nucleic acid bases, at least, or at most, about 19 nucleic acid bases, at least, or at most, about 20 nucleic acid bases, at least, or at most, about 21 nucleic acid bases, at least, or at most, about 22 nucleic acid bases, at least, or at most, about 23 nucleic acid bases, at least, or at most, about 24 nucleic acid bases, at least, or at most, about 25 nucleic acid bases, at least, or at most, about 26 nucleic acid bases, at least, or at most, about 27 nucleic acid bases, at least, or at most, about 28 nucleic acid bases, at least, or at most, about 29 nucleic acid bases, at least, or at most, about 30 nucleic acid bases, at least, or at most, about 31 nucleic acid bases, at least, or at most, about 32 nucleic acid bases, at least, or at most, about 33 nucleic acid bases, at least, or at most, about 34 nucleic acid bases, at least, or at most, about 35 nucleic acid bases, at least, or at most, about 36 nucleic acid bases, at least, or at most, about 37 nucleic acid bases, at least, or at most, about 38 nucleic acid bases, at least, or at most, about 39 nucleic acid bases, at least, or at most, about 40 nucleic acid bases, at least, or at most, about 41 nucleic acid bases, at least, or at most, about 42 nucleic acid bases, at least, or at most, about 43 nucleic acid bases, at least, or at most, about 44 nucleic acid bases, at least, or at most, about 45 nucleic acid bases, at least, or at most, about 46 nucleic acid bases, at least, or at most, about 47 nucleic acid bases, at least, or at most, about 48 nucleic acid bases, at least, or at most, about 49 nucleic acid bases, at least,...or at most about 50 nucleobases, at least, or at most about 51 nucleobases, at least, or at most about 52 nucleobases, at least, or at most about 53 nucleobases, at least, or at most about 54 nucleobases, at least, or at most about 55 nucleobases, at least, or at most about 56 nucleobases, at least, or at most about 57 nucleobases, at least, or at most about 58 nucleobases, at least, or at most about 59 nucleobases, at least, or at most about 60 nucleobases, at least, or at most about 65 nucleobases, at least, or at most about 70 nucleobases, 75 nucleobases, at least, or at most about 80 nucleobases, at least, or at most about 85 nucleobases, at least, or at most about 90 nucleobases, at least, or at most about 95 nucleobases, or at least, or at most about 100 nucleobases and may be.

[0093] Optionally, at least one edit can be made to the polyX sequence. The edits made to the polyX sequence can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, or more. The edits made to the polyX sequence can be at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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. The edit to the polyX sequence can be an insertion. Alternatively or additionally, the edit to the polyX sequence can be a deletion. Alternatively or additionally, the edit to the polyX sequence can be an excision of the polyX sequence. The excision of the polyX sequence can be achieved using two cleavage sites adjacent to the polyX sequence. The edit to the polyX sequence can utilize various forms of nucleic acid repair mechanisms such as homology-directed repair (HDR), non-homologous end joining (NHEJ) repair, and microhomology-mediated end joining (MMEJ) repair, but is not limited thereto.

[0094] In some cases, at least one edit can be made to the polyT sequence. The edits made to the polyT sequence can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, or more. The edits made to the polyT sequence can be at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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. The edit to the polyT sequence can be an insertion. Alternatively or additionally, the edit to the polyT sequence can be a deletion. Alternatively or additionally, the edit to the polyT sequence can be excision of the polyT sequence. Excision of the polyT sequence can be achieved using two cleavage sites adjacent to the polyT sequence. The edit to the polyT sequence can utilize various forms of nucleic acid repair mechanisms such as homology-directed repair (HDR), non-homologous end joining (NHEJ) repair, and microhomology-mediated end joining (MMEJ) repair, but is not limited thereto.

[0095] Edits to the polyX sequence in the gNA (e.g., sgRNA) can affect the expression of the guide nucleic acid molecule from the polynucleotide sequence. The edits to the polyX sequence can enhance, decrease, or stop the expression of the gNA molecule from the polynucleotide sequence.

[0096] In some cases, the modification of the polyX sequence can reduce the expression and / or activity level of the guide nucleic acid molecule 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 modification of the polyX sequence can reduce the expression and / or activity level of the guide nucleic acid molecule 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.

[0097] In some cases, modification of the polyX sequence can increase the expression and / or activity level of the guide nucleic acid molecule 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%, at least about 1,000,000%, or more.Modification of the polyX array can increase the expression and / or activity level of the guide nucleic acid molecule by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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.

[0098] In some cases, modification of the polyX sequence can reduce the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the polyX array can reduce the expression and / or activity level of the guide nucleic acid molecule by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of the equivalent guide nucleic acid.

[0099] In some cases, modification of the polyX array can increase the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold, compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the polyX sequence can increase the expression and / or activity level of the guide nucleic acid molecule by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold as compared to the control expression and / or activity level of the equivalent guide nucleic acid.

[0100] Editing of the polyT sequence in the gNA can affect the expression of the guide nucleic acid molecule from the polynucleotide sequence. Editing of the polyT sequence can enhance, decrease, or stop the expression of the gNA molecule from the polynucleotide sequence.

[0101] In some cases, the modification of the polyT sequence can reduce the expression and / or activity level of the guide nucleic acid molecule 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 modification of the polyT sequence can reduce the expression and / or activity level of the guide nucleic acid molecule 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.

[0102] In some cases, the modification of the polyT sequence can increase the expression and / or activity level of the guide nucleic acid molecule 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%, at least about 1,000,000%, or more.Modification of the polyT array can increase the expression and / or activity level of the guide nucleic acid molecule by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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, the modification of the polyT array can reduce the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the polyT array can reduce the expression and / or activity level of the guide nucleic acid molecule by at least or maximally about 10,000-fold, at least or maximally about 5,000-fold, at least or maximally about 1,000-fold, at least or maximally about 500-fold, at least or maximally about 100-fold, at least or maximally about 90-fold, at least or maximally about 80-fold, at least or maximally about 70-fold, at least or maximally about 60-fold, at least or maximally about 50-fold, at least or maximally about 40-fold, at least or maximally about 30-fold, at least or maximally about 20-fold, at least or maximally about 10-fold, at least or maximally about 9-fold, at least or maximally about 8-fold, at least or maximally about 7-fold, at least or maximally about 6-fold, at least or maximally about 5-fold, at least or maximally about 4-fold, at least or maximally about 3-fold, at least or maximally about 2-fold, at least or maximally about 1-fold, at least or maximally about 0.9-fold, at least or maximally about 0.8-fold, at least or maximally about 0.7-fold, at least or maximally about 0.6-fold, at least or maximally about 0.5-fold, at least or maximally about 0.4-fold, at least or maximally about 0.3-fold, at least or maximally about 0.2-fold, at least or maximally about 0.1-fold, when compared to the control expression and / or activity level of the equivalent guide nucleic acid.

[0104] In some cases, modification of the polyT array can increase the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the polyT sequence can increase the expression and / or activity level of the guide nucleic acid molecule by up to or at least about 10,000-fold, up to or at least about 5,000-fold, up to or at least about 1,000-fold, up to or at least about 500-fold, up to or at least about 100-fold, up to or at least about 90-fold, up to or at least about 80-fold, up to or at least about 70-fold, up to or at least about 60-fold, up to or at least about 50-fold, up to or at least about 40-fold, up to or at least about 30-fold, up to or at least about 20-fold, up to or at least about 10-fold, up to or at least about 9-fold, up to or at least about 8-fold, up to or at least about 7-fold, up to or at least about 6-fold, up to or at least about 5-fold, up to or at least about 4-fold, up to or at least about 3-fold, up to or at least about 2-fold, up to or at least about 1-fold, up to or at least about 0.9-fold, up to or at least about 0.8-fold, up to or at least about 0.7-fold, up to or at least about 0.6-fold, up to or at least about 0.5-fold, up to or at least about 0.4-fold, up to or at least about 0.3-fold, up to or at least about 0.2-fold, up to or at least about 0.1-fold compared to the control expression and / or activity level of an equivalent guide nucleic acid.

[0105] Editing of the polyX sequence in the gNA (e.g., sgRNA) can regulate the expression or activity of the target gene by affecting the expression of the guide nucleic acid molecule from the polynucleotide sequence. Editing of the polyX sequence can enhance, decrease, or abolish the expression of the target gene.

[0106] In some cases, modification of the polyX sequence can reduce 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. Modification of the polyX sequence can reduce the expression and / or activity level 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.

[0107] In some cases, modification of the polyX array can increase 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%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000%, or more.Modification of the polyX array can increase the expression and / or activity level of the target gene by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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.

[0108] In some cases, the modification of the polyX sequence can reduce the expression and / or activity level of the target gene by at least, or up to approximately 0.1-fold, at least, or up to approximately 0.2-fold, at least, or up to approximately 0.3-fold, at least, or up to approximately 0.4-fold, at least, or up to approximately 0.5-fold, at least, or up to approximately 0.6-fold, at least, or up to approximately 0.7-fold, at least, or up to approximately 0.8-fold, at least, or up to approximately 0.9-fold, at least, or up to approximately 1-fold, at least, or up to approximately 2-fold, at least, or up to approximately 3-fold, at least, or up to approximately 4-fold, at least, or up to approximately 5-fold, at least, or up to approximately 6-fold, at least, or up to approximately 7-fold, at least, or up to approximately 8-fold, at least, or up to approximately 9-fold, at least, or up to approximately 10-fold, at least, or up to approximately 20-fold, at least, or up to approximately 30-fold, at least, or up to approximately 40-fold, at least, or up to approximately 50-fold, at least, or up to approximately 60-fold, at least, or up to approximately 70-fold, at least, or up to approximately 80-fold, at least, or up to approximately 90-fold, at least, or up to approximately 100-fold, at least, or up to approximately 500-fold, at least, or up to approximately 1,000-fold, at least, or up to approximately 5,000-fold, or at least, or up to approximately 10,000-fold compared to the control expression and / or activity level of the equivalent gene.Modification of the polyX array can reduce the expression and / or activity level of the target gene by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of the equivalent gene.

[0109] In some cases, modification of the polyX sequence can increase the expression and / or activity level of the target gene by at least, or up to approximately 0.1-fold, at least, or up to approximately 0.2-fold, at least, or up to approximately 0.3-fold, at least, or up to approximately 0.4-fold, at least, or up to approximately 0.5-fold, at least, or up to approximately 0.6-fold, at least, or up to approximately 0.7-fold, at least, or up to approximately 0.8-fold, at least, or up to approximately 0.9-fold, at least, or up to approximately 1-fold, at least, or up to approximately 2-fold, at least, or up to approximately 3-fold, at least, or up to approximately 4-fold, at least, or up to approximately 5-fold, at least, or up to approximately 6-fold, at least, or up to approximately 7-fold, at least, or up to approximately 8-fold, at least, or up to approximately 9-fold, at least, or up to approximately 10-fold, at least, or up to approximately 20-fold, at least, or up to approximately 30-fold, at least, or up to approximately 40-fold, at least, or up to approximately 50-fold, at least, or up to approximately 60-fold, at least, or up to approximately 70-fold, at least, or up to approximately 80-fold, at least, or up to approximately 90-fold, at least, or up to approximately 100-fold, at least, or up to approximately 500-fold, at least, or up to approximately 1,000-fold, at least, or up to approximately 5,000-fold, or at least, or up to approximately 10,000-fold compared to the control expression and / or activity level of the equivalent gene.Modification of the polyX sequence can increase the expression and / or activity level of the target gene by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of the equivalent gene.

[0110] Editing of the polyT sequence in the gNA (e.g., sgRNA) can regulate the expression or activity of the target gene by affecting the expression of the guide nucleic acid molecule from the polynucleotide sequence. Editing of the polyT sequence can enhance, decrease, or stop the expression of the target gene.

[0111] In some cases, the modification of the polyT sequence can reduce 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 modification of the polyT sequence can reduce the expression and / or activity level 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.

[0112] In some cases, modification of the polyT array can increase 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%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1,000%, at least about 2,000%, at least about 3,000%, at least about 4,000%, at least about 5,000%, at least about 6,000%, at least about 7,000%, at least about 8,000%, at least about 9,000%, at least about 10,000%, at least about 100,000%, at least about 1,000,000%, or more.Modification of the polyT sequence can increase the expression and / or activity level 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 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 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%, up to approximately 1%, up to approximately 0.9%, up to approximately 0.8%, up to approximately 0.7%, up to approximately 0.6%, up to approximately 0.5%, up to approximately 0.4%, up to approximately 0.3%, up to approximately 0.2%, up to approximately 0.1%, or less.

[0113] In some cases, the modification of the polyT array can reduce the expression and / or activity level of the target gene by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold compared to the control expression and / or activity level of the equivalent gene.Modification of the polyT sequence can reduce the expression and / or activity level of the target gene by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of the equivalent gene.

[0114] In some cases, the modification of the polyT array can increase the expression and / or activity level of the target gene by at least, or up to approximately 0.1-fold, at least, or up to approximately 0.2-fold, at least, or up to approximately 0.3-fold, at least, or up to approximately 0.4-fold, at least, or up to approximately 0.5-fold, at least, or up to approximately 0.6-fold, at least, or up to approximately 0.7-fold, at least, or up to approximately 0.8-fold, at least, or up to approximately 0.9-fold, at least, or up to approximately 1-fold, at least, or up to approximately 2-fold, at least, or up to approximately 3-fold, at least, or up to approximately 4-fold, at least, or up to approximately 5-fold, at least, or up to approximately 6-fold, at least, or up to approximately 7-fold, at least, or up to approximately 8-fold, at least, or up to approximately 9-fold, at least, or up to approximately 10-fold, at least, or up to approximately 20-fold, at least, or up to approximately 30-fold, at least, or up to approximately 40-fold, at least, or up to approximately 50-fold, at least, or up to approximately 60-fold, at least, or up to approximately 70-fold, at least, or up to approximately 80-fold, at least, or up to approximately 90-fold, at least, or up to approximately 100-fold, at least, or up to approximately 500-fold, at least, or up to approximately 1,000-fold, at least, or up to approximately 5,000-fold, or at least, or up to approximately 10,000-fold, compared to the control expression and / or activity level of the equivalent gene.Modification of the polyT sequence can increase the expression and / or activity level of the target gene by up to or at least about 10,000-fold, up to or at least about 5,000-fold, up to or at least about 1,000-fold, up to or at least about 500-fold, up to or at least about 100-fold, up to or at least about 90-fold, up to or at least about 80-fold, up to or at least about 70-fold, up to or at least about 60-fold, up to or at least about 50-fold, up to or at least about 40-fold, up to or at least about 30-fold, up to or at least about 20-fold, up to or at least about 10-fold, up to or at least about 9-fold, up to or at least about 8-fold, up to or at least about 7-fold, up to or at least about 6-fold, up to or at least about 5-fold, up to or at least about 4-fold, up to or at least about 3-fold, up to or at least about 2-fold, up to or at least about 1-fold, up to or at least about 0.9-fold, up to or at least about 0.8-fold, up to or at least about 0.7-fold, up to or at least about 0.6-fold, up to or at least about 0.5-fold, up to or at least about 0.4-fold, up to or at least about 0.3-fold, up to or at least about 0.2-fold, up to or at least about 0.1-fold, when compared to the control expression and / or activity level of the equivalent gene.

[0115] In some cases, termination of Pol-III controlled transcription can occur at non-standard sequences. The non-standard sequences can be in the form of UUAUUU (SEQ ID NO: 1), which can also be denoted as its DNA complement, e.g., TTATTT, i.e., T2AT3 (SEQ ID NO: 2). The non-standard sequences can be T3AT2 (SEQ ID NO: 3), T3CT2 (SEQ ID NO: 4), T2CT3 (SEQ ID NO: 5), T3GT2 (SEQ ID NO: 6), T2GT3 (SEQ ID NO: 7), T3AT (SEQ ID NO: 8), TAT3 (SEQ ID NO: 9), T3CT (SEQ ID NO: 10), TCT3 (SEQ ID NO: 11), T3GT (SEQ ID NO: 12), TGT3 (SEQ ID NO: 13), T2AT2 (SEQ ID NO: 14), T2CT2 (SEQ ID NO: 15), or T2GT2 (SEQ ID NO: 16). In some cases, the disrupted non-standard termination sequence can be in the form of UUAAUUU (SEQ ID NO: 3).

[0116] Optionally, the non-standard termination sequence comprises at least, or at most about 40%, at least, or at most about 45%, 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 a polynucleotide sequence of one or more members selected from the group consisting of array numbers 1-16, 36, and 45, or a complementary sequence thereof, or may consist essentially of this.

[0117] Optionally, the polynucleotide sequence (or its complementary sequence) comprising the non-standard termination sequence has the following structure (I): T a NT b may have wherein (i) "T" is a thymine nucleobase, (ii) "a" is an integer of 2 or more, (iii) "b" is an integer of 2 or more, and (iv) "N" is one or more nucleobases containing at least one nucleobase other than T. The provided structure (I) may be a continuous sequence. Structure (I) may be a DNA sequence provided from 5' to 3'.

[0118] In structure (I), "a" and "b" may be the same number. Alternatively, "a" and "b" may not be the same number. For example, "a" may be at least, or at most about 1, at least, or at most about 2, at least, or at most about 3, at least, or at most about 4, at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, or at least, or at most about 10 greater than "b". In another example, "b" may be at least, or at most about 1, at least, or at most about 2, at least, or at most about 3, at least, or at most about 4, at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, or at least, or at most about 10 greater than "a".

[0119] In structure (I), both "a" and "b" can be at least, or at most about 3, at least, or at most about 4, at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, at least, or at most about 10, at least, or at most about 11, at least, or at most about 12, at least, or at most about 13, at least, or at most about 14, at least, or at most about 15, at least, or at most about 20.

[0120] In structure (I), when N is 1 or 2, N may not include (or alternatively, may consist of) A, G, and / or C.

[0121] In structure (I), when N is 3 or more, (i) the 5' terminal nucleobase of N (e.g., directly adjacent to T a and (e.g., T bThe 3'-terminal nucleobase (immediately adjacent to it) may not be T, and (ii) one or more nucleobases (e.g., the "core region of N") arranged between the 5'-terminal nucleobase and the 3'-terminal nucleobase of N may be any of the nucleobases A, C, G, and / or T. Optionally, the core region of N may not contain a continuous polyT sequence (e.g., TT, TTT, TTTT, TTTTT, etc.). The core region of N may have a length of at least, or up to about 1, at least, or up to about 2, at least, or up to about 3, at least, or up to about 4, at least, or up to about 5, at least, or up to about 6, at least, or up to about 7, at least, or up to about 8, at least, or up to about 9, at least, or up to about 10, at least, or up to about 11, at least, or up to about 12, at least, or up to about 13, at least, or up to about 14, at least, or up to about 15, at least, or up to about 16, at least, or up to about 17, at least, or up to about 18, at least, or up to about 19, at least, or up to about 20, at least, or up to about 21, at least, or up to about 22, at least, or up to about 23, at least, or up to about 24, at least, or up to about 25, at least, or up to about 30, at least, or up to about 40, at least, or up to about 50 nucleobases.

[0122] Optionally, a polynucleotide sequence (or its complementary sequence) containing a non-standard termination sequence has the following structure (II): M-T a NT b -M' and may have wherein (i) T a NT bis as described above in Structure (I), (ii) M and M’ are polynucleotide sequences that are at least partially complementary to each other, and (iii) “-” is a polynucleotide linker or is absent. Optionally, M and M’ can be targeted by the same gene editing moiety (e.g., a Cas protein complexed with a guide RNA). For example, Structure (II) can be part of a double-stranded vector, and a guide RNA containing the same spacer sequence can, by (1) creating a cleavage within M and creating additional cleavage within the reverse / complementary strand of M’, or (2) creating a cleavage within the reverse / complementary strand of M and creating additional cleavage within M’, via one or more endogenous polynucleotide repair mechanisms such as MMEJ, at least the 3’ portion of M (e.g., the portion closer to T a ), substantially all of T a NT b and at least the 5’ portion of M’ (e.g., T bThe closer part can be removed. Optionally, the number of nucleobases from which M is removed and the number of nucleobases from which M’ is removed can be the same or different. Optionally, the number of nucleobases from which M and / or M’ are removed can be at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, at least, or at most about 10, at least, or at most about 11, at least, or at most about 12, at least, or at most about 13, at least, or at most about 14, at least, or at most about 15, at least, or at most about 16, at least, or at most about 17, at least, or at most about 18, at least, or at most about 19, at least, or at most about 20, at least, or at most about 21, at least, or at most about 22, at least, or at most about 23, at least, or at most about 24, at least, or at most about 25, at least, or at most about 26, at least, or at most about 27, at least, or at most about 28, at least, or at most about 29, or at least, or at most about 30. As provided herein, the remaining (e.g., non-removed) portions of M and M’ can form part of the scaffold sequence of the functional guide nucleic acid.

[0123] Optionally, a polynucleotide sequence (or its complementary sequence) comprising a non-standard termination sequence has the following structure (II): M-T’-M’ can have, wherein (i) T’ is a non-standard termination sequence provided herein (e.g., polyT), and (ii) M and M’ are as described above in structure (II).

[0124] Optionally, in a pair comprising M and M’ as shown in structure (II) and / or structure (III), the pair can form an insulator sequence provided herein. Alternatively, the pair can form a stem sequence provided herein.

[0125] In some cases, in pairs containing M and M’ shown in structure (II) and / or structure (III), the polynucleotide sequence M and the further polynucleotide sequence M’ are each selected from the group consisting of (1) SEQ ID NO: 17 and SEQ ID NO: 54, (2) SEQ ID NO: 18 and SEQ ID NO: 55, (3) SEQ ID NO: 19 and SEQ ID NO: 56, (4) SEQ ID NO: 20 and SEQ ID NO: 57, (5) SEQ ID NO: 21 and SEQ ID NO: 58, (6) SEQ ID NO: 22 and SEQ ID NO: 59, (7) SEQ ID NO: 23 and SEQ ID NO: 60, (8) SEQ ID NO: 24 and SEQ ID NO: 61, (9) SEQ ID NO: 26 and SEQ ID NO: 62, (10) SEQ ID NO: 27 and SEQ ID NO: 63, (11) SEQ ID NO: 28 and SEQ ID NO: 64, (12) SEQ ID NO: 29 and SEQ ID NO: 65, (13) SEQ ID NO: 30 and SEQ ID NO: 66, (14) SEQ ID NO: 31 and SEQ ID NO: 67, (15) SEQ ID NO: 32 and SEQ ID NO: 68, (16) SEQ ID NO: 33 and SEQ ID NO: 69, (17) SEQ ID NO: 34 and SEQ ID NO: 70, and (18) SEQ ID NO: 35 and SEQ ID NO: 71, or pairs of their complementary sequences, and can show at least, or up to about 40%, at least, or up to about 45%, at least, or up to about 50%, at least, or up to about 55%, at least, or up to about 60%, at least, or up to about 65%, at least, or up to about 70%, at least, or up to about 75%, at least, or up to about 80%, at least, or up to about 85%, at least, or up to about 86%, at least, or up to about 87%, at least, or up to about 88%, at least, or up to about 89%, at least, or up to about 90%, at least, or up to about 91%, at least, or up to about 92%, at least, or up to about 93%, at least, or up to about 94%, at least, or up to about 95%, at least, or up to about 96%, at least, or up to about 97%, at least, or up to about 98%, at least, or up to about 99%, or substantially about 100% sequence identity.

[0126] Non-standard disruption arrays, also known as non-standard arrays or non-standard termination arrays, can cause premature termination. Non-standard termination arrays can be modified by an endonuclease (e.g., Cas9 endonuclease) to disrupt the non-standard termination array by inserting at least one nucleotide. The non-standard termination array can be altered by inserting at least, or up to about 1, at least, or up to about 2, at least, or up to about 3, at least, or up to about 4, at least, or up to about 5, at least, or up to about 6, at least, or up to about 7, at least, or up to about 8, at least, or up to about 9, or at least, or up to about 10 nucleotides. Alternatively or additionally, non-standard termination arrays can be modified by an endonuclease (e.g., Cas9 endonuclease) to disrupt the non-standard termination array by deleting at least one nucleotide.The non-standard termination array can be altered by deletion of at least, or maximally about 1, at least, or maximally about 2, at least, or maximally about 3, at least, or maximally about 4, at least, or maximally about 5, at least, or maximally about 6, at least, or maximally about 7, at least, or maximally about 8, at least, or maximally about 9, at least, or maximally about 10, at least, or maximally about 12, at least, or maximally about 13, at least, or maximally about 14, at least, or maximally about 15, at least, or maximally about 16, at least, or maximally about 17, at least, or maximally about 18, at least, or maximally about 19, at least, or maximally about 20, at least, or maximally about 25, at least, or maximally about 20, at least, or maximally about 25, at least, or maximally about 30, at least, or maximally about 35, at least, or maximally about 40, at least, or maximally about 45, at least, or maximally about 50, at least, or maximally about 55, at least, or maximally about 60, at least, or maximally about 65, at least, or maximally about 70, at least, or maximally about 75, at least, or maximally about 80, at least, or maximally about 90, or at least, or maximally about 100 nucleotides.

[0127] In some cases, the non-standard termination sequence can be altered by deletion of at least, or up to approximately 1%, at least, or up to approximately 2%, at least, or up to approximately 3%, at least, or up to approximately 4%, at least, or up to approximately 5%, at least, or up to approximately 6%, at least, or up to approximately 7%, at least, or up to approximately 8%, at least, or up to approximately 9%, at least, or up to approximately 10%, at least, or up to approximately 15%, at least, or up to approximately 20%, at least, or up to approximately 25%, at least, or up to approximately 30%, at least, or up to approximately 35%, at least, or up to approximately 40%, at least, or up to approximately 45%, at least, or up to approximately 50%, at least, or up to approximately 55%, at least, or up to approximately 60%, at least, or up to approximately 65%, at least, or up to approximately 70%, at least, or up to approximately 75%, at least, or up to approximately 80%, at least, or up to approximately 85%, at least, or up to approximately 90%, at least, or up to approximately 91%, at least, or up to approximately 92%, at least, or up to approximately 93%, at least, or up to approximately 94%, at least, or up to approximately 95%, at least, or up to approximately 96%, at least, or up to approximately 97%, at least, or up to approximately 98%, at least, or up to approximately 99%, or substantially about 100% thereof, thereby enabling the expression of a functional variant of the guide nucleic acid molecule. For example, two ends of a desired portion of the non-standard termination sequence (e.g., the 5' upstream stem and the 3' downstream stem disposed adjacent to the 5' end and the 3' end of the polyT non-standard termination sequence, as shown in FIGS. 22A and 22B) can be specifically targeted (e.g., via a Cas / guide nucleic acid complex) and cleaved at or adjacent to the 5' end and the 3' end of the polyT non-standard termination sequence, thereby removing at least a portion or all of the polyT non-standard termination sequence.

[0128] In some cases, the non-standard termination sequence may be located within the RNA (e.g., not at the end). In some cases, the non-standard termination sequence may be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 3' end of the polynucleotide sequence. In some cases, the non-standard termination sequence may be located at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 bases away from the 5' end of the polynucleotide sequence. In some cases, the non-standard termination sequence may be located at the end of the nucleic acid sequence.

[0129] Optionally, at least one edit can be made to the non-standard termination sequence. The edits made to the polyX sequence can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, or more. The edits made to the non-standard termination sequence can be at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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. The edits to the non-standard termination sequence can be insertions. Alternatively or additionally, the edits to the non-standard termination sequence can be deletions. Alternatively or additionally, the edits to the non-standard termination sequence can be excision of the non-standard termination sequence. Excision of the non-standard termination sequence can be achieved using two cleavage sites flanking the non-standard termination sequence. The edits to the non-standard termination sequence can utilize various forms of nucleic acid repair mechanisms such as homology-directed repair (HDR), non-homologous end joining (NHEJ) repair, and microhomology-mediated end joining (MMEJ) repair, but are not limited thereto.

[0130] Optionally, at least one edit can be made to the non-standard termination sequence. The edits made to the non-standard termination sequence can be 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 11, at least about 12, at least about 13, at least about 14, at least about 15, or more. The edits made to the non-standard termination sequence can be at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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. The edits to the non-standard termination sequence can be insertions. Alternatively or additionally, the edits to the non-standard termination sequence can be deletions. The edits to the non-standard termination sequence can utilize various forms of nucleic acid repair mechanisms such as homology-directed repair (HDR), non-homologous end joining (NHEJ) repair, and microhomology-mediated end joining (MMEJ) repair, but are not limited thereto.

[0131] In some cases, modification of the non-standard termination sequence can reduce the expression and / or activity level of the guide nucleic acid molecule by at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more. Modification of the non-standard termination sequence can reduce the expression and / or activity level of the guide nucleic acid molecule 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.

[0132] In some cases, modification of the non-standard termination sequence can increase the expression and / or activity level of the guide nucleic acid molecule 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%, at least about 1,000,000%, or more.Modification of the non-standard termination array can increase the expression and / or activity level of the guide nucleic acid molecule by up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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.

[0133] In some cases, modification of the non-standard termination sequence can reduce the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the polyX array can reduce the expression and / or activity level of the guide nucleic acid molecule by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of an equivalent guide nucleic acid.

[0134] In some cases, modification of the non-standard termination sequence can increase the expression and / or activity level of the guide nucleic acid molecule by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold, compared to the control expression and / or activity level of the equivalent guide nucleic acid.Modification of the non-standard termination sequence can increase the expression and / or activity level of the guide nucleic acid molecule by at least or up to about 10,000-fold, at least or up to about 5,000-fold, at least or up to about 1,000-fold, at least or up to about 500-fold, at least or up to about 100-fold, at least or up to about 90-fold, at least or up to about 80-fold, at least or up to about 70-fold, at least or up to about 60-fold, at least or up to about 50-fold, at least or up to about 40-fold, at least or up to about 30-fold, at least or up to about 20-fold, at least or up to about 10-fold, at least or up to about 9-fold, at least or up to about 8-fold, at least or up to about 7-fold, at least or up to about 6-fold, at least or up to about 5-fold, at least or up to about 4-fold, at least or up to about 3-fold, at least or up to about 2-fold, at least or up to about 1-fold, at least or up to about 0.9-fold, at least or up to about 0.8-fold, at least or up to about 0.7-fold, at least or up to about 0.6-fold, at least or up to about 0.5-fold, at least or up to about 0.4-fold, at least or up to about 0.3-fold, at least or up to about 0.2-fold, at least or up to about 0.1-fold, when compared to the control expression and / or activity level of the equivalent guide nucleic acid.

[0135] Optionally, the sgRNA includes an additional termination sequence. The sgRNA can include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 termination sequences.

[0136] Optionally, the sgRNA comprises a first termination sequence and a second termination sequence. Optionally, the first termination sequence is a polyX sequence and the second termination sequence is a polyX sequence. Optionally, the first termination sequence is a polyX sequence and the second termination sequence is a polyT sequence. Optionally, the first termination sequence is a polyX sequence and the second termination sequence is a non-standard termination sequence. Optionally, the first termination sequence is a polyT sequence and the second termination sequence is a polyX sequence. Optionally, the first termination sequence is a polyT sequence and the second termination sequence is a polyT sequence. Optionally, the first termination sequence is a polyT sequence and the second termination sequence is a non-standard termination sequence. Optionally, the first termination sequence is a non-standard termination sequence and the second termination sequence is a polyX sequence. Optionally, the first termination sequence is a non-standard termination sequence and the second termination sequence is a polyT sequence. Optionally, the first termination sequence is a non-standard termination sequence and the second termination sequence is a non-standard termination sequence.

[0137] Optionally, the two termination sequences are adjacent to each other. Alternatively or additionally, the two termination sequences can be separated by 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, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 30, at least about 40, or at least about 50 nucleotides.

[0138] Optionally, the sgRNA comprises a first polyX sequence (e.g., a polyT sequence) and a second polyX sequence (e.g., a polyT sequence). Optionally, the first polyX sequence and the second polyX sequence are the same. Alternatively, optionally, the first polyX sequence and the second polyX sequence are different. Optionally, the nucleobase length of the first polyX sequence and the nucleobase length of the second polyX sequence are the same. Alternatively, optionally, the nucleobase length of the first polyX sequence and the nucleobase length of the second polyX sequence are different. Optionally, the first polyX sequence and the second polyX sequence are separated by a non-polyX sequence (or non-terminating sequence). Optionally, the non-polyX sequence between which the first polyX sequence and the second polyX sequence are adjacent (e.g., disposed therebetween) is at least about 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleobases in length. Optionally, the non-polyX sequence between which the first polyX sequence and the second polyX sequence are adjacent (e.g., disposed therebetween) is at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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 nucleobase in length.

[0139] Optionally, the sgRNA comprises a first polyT sequence and a second polyT sequence. Optionally, the first polyT sequence and the second polyT sequence are the same. Alternatively, optionally, the first polyT sequence and the second polyT sequence are different. Optionally, the first polyT sequence and the second polyT sequence are separated by a non-polyT sequence. Optionally, the non-polyT sequence adjacent to the polyT sequence is at least about 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 bases in length. Optionally, the non-polyT sequence adjacent to the polyT sequence is at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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 base in length.

[0140] Optionally, the sgRNA includes a first non-standard termination sequence and a second non-standard termination sequence. Optionally, the first non-standard termination sequence and the second non-standard termination sequence are the same. Alternatively, optionally, the first non-standard termination sequence and the second non-standard termination sequence are different. Optionally, the first non-standard termination sequence and the second non-standard termination sequence are separated by a sequence that is not a non-standard termination sequence (e.g., a non-polyX sequence, a non-polyT sequence, etc.). Optionally, the sequence that is not a non-standard termination sequence and is adjacent to the non-standard termination sequence is at least about 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 bases in length. Optionally, the sequence that is not a non-standard termination sequence and is adjacent to the non-standard termination sequence is at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, 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 base in length.

[0141] When a guide nucleic acid molecule such as a guide RNA (or sgRNA) is described as including components (e.g., one or more termination sequences, one or more polyX sequences, etc.), this description may refer to the expression (e.g., transcription) form of the guide nucleic acid molecule, or alternatively, may refer to a polynucleotide sequence encoding such a guide nucleic acid molecule, such as a vector or plasmid. Optionally, when a polynucleotide sequence encoding an activatable guide nucleic acid molecule (e.g., including polyT) is described, such an activatable guide nucleic acid molecule may be referred to as a "guide nucleic acid molecule" or a "guide RNA".

[0142] Optionally, the polynucleotide sequence encoding the guide nucleic acid molecule may contain a domain comprising polyT, which domain is located between two cleavage sites (e.g., the upstream stem site and the downstream stem site provided herein) to allow removal of such domain for activation of the guide nucleic acid molecule. The domain can be a continuous polynucleotide sequence. The domain can contain a polyT sequence and a non-polyT sequence. The domain can have a length of at least, or up to about 6 nucleobases, at least, or up to about 8 nucleobases, at least, or up to about 10 nucleobases, at least, or up to about 12 nucleobases, at least, or up to about 15 nucleobases, at least, or up to about 20 nucleobases, at least, or up to about 25 nucleobases, at least, or up to about 30 nucleobases, at least, or up to about 35 nucleobases, at least, or up to about 40 nucleobases, at least, or up to about 45 nucleobases, at least, or up to about 50 nucleobases, at least, or up to about 55 nucleobases, at least, or up to about 60 nucleobases, at least, or up to about 65 nucleobases, at least, or up to about 70 nucleobases, at least, or up to about 75 nucleobases, at least, or up to about 80 nucleobases, at least, or up to about 85 nucleobases, at least, or up to about 90 nucleobases, at least, or up to about 95, or at least, or up to about 100 nucleobases.The percentage of the polyT sequence within the domain can be at least, or at most approximately 510%, at least, or at most approximately 15%, at least, or at most approximately 20%, at least, or at most approximately 25%, at least, or at most approximately 30%, at least, or at most approximately 35%, at least, or at most approximately 40%, at least, or at most approximately 45%, at least, or at most approximately 50%, at least, or at most approximately 55%, at least, or at most approximately 60%, at least, or at most approximately 65%, at least, or at most approximately 70%, at least, or at most approximately 75%, at least, or at most approximately 80%, at least, or at most approximately 85%, at least, or at most approximately 90%, or at least, or at most approximately 95%. The percentage of the non-polyT sequence within the domain can be at least, or at most approximately 510%, at least, or at most approximately 15%, at least, or at most approximately 20%, at least, or at most approximately 25%, at least, or at most approximately 30%, at least, or at most approximately 35%, at least, or at most approximately 40%, at least, or at most approximately 45%, at least, or at most approximately 50%, at least, or at most approximately 55%, at least, or at most approximately 60%, at least, or at most approximately 65%, at least, or at most approximately 70%, at least, or at most approximately 75%, at least, or at most approximately 80%, at least, or at most approximately 85%, at least, or at most approximately 90%, or at least, or at most approximately 95%.

[0143] Optionally, the polynucleotide sequence further comprises a region encoding an endonuclease recognition site. The endonuclease recognition site can be located adjacent to the region encoding the gNA molecule. The endonuclease recognition site can be located 5' to the region encoding the gNA molecule. The endonuclease recognition site can be located 3' to the region encoding the gNA molecule.

[0144] Optionally, the polynucleotide sequence may include a filler sequence adjacent to the region encoding the gNA molecule. Optionally, the polynucleotide sequence may include a filler sequence that is 5' of the region encoding the gNA molecule. Optionally, the polynucleotide sequence may include a filler sequence that is 3' of the region encoding the gNA molecule. Optionally, the polynucleotide sequence may include a region encoding a gNA molecule adjacent to which the filler sequence is located. The filler sequence may be 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, or more bases in length. The filler sequence may be at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 15, at most about 10, or less bases in length.

[0145] Optionally, the polynucleotide sequence further includes an insulator region. The insulator region may be an additional sequence that provides stability to the gNA molecule. The insulator region may be a sequence that includes a sequence targetable by a gene editing moiety. For example, the insulator region may include a PAM sequence targetable by a Cas endonuclease.

[0146] The insulator region may include one PAM sequence. Alternatively, the insulator region may include multiple PAM sequences. The insulator region may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 PAM regions. The insulator region may have at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 PAM region. The insulator region may have PAM sequences that face the same direction (e.g., PAM sequences in the direction from 5' to 3'). Alternatively, the insulator region may have PAM sequences that face opposite directions (e.g., PAM sequences in both the direction from 5' to 3' and the direction from 3' to 5').

[0147] The insulator region can be located between the transcription terminator region and the hairpin region of the gNA. The insulator region can be adjacent to the transcription terminator region (e.g., the polyU region). Alternatively, the insulator region may not be adjacent to the transcription terminator region. The insulator region can be downstream of the transcription terminator region (e.g., the polyU region). The insulator region can be immediately downstream of the transcription terminator region (e.g., the polyU region). Alternatively, the insulator region can be upstream of the transcription terminator region (e.g., the polyU region). The insulator region can be immediately upstream of the transcription terminator region (e.g., the polyU region).

[0148] Optionally, the insulator region does not include a polyX region (e.g., the polyU region). Alternatively, the insulator region can include a polyX region. Optionally, the insulator region sequence is precisely defined. Alternatively, optionally, the insulator region sequence is agnostic.

[0149] As seen in FIG. 5A, the insulator region can include a completely complementary sequence (I). Alternatively or additionally, the insulator region can include an array including a stem (S) also described as a non-complementary bubble region. Optionally, the insulator region can include an array (SI) including a complementary region following a non-complementary stem. Optionally, the insulator region can include an array (IS) including a non-complementary stem following a complementary region. Optionally, the insulator region can include an array (ISI) including a non-complementary stem adjacent to a complementary region.

[0150] Optionally, the insulator region may have multiple non-complementary stem regions. The insulator region may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 non-complementary stems. The insulator region may have up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 stem.

[0151] The further sequence of the insulator region may be at least about 10, at least about 12, at least about 14, at least about 15, at least about 20, at least about 20, 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 150, or at least about 200 nucleotides in length. The further sequence of the insulator region may be up to about 200, up to about 150, 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, or up to about 10 nucleotides in length.

[0152] Optionally, the addition of the insulator region may result in a gNA with increased stability after modification by the gene editing moiety, compared to a gNA that does not contain the insulator region. Optionally, the addition of a fully complementary insulator region may result in a gNA with increased stability after modification by the gene editing moiety, compared to a gNA that contains a stem region. Alternatively, the addition of one or more stem regions may result in a gNA with increased stability after modification by the gene editing moiety, compared to a gNA that contains a fully complementary insulator region.

[0153] In some cases, the addition of an insulator region can result in a gNA with reduced stability after modification by the gene editing portion, compared to a gNA that does not include the insulator region. In some cases, the addition of a fully complementary insulator region can result in a gNA with reduced stability after modification by the gene editing portion, compared to a gNA that includes a stem region. Alternatively, the addition of one or more stem regions can result in a gNA with reduced stability after modification by the gene editing portion, compared to a gNA that includes a fully complementary insulator region.

[0154] In some cases, the system of the present disclosure may further include an endonuclease capable of forming a complex with a gNA molecule. In some cases, the gNA-endonuclease complex may affect the regulation of the expression or activity of a target gene. The endonuclease can be a type I endonuclease, a type II endonuclease, or a type III endonuclease. The endonuclease can be a Cas endonuclease (e.g., Cas9, Cas10, Cas12, Cas13, Cas14, dCas).

[0155] In some cases, a guide nucleic acid molecule (gNA) (e.g., a functional gNA) expressed by a second gate unit can cause a modification to at least a portion of the first gate unit upon activation. For example, the activated gNA of the second gate unit can cause a modification to the polynucleotide sequence of the first gate unit that encodes the gNA (e.g., an activatable gNA), or to the promoter sequence of the first gate unit operably linked to such gNA of the same first gate unit. Such a modification can render the gNA of the first gate unit inoperable when expressed (e.g., reduce or inhibit specific binding to a target gene). Alternatively, the modification can reduce (e.g., inhibit) the expression of the gNA of the first gate unit.

[0156] Optionally, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be caused by a single-strand break that has discontinuity in one nucleotide strand. Inactivation of a polynucleotide sequence or a target gene can be caused by 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, or more single-strand breaks. Optionally, gene inactivation can be caused by 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 single-strand break.

[0157] Optionally, the gNA can have a size of at least, or up to about 60 nucleotides, at least, or up to about 70 nucleotides, at least, or up to about 80 nucleotides, at least, or up to about 85 nucleotides, at least, or up to about 90 nucleotides, at least, or up to about 95 nucleotides, at least, or up to about 100 nucleotides, at least, or up to about 105 nucleotides, at least, or up to about 110 nucleotides, at least, or up to about 120 nucleotides, at least, or up to about 130 nucleotides, at least, or up to about 140 nucleotides, at least, or up to about 150 nucleotides, or at least, or up to about 200 nucleotides (e.g., including both a spacer sequence and a scaffold sequence).

[0158] Optionally, the scaffold sequence of the gNA can have a size of at least, or up to about 30 nucleotides, at least, or up to about 35 nucleotides, at least, or up to about 40 nucleotides, at least, or up to about 45 nucleotides, at least, or up to about 50 nucleotides, at least, or up to about 55 nucleotides, at least, or up to about 60 nucleotides, at least, or up to about 65 nucleotides, at least, or up to about 70 nucleotides, at least, or up to about 75 nucleotides, at least, or up to about 80 nucleotides, at least, or up to about 85 nucleotides, at least, or up to about 90 nucleotides, at least, or up to about 95 nucleotides, at least, or up to about 100 nucleotides, at least, or up to about 100 nucleotides, at least, or up to about 120 nucleotides, at least, or up to about 130 nucleotides, at least, or up to about 140 nucleotides, or at least, or up to about 150 nucleotides.

[0159] Optionally, the spacer sequence of the gNA can have a size of at least, or up to about 10 nucleotides, at least, or up to about 11, at least, or up to about 12, at least, or up to about 13, at least, or up to about 14, at least, or up to about 15, at least, or up to about 16, at least, or up to about 17, at least, or up to about 18, at least, or up to about 19, at least, or up to about 20, at least, or up to about 21, at least, or up to about 22, at least, or up to about 23, at least, or up to about 24, at least, or up to about 25, at least, or up to about 26, at least, or up to about 27, at least, or up to about 28, at least, or up to about 29, or at least, or up to about 30 nucleotides.

[0160] In some cases, the systems and methods of the present disclosure can utilize a single endonuclease system (e.g., a Cas inhibitor) to achieve both (i) polynucleotide cleavage (e.g., for activating / inactivating a gate portion and / or a gene regulatory portion) and (ii) regulation of target gene expression. When using a single endonuclease transcriptional regulator system, unique guide nucleic acid molecules (gNAs) with different spacer sequence lengths can be used to determine whether the single endonuclease transcriptional regulator system can (i) hybridize to a polynucleotide sequence to induce Cas-mediated nuclease activity of the polynucleotide sequence, or (ii) hybridize to a target gene (e.g., genomic DNA) to regulate the expression and / or activity level of the target gene by the action of a transcriptional activator without mediating Cas nuclease activity as desired by individual non-homologous genetic circuits. For example, the use of gNAs with different spacer sequence lengths that bind to different targets can enable the second gate unit provided herein to induce inactivation of the activated first gate unit and / or to induce individual regulation of the second target gene.

[0161] As described above, the length of the spacer array of the gNA can affect the ability of the gNA to mediate Cas nuclease activity. Optionally, gNAs having spacer arrays of different lengths can be used in the same non-homologous genetic circuit to affect the cleavage, activation, inactivation, and / or regulation of different types of one or more target nucleic acids. Optionally, a gNA spacer array shorter than a threshold length (e.g., about 16 nucleotides) can mediate DNA binding for transcriptional regulation of a target gene while eliminating the nuclease activity of the Cas transcriptional regulator. Optionally, a gNA spacer array 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 mediate DNA binding while eliminating the nuclease activity of the Cas protein.

[0162] For example, a gNA comprising a 20-nucleotide spacer array (e.g., a gNA encoded by a gate portion to target a gene regulatory portion plasmid) may be sufficient to promote the nuclease activity of an endonuclease (e.g., Cas or a Cas transcriptional regulator fusion protein) in a target polynucleotide sequence. Alternatively or additionally, a gNA comprising a 14-nucleotide spacer array (e.g., a gNA encoded by a gene regulatory portion) can hybridize to DNA but may not be long enough to mediate nuclease activity and can only promote endonuclease binding to cognate DNA sequences. Thus, shorter gNAs can selectively enable transcriptional regulation of a target gene through the use of an endonuclease transcriptional regulator system (e.g., a Cas activator system, a Cas repressor system) without cleaving the target gene.

[0163] Optionally, the modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be caused by a double-strand break in which discontinuities exist in both nucleotide strands. Optionally, the number of such double-strand breaks (e.g., required for such modification) can be at least, or at most about 1, at least, or at most about 2, at least, or at most about 3, at least, or at most about 4, at least, or at most about 5, at least, or at most about 6, at least, or at most about 7, at least, or at most about 8, at least, or at most about 9, or at least, or at most about 10. Optionally, the modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be caused by an indel, also known as an insertion-deletion mutation. Indel mutations can include frameshift mutations or non-frameshift mutations. Indel mutations can include point mutations, also called base substitutions, in which only one base or base pair is modified. Indel mutations can include lengths 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, 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. Indel mutations can include lengths of at most about 2000, at most about 1000, at most about 900, at most about 800, at most about 700, at most about 600, at most about 500, at most about 400, at most about 300, at most about 200, at most about 100, at most about 90, at most about 80, at most about 70, at most about 60, at most about 50, at most about 40, at most about 30, at most about 20, at most about 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 base or base pair.

[0164] In some cases, modification of a polynucleotide sequence (e.g., as a component of a gate unit such as a gate portion) or a target gene can be achieved without cleaving the polynucleotide sequence or the target gene. For example, a gene regulatory moiety (e.g., a nucleic acid molecule such as a complex comprising a CRISPR / Cas protein and a guide nucleic acid molecule and / or an endonuclease) can specifically bind to a polynucleotide sequence or a target gene such that the expression and / or activity of the polynucleotide sequence or the target gene is modified. The gene regulatory moiety can include a transcriptional repressor or transcriptional activator provided herein. Alternatively or additionally, the gene regulatory moiety can induce an epigenetic modification (or epigenetic modification) provided herein.

[0165] In some cases, modification of a polynucleotide sequence or a target gene provided herein can inactivate the polynucleotide sequence or the target gene. For example, modification of a polynucleotide sequence or a target gene can suppress or reduce the expression and / or activity level of the polynucleotide sequence or the target gene. In some cases, modification of a polynucleotide sequence or a target gene provided herein can activate the polynucleotide sequence or the target gene. For example, modification of a polynucleotide sequence or a target gene can increase the expression and / or activity level of the polynucleotide sequence or the target gene.

[0166] In some cases, the modification of the polynucleotide sequences or target genes provided herein may reduce the expression and / or activity levels of the polynucleotide sequences or target genes by at least, or up to about 0.1%, at least, or up to about 0.2%, at least, or up to about 0.3%, at least, or up to about 0.4%, at least, or up to about 0.5%, at least, or up to about 1%, at least, or up to about 2%, at least, or up to about 3%, at least, or up to about 4%, at least, or up to about 5%, at least, or up to about 10%, at least, or up to about 15%, at least, or up to about 20%, at least, or up to about 30%, at least, or up to about 40%, at least, or up to about 50%, at least, or up to about 60%, at least, or up to about 70%, at least, or up to about 80%, at least, or up to about 90%, at least, or up to about 95%, at least, or up to about 99%, or about 100% (e.g., as compared to a control without the modification).

[0167] In some cases, the modification of the polynucleotide sequences or target genes provided herein can reduce the expression and / or activity levels of the polynucleotide sequences or target genes by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 1.5-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 11-fold, at least, or up to about 12-fold, at least, or up to about 13-fold, at least, or up to about 14-fold, at least, or up to about 15-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, or at least, or up to about 100-fold (e.g., as compared to a control without the modification).

[0168] Optionally, the modification of the polynucleotide sequence or target gene provided herein can increase the expression and / or activity level of the polynucleotide sequence or target gene by at least, or up to about 0.1%, at least, or up to about 0.2%, at least, or up to about 0.3%, at least, or up to about 0.4%, at least, or up to about 0.5%, at least, or up to about 1%, at least, or up to about 2%, at least, or up to about 3%, at least, or up to about 4%, at least, or up to about 5%, at least, or up to about 10%, at least, or up to about 15%, at least, or up to about 20%, at least, or up to about 30%, at least, or up to about 40%, at least, or up to about 50%, at least, or up to about 60%, at least, or up to about 70%, at least, or up to about 80%, at least, or up to about 90%, at least, or up to about 100%, at least, or up to about 150%, at least, or up to about 200%, at least, or up to about 300%, at least, or up to about 400%, or at least, or up to about 500% (e.g., compared to a control without the modification).

[0169] In some cases, the modification of the polynucleotide sequences or target genes provided herein can increase the expression and / or activity levels of the polynucleotide sequences or target genes by at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 1.5-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 11-fold, at least, or up to about 12-fold, at least, or up to about 13-fold, at least, or up to about 14-fold, at least, or up to about 15-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 100-fold, at least, or up to about 200-fold, at least, or up to about 300-fold, at least, or up to about 400-fold, at least, or up to about 500-fold, or at least, or up to about 1,000-fold (e.g., compared to a control without the modification).

[0170] Optionally, the control expression and / or activity level of an equivalent guide nucleic acid disclosed herein may refer to the expression and / or activity level of a guide nucleic acid molecule that is derived from the same polynucleotide sequence but does not contain a modification of a polyX sequence such as a polyT sequence within the polynucleotide sequence. Optionally, the control expression and / or activity level of an equivalent guide nucleic acid disclosed herein may refer to the expression and / or activity level of an equivalent guide nucleic acid molecule from a control polynucleotide sequence encoding the equivalent guide nucleic acid molecule, wherein the domain of the control polynucleotide sequence corresponding to the tetraloop region of the equivalent guide nucleic acid molecule does not contain the polyX sequence (e.g., polyT sequence) provided herein.

[0171] When a non-homologous genetic circuit is activated as provided herein to induce multiple individual modulations of a target gene as provided herein, the multiple individual modulations of the target gene can be different (e.g., the degree of change in the expression and / or activity level of the target gene is different). For example, the first modulation exerted by the first gate unit and the second modulation exerted by the second gate 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% different. The first modulation and the second modulation can be at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, or at most about 0.1% different. Alternatively or additionally, the individual modulations of the target gene can be substantially the same (e.g., the same).

[0172] The multiple individual modulations can each be sufficient to induce a desired change in the expression and / or activity level of the target gene. Alternatively, the individual modulations can each be insufficient to induce a desired change in the expression and / or activity level of the target gene.

[0173] One or more target genes disclosed herein may include one or more endogenous genes (e.g., genomic DNA, mRNA, mitochondrial DNA, etc.), exogenous genes, transgenes, or combinations thereof.

[0174] One or more target genes disclosed herein may include cell differentiation regulators, molecular function regulators, binding factors, fusion factors, protein folding chaperones, protein tags, RNA folding chaperones, cell signaling factors, immune response factors, sensory receptors, cell structure factors, protein binding factors, cargo receptors, catalytic factors, or small molecule sensors.

[0175] Optionally, the target gene may be subjected to at least two separate regulations including a first regulation and a second regulation. The timing of the first regulation and the second regulation is controllable (e.g., as predetermined by the design of a heterologous genetic circuit). For example, the start of the second regulation (e.g., by at least a part of a second gate unit such as a second gene regulation part) may occur at least about 1 second, at least about 2 seconds, at least about 3 seconds, at least about 4 seconds, at least about 5 seconds, at least about 6 seconds, at least about 7 seconds, at least about 8 seconds, at least about 9 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days after the start of the first regulation (e.g., by at least a part of a first gate unit such as a first gene regulation part).The start of this second regulation (e.g., by at least a part of a second gate unit such as a second gene regulation part) can occur 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, up to about 1 day, up to about 20 hours, up to about 10 hours, up to about 9 hours, up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 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 after the start of the first regulation (e.g., by at least a part of a first gate unit such as a first gene regulation part).

[0176] In some cases, the number of gate units that need to be activated (e.g., continuously 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 at least partially determine (e.g., substantially determine) the timing between the first regulation and the second regulation. When the first regulation of the target gene is activated by the first gate unit, in order to activate the second gate unit to induce the second regulation, it may be necessary to activate (e.g., continuously activate) 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. When the first regulation of the target gene is activated by the first gate unit, in order to activate the second gate unit to induce the second regulation, it may be necessary to activate (e.g., continuously activate) a maximum of about 50, a maximum of about 40, a maximum of about 30, a maximum of about 20, a maximum of about 15, a maximum of about 10, a maximum of about 9, a maximum of about 8, a maximum of about 7, a maximum of about 6, a maximum of about 5, a maximum of about 4, a maximum of about 3, a maximum of about 2, or a maximum of about 1 additional gate unit.

[0177] Cell fate can involve the regulation of multiple target genes. For example, fate can involve the regulation of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or more target genes. Fate can involve the 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 of the genes disclosed herein can be subjected 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 regulatory events. Each of the genes disclosed herein can be subjected 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 regulatory event. One or more regulations of a target gene (e.g., an endogenous gene) induced by a heterologous genetic circuit of the present disclosure may be artificial regulation (or heterologous regulation), and otherwise may not occur in a cell in the absence of (i) the heterologous genetic circuit and / or (ii) the activating portion of the heterologous genetic circuit.

[0178] The plurality of gate units can operate sequentially (e.g., each of the plurality of gate units is sequentially activated). For example, the plurality of gate units are activated to activate the next plurality of gate units. The sequential operation of the gate units can be linear. Alternatively, the sequential operation of the gate units can be routed to feed back to each other as inputs forming a loop. For example, the plurality of gate units can induce a feedback loop such as a positive feedback loop or a negative feedback loop.

[0179] In some embodiments of any one of the systems disclosed herein, to induce a first individual regulation, the first gate unit may include a first gene regulatory moiety that can be activated to exhibit specific binding to a target gene. Alternatively or additionally, to induce a first individual regulation, the first gate unit may include a first gene regulatory moiety that can be activated to exhibit non-specific binding to a target gene.

[0180] The first individual regulation can induce a change (e.g., increase or decrease) of 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 in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the first individual regulation. The first individual regulation can induce a change (e.g., increase or decrease) of at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 9%, at most about 8%, at most about 7%, at most about 6%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, at most about 0.9%, at most about 0.8%, at most about 0.7%, at most about 0.6%, at most about 0.5%, at most about 0.4%, at most about 0.3%, at most about 0.2%, at most about 0.1%, or less in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the first individual regulation.

[0181] The first individual regulation disclosed herein (e.g., induced by the first gate unit) can induce a change (e.g., an increase or decrease) of at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the first individual regulation.The first individual regulation can induce a change (e.g., increase or decrease) of at least about 0.1-fold, at least about 0.2-fold, at least about 0.3-fold, at least about 0.4-fold, at least about 0.5-fold, at least about 0.6-fold, at least about 0.7-fold, at least about 0.8-fold, at least about 0.9-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 500-fold, at least about 1,000-fold, at least about 5,000-fold, or at least about 10,000-fold as compared to the control expression and / or activity level of a gene not targeted by the first individual regulation in the expression and / or activity level of the target gene.

[0182] Optionally, as disclosed herein, the control expression and / or activity level of a gene not targeted by a first individual regulation may refer to the expression and / or activity level of a housekeeping gene (e.g., a constitutive gene that controls basal cell function). Optionally, as disclosed herein, the control expression and / or activity level of a gene not targeted by a first individual regulation may refer to the expression and / or activity level of a gene controlled by a second individual regulation. Optionally, as disclosed herein, the control expression and / or activity level of a gene not targeted by a first individual regulation may refer to the expression and / or activity level of a gene controlled by a second genetic circuit. Optionally, as disclosed herein, the control expression and / or activity level of a gene not targeted by a first individual regulation may refer to the expression and / or activity level of a gene acting in the same metabolic pathway as the target gene. Alternatively, as disclosed herein, the control expression and / or activity level of a gene not targeted by a first individual regulation may refer to the expression and / or activity level of a gene not acting in the same metabolic pathway as the target gene.

[0183] Subsequently, the second individual regulation disclosed herein (e.g., induced by a second gate unit) induces a further change (e.g., an increase, a decrease, or a selective attenuation) of 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% in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the second individual regulation.The second individual regulation can induce a further change (e.g., increase or decrease) of up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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% in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the second individual regulation.

[0184] A further change due to the second individual regulation is at least, or up to about 0.1-fold, at least, or up to about 0.2-fold, at least, or up to about 0.3-fold, at least, or up to about 0.4-fold, at least, or up to about 0.5-fold, at least, or up to about 0.6-fold, at least, or up to about 0.7-fold, at least, or up to about 0.8-fold, at least, or up to about 0.9-fold, at least, or up to about 1-fold, at least, or up to about 2-fold, at least, or up to about 3-fold, at least, or up to about 4-fold, at least, or up to about 5-fold, at least, or up to about 6-fold, at least, or up to about 7-fold, at least, or up to about 8-fold, at least, or up to about 9-fold, at least, or up to about 10-fold, at least, or up to about 20-fold, at least, or up to about 30-fold, at least, or up to about 40-fold, at least, or up to about 50-fold, at least, or up to about 60-fold, at least, or up to about 70-fold, at least, or up to about 80-fold, at least, or up to about 90-fold, at least, or up to about 100-fold, at least, or up to about 500-fold, at least, or up to about 1,000-fold, at least, or up to about 5,000-fold, or at least, or up to about 10,000-fold further change (e.g., increase or decrease) in the expression and / or activity level of the target gene as compared to the control expression and / or activity level of genes not targeted by the second individual regulation.The second individual regulation can induce a further change (e.g., increase or decrease) of at least about 10,000-fold, at least about 5,000-fold, at least about 1,000-fold, at least about 500-fold, at least about 100-fold, at least about 90-fold, at least about 80-fold, at least about 70-fold, at least about 60-fold, at least about 50-fold, at least about 40-fold, at least about 30-fold, at least about 20-fold, at least about 10-fold, at least about 9-fold, at least about 8-fold, at least about 7-fold, at least about 6-fold, at least about 5-fold, at least about 4-fold, at least about 3-fold, at least about 2-fold, at least about 1-fold, at least about 0.9-fold, at least about 0.8-fold, at least about 0.7-fold, at least about 0.6-fold, at least about 0.5-fold, at least about 0.4-fold, at least about 0.3-fold, at least about 0.2-fold, or at least about 0.1-fold, compared to the control expression and / or activity level of genes not targeted by the second individual regulation, in the expression and / or activity level of the target gene.

[0185] The further change by the second individual regulation can occur when the expression and / or activity level of the target gene reaches the target level, for example, by the action of the first individual regulation by the design of a non-homologous genetic circuit.

[0186] A further change due to the second individual regulation is that the expression and / or activity level of the target gene is at least, or maximally about 0.1-fold, at least, or maximally about 0.2-fold, at least, or maximally about 0.3-fold, at least, or maximally about 0.4-fold, at least, or maximally about 0.5-fold, at least, or maximally about 0.6-fold, at least, or maximally about 0.7-fold, at least, or maximally about 0.8-fold, at least, or maximally about 0.9-fold, at least, or maximally about 1-fold, at least, or maximally about 2-fold, at least, or maximally about 3-fold, at least, or maximally about 4-fold, at least, or maximally about 5-fold, at least, or maximally about 6-fold, at least, or maximally about 7-fold, at least, or maximally about 8-fold, at least, or maximally about 9-fold, at least, or maximally about 10-fold, at least, or maximally about 20-fold, at least, or maximally about 30-fold, at least, or maximally about 40-fold, at least, or maximally about 50-fold, at least, or maximally about 60-fold, at least, or maximally about 70-fold, at least, or maximally about 80-fold, at least, or maximally about 90-fold, at least, or maximally about 100-fold, at least, or maximally about 500-fold, at least, or maximally about 1,000-fold, at least, or maximally about 5,000-fold, or at least, or maximally about 10,000-fold changed (e.g., increased or decreased) compared to the control expression and / or activity level of a gene not targeted by the second individual regulation by the action of the first individual regulation.A further change by the second individual regulation is that the expression and / or activity level of the target gene is maximally or at least about 10,000-fold, maximally or at least about 5,000-fold, maximally or at least about 1,000-fold, maximally or at least about 500-fold, maximally or at least about 100-fold, maximally or at least about 90-fold, maximally or at least about 80-fold, maximally or at least about 70-fold, maximally or at least about 60-fold, maximally or at least about 50-fold, maximally or at least about 40-fold, maximally or at least about 30-fold, maximally or at least about 20-fold, maximally or at least about 10-fold, maximally or at least about 9-fold, maximally or at least about 8-fold, maximally or at least about 7-fold, maximally or at least about 6-fold, maximally or at least about 5-fold, maximally or at least about 4-fold, maximally or at least about 3-fold, maximally or at least about 2-fold, maximally or at least about 1-fold, maximally or at least about 0.9-fold, maximally or at least about 0.8-fold, maximally or at least about 0.7-fold, maximally or at least about 0.6-fold, maximally or at least about 0.5-fold, maximally or at least about 0.4-fold, maximally or at least about 0.3-fold, maximally or at least about 0.2-fold, maximally or at least about 0.1-fold changed (e.g., increased or decreased) compared to the control expression and / or activity level of a gene not targeted by the second individual regulation by the action of the first individual regulation.

[0187] Alternatively or additionally, the second individual regulation disclosed herein (e.g., induced by a second gate unit) can induce a change (e.g., an increase or decrease) of 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% in the expression and / or activity level of a further target gene compared to the control expression and / or activity level of a gene not targeted by the second individual regulation.The second individual regulation can induce a change (e.g., an increase or decrease) of up to about 1,000,000%, up to about 100,000%, up to about 9,000%, up to about 8,000%, up to about 7,000%, up to about 6,000%, up to about 5,000%, up to about 4,000%, up to about 3,000%, up to about 2,000%, up to about 1,000%, up to about 900%, up to about 800%, up to about 700%, up to about 600%, up to about 500%, up to about 400%, up to about 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% in the expression and / or activity level of the additional target gene as compared to the control expression and / or activity level of the gene not targeted by the second individual regulation.

[0188] Optionally, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of housekeeping genes (e.g., constitutive genes that control basal cell function). Optionally, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of genes controlled by the first individual regulation. Optionally, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of genes controlled by the third individual regulation. Optionally, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of genes controlled by the second genetic circuit. Optionally, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of genes acting in the same metabolic pathway as the target gene. Alternatively, as disclosed herein, the control expression and / or activity levels of genes not targeted by the second individual regulation may refer to the expression and / or activity levels of genes not acting in the same metabolic pathway as the target gene.

[0189] The cell may include a prokaryotic cell, a eukaryotic cell, or an artificial cell. The cell may be a fungal cell, a plant cell, or an animal cell (e.g., a mammalian cell). The cell (e.g., the initial cell modified to the engineered cell disclosed herein, the final cell product generated from the engineered cell disclosed herein, etc.) may include a muscle cell, an immune cell, a neuron, an osteoblast, an endothelial cell, a mesenchymal cell, an epithelial cell, a stem cell, a secretory cell, a blood cell, a germ cell, a nurse cell, a storage cell, an enteroendocrine cell, a pituitary cell, a neurosecretory cell, a duct cell, an odontoblast, a cementoblast, a glial cell, or a stromal cell.

[0190] Non-limiting examples of such cells include lymphoid cells such as B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, cytokine-induced killer (CIK) cells (see, e.g., U.S. Patent Application Publication No. 20080241194), myeloid cells such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes / hyper-segmented neutrophils), monocytes / macrophages, erythrocytes (reticulocytes), mast cells, monocytes / macrophages, dendritic cells, cells derived from the endocrine system including thyroid (thyroid epithelial cells, follicular cells), parathyroid (parathyroid chief cells, oxyphil cells), adrenal (chromaffin cells), pineal (pinealocytes) cells, glial cells (astrocytes, microglia), megakaryocytic neurosecretory cells, stellate cells, Betz cells, and cells of the nervous system including pituitary (gonadotropin-secreting cells, adrenocorticotropic hormone-secreting cells, thyroid-stimulating hormone-producing cells, growth hormone-producing cells, mammotropic hormone-secreting cells), lung cells (type I pneumocytes, type II pneumocytes), Clara cells, goblet cells, cells of the respiratory system including dust cells, cells of the circulatory system including cardiomyocytes and pericytes, cells of the digestive system including stomach (chief cells, parietal cells), goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enterochromaffin cells, APUD cells, liver (hepatocytes, Kupffer cells), enteric endocrine cells including cartilage / bone / muscle, osteoblasts, osteocytes, osteoclasts, cells of bone including teeth (cementoblasts, ameloblasts), chondrocytes including chondroblasts and chondrocytes (Chondrocyte), cells of the skin including hair follicle cells, keratinocytes, melanocytes (nevus cells), muscle cells including myocytes, podocytes, juxtaglomerular cells, mesangial cells within the glomerulus / mesangial cells outside the glomerulus, renal proximal tubule brush border cells, cells of the urinary system including macula densa cells, germ cells including sperm, Sertoli cells, Leydig cells, ova, as well as adipocytes, fibroblasts, tendon cells, epithelial keratinocytes (differentiated epithelial cells), epithelial basal cells (stem cells), keratinocytes of the fingernails and toenails, nail bed basal cells (stem cells), medullary hair shaft cells, cortical hair shaft cells, reticular hair shaft cells, reticular hair root sheath cells, hair root sheath cells of the cuticle layer, hair root sheath cells of the Henle layer, outer hair root sheath cells, hair matrix cells (stem cells), wet stratifiedbarrier) surface epithelial cells of stratified squamous epithelium of epithelial cells, cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, basal cells (stem cells) of the epithelium of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, and vagina, urothelial cells (covering the bladder and ureters), exocrine epithelial cells, salivary gland mucous cells (secretion rich in polysaccharides), salivary gland serous cells (secretion rich in glycoprotein enzymes), von Ebner's gland cells of the tongue (washing taste buds), mammary gland cells (milk secretion), lacrimal gland cells (tear secretion), skin gland cells of the ear (wax secretion), dark cells of eccrine sweat glands (glycoprotein secretion), clear cells of eccrine sweat glands (secretion of small molecules), and other cells including apocrine sweat gland cells (odoriferous secretion, sex hormone sensitive), Moll gland cells of the eyelid (specialized sweat glands), sebaceous gland cells (secretion of lipid-rich sebum), Bowman's gland cells of the nose (washing olfactory epithelium), Brunner's gland cells of the duodenum (enzymes and alkaline mucus), seminal vesicle cells (secreting semen components, including fructose necessary for sperm motility), prostate gland cells (secreting semen components), bulbourethral gland cells (mucus secretion), Bartholin gland cells (secreting vaginal lubricant), Littre gland cells (mucus secretion), endometrial cells (carbohydrate secretion), goblet cells separated from the respiratory and digestive tracts (mucus secretion), gastric inner layer mucous cells (mucus secretion), gastric gland enzyme-producing cells (pepsinogen secretion), gastric gland acid-secreting cells (hydrochloric acid secretion), pancreatic acinar cells (secretion of bicarbonate and digestive enzymes), Paneth cells of the small intestine (lysozyme secretion), type II alveolar epithelial cells of the lung (surfactant secretion), Clara cells of the lung, hormone-secreting cells, anterior pituitary cells, growth hormone-secreting cells, mammotropic cells, thyroid-stimulating cells, gonadotropic cells, adrenocorticotropic cells, intermediate pituitary cells, magnocellular neurosecretory cells, cells of the digestive and respiratory systems, thyroid cells, thyroid epithelial cells, parafollicular cells, parathyroid cells, chief cells of the parathyroid, acid-secreting cells, adrenal cells, chromaffin cells, Leydig cells of the testis, theca interna cells of the follicle, luteal cells of the ruptured follicle, granulosa lutein cells, theca lutein cells, juxtaglomerular cells (renin secretion), macula densa cells of the kidney, metabolic and storage cells, barrier function cells (lung, digestive tract, exocrine glands, and urogenital tract), the kidney, type I alveolar epithelial cells (inner layer of the airspace of the lung), pancreatic duct cells (centroacinar cells), nonstriated duct cells (Nonstriated ductcells (such as sweat glands, salivary glands, mammary glands), duct cells (such as seminal vesicles, prostate glands), inner epithelial cells of closed internal body cavities, ciliated cells with propulsion function, extracellular matrix-secreting cells, contractile cells, skeletal muscle cells, stem cells, cardiomyocytes, blood and immune system cells, red blood cells, megakaryocytes (platelet precursors), monocytes, connective tissue macrophages (various types), epidermal Langerhans cells, osteoclasts (in bone), dendritic cells (in lymphoid tissues), microglial cells (in the central nervous system), neutrophil granulocytes, eosinophil granulocytes, basophil granulocytes, mast cells, helper T cells, suppressor T cells, cytotoxic T cells, natural killer T cells, B cells, natural killer cells, reticulocytes, blood and immune system stem cells and committed progenitors (various types), pluripotent stem cells, totipotent stem cells, induced pluripotent stem cells, adult stem cells, sensory transmission cells, autonomic nerve cells, sensory organ and peripheral nerve support cells, central nervous system neurons and glial cells, lens cells, pigment cells, melanocytes, retinal pigment epithelial cells, germ cells, oogonia / oocytes, spermatids, spermatocytes, spermatogonia (stem cells of spermatocytes), sperm, nurse cells, ovarian follicle cells, Sertoli cells (in the testis), thymic epithelial cells, interstitial cells, and interstitial kidney cells may be mentioned.

[0191] The present disclosure also provides a composition comprising an engineered gene regulator and / or an engineered genetic circuit disclosed herein. The composition may further comprise an activator of a non-homologous genetic circuit. The present disclosure also provides a kit comprising the composition. The kit may further comprise an activator of a non-homologous genetic circuit. The activator may be in the same composition as the engineered gene regulator and / or the engineered genetic circuit. Alternatively or additionally, the activator may be in a separate composition different from the engineered gene regulator and / or the engineered genetic circuit.

Examples

[0192] Example 1: Inactivation of sgRNA Activity

[0193] This example shows that the RNA polymerase III transcription termination sequence (polyT tract) is sufficient to inactivate sgRNA activity. When inactivating sgRNA, the ribozymal activity and polyU effect are compared.

[0194] In vitro RNA analysis was performed to determine the catalytic ability of ribozymes that introduce various modifications to secondary structures. Figures 1A - 1B show exemplary ribozyme sgRNAs, and Figures 2A - 2D show examples of deformed secondary RNA structures. Figure 2E shows that ribozyme activity was not inhibited by specific modifications to Stem I and Stem III, but was disrupted by the elongation of Stem II.

[0195] Next, various modifications were tested for their ability to inactivate guide nucleic acids (Figure 3). PG3 is a gNA with a stem, GFP spacer, and a hairpin with a modified ribozyme and 6U, Rz is a gNA with a modified ribozyme, 6xU is a gNA with a 6U polyU sequence, FL4 is a gNA with a full - length ribozyme, FL4 + 6xU is a gNA with a full - length ribozyme and a 6U polyU sequence, FL5 is a gNA with an extended full - length ribozyme, and FL6 is a different gNA with an extended full - length ribozyme. Both an sgRNA that directly targets GFP (sgRNA) and a transfection control (Trnfx) where neither Cas9 nor sgRNA was administered to the cells were used as controls. ag+ indicates a sample administered with an activated guide nucleic acid (gNA), and ag− indicates a sample not administered with an activated gNA.

[0196] The polyU termination sequence was shown to be sufficient to inactivate guide nucleic acids. The polyU sequence with increased length (polyT sequence in DNA) was sufficient to inactivate gNA both when located in the hairpin (Figure 4A) and when located in the tetraloop (Figure 4B). Additionally, the longer the polyU sequences, the more efficient they are in their termination efficiency, capping around the 8T position (Figure 4C).

[0197] When the inactivation array is adjacent to the insulator region and / or the stem region on each side, the orientation of the insulator sequence / stem sequence in the DNA can be arranged so that the RNA can form a secondary structure. When the same DNA sequence is arranged in the direct repeat direction at two positions, the RNA forms a non-complementary bubble structure indicated by the stem (S). When the DNA sequence is arranged in the inverted repeat direction, the RNA can form a complementary structure indicated by the insulator (I). When the DNA sequence of each site is a mixture of the direct repeat direction and the inverted repeat direction, an RNA structure composed of complementary regions and non-complementary bubble structures indicated by SI, IS, and ISI at different positions can be formed. These abbreviations I, S, SI, IS, ISI are used in FIGS. 5B, 5C, and FIGS. 6A, 6B.

[0198] The most significant conversion from the inactive proGuide to the active matureGuidematureGuide occurred when the polyT tract was flanked by a stem sequence oriented in the inverted repeat sequence (I_U) either when the proUnit was located at the hairpin 1 (FIG. 5B) or the tetraloop (FIG. 5C) position within the gNA. The lowest level of activation occurred when the stem sequence was arranged in the direct repeat direction (S_U) in the hairpin 1 (FIG. 5B) and tetraloop (FIG. 5C) mutants.

[0199] Compared with the efficiency of inactivation of the insulator region when paired with a ribozyme instead of the polyU region, both those with a stem before the ribozyme (S_Rz) or those with a complementary sequence after the stem (SI_Rz) enhanced inactivation the most when the ribozyme was located at the tetraloop (FIG. 6A) and were at a level equivalent to polyU (FIG. 6B). However, in the S and SI orientations, the conversion efficiency to the active matureGuide was the weakest (black bars), and polyU was significantly effective in inactivating the proGuide in the ISI and I orientations.

[0200] These experiments demonstrated that the polyT termination sequence is sufficient to act as an sgRNA inactivation module. Furthermore, the secondary structure resulting from the orientation of the sequence adjacent to the polyT sequence can modulate its effect on the efficiency of termination, similar to the length of polyT itself. The conversion to active mature Guide RNA is also affected by the orientation of the sequence adjacent to polyT.

[0201] Example 2: Optimization of sgRNA inactivation

[0202] In this predictive example, the effect of the sequence adjacent to the polyT tract is examined in the case where it may undergo read-through transcription by RNA Pol III for synthesizing the complete guide RNA from the proGuide DNA template. In the insulator (I) configuration with a single polyT tract, the read-through transcription event generates a proGuide with the extension of a tetraloop or a hairpin (Figure 7). This extension can be predicted to form a stable guide RNA that can function with Cas (e.g., Cas9) or its variants. In the insulator-stem (IS) orientation, read-through transcription generates a proGuide with a longer extension at the end of the tetraloop, and the longer the extension, the more complex secondary structure it may have (Figure 8). A more complex secondary structure can be predicted to inhibit the activity of Cas (e.g., Cas9) or its variants and reduce the residual activity of the proGuide before it is converted to the active state by removing the stem and polyT tract. However, in some cases, the presence of a polyT track that sufficiently terminates the read-through (e.g., transcription) of the complete guide RNA may be more efficient in reducing (or preventing) changes in the formation of the complex with the Cas protein, thereby being more efficient in inhibiting the activity of the Cas protein and reducing the residual activity.

[0203] Example 3: Conversion from inactive proGuide to active matureGuide

[0204] The systems and methods provided herein disclose the conversion of nucleic acid molecules from an inactive state to an active state. In some embodiments, the nucleic acid molecule is a proGuide that can be converted from an inactive state to an active state. In this example, as shown by the lack of GFP disruption when enzymatically inactive dCas9 was used, the genetic circuit utilized an sgRNA or a variant thereof to disrupt the GFP output that requires Cas9 endonuclease activity (Figure 9). The significance of the GFP disruption data is to show the conversion from an inactive proGuide where the spacer targets GFP to an active matureGuide state that mutates the genomic transgene (e.g., EGFP). The conversion is caused by Cas9 activity at the proGuide cleavage site by the activating guide sgRNA (aGuide).

[0205] Results

[0206] The conversion of proGuide using a polyT tract for inactivation was examined using several proGuide variants that have the same spacer targeting GFP but different inactivation moieties. Figure 10A shows the activity of proGuide converted to matureGuide by aGuide for variants in which a ribozyme (Rz) or polyT tract (U) or both were inserted at either the hairpin 1 (H) or tetraloop (T) site. Note that the cleavage site (e.g., VPS16) for each of the variants is the same and in the same orientation. This experiment shows that proGuides with different inactivation sequences but the same cleavage site sequence and orientation exhibited the same activity as the matureGuide. MatureGuides derived from some insertions (e.g., tetraloop insertion) showed higher activity than those derived from other insertions (e.g., hairpin 1 insertion). This experiment also showed that each of these matureGuides was less active (fewer GFP-negative cells) than the sgRNA control targeting GFP in cells.

[0207] Figure 10B shows that the change in the concentration of proGuide relative to aGuide in the transfection mixture had relatively little effect on the frequency of GFP disruption in cells. In this experiment, proGuide (PG) 0% represents the level of GFP-negative cells with transfection of aGuide but without proGuide. 100% represents the level of GFP-negative cells with transfection of proGuide but without aGuide. The fact that the level of activity from proGuides with some insertions (e.g., tetraloop insertions) is higher than that from proGuides with other insertions (e.g., hairpin insertions) indicates that the activity cap is not caused by the level of guide RNA in the cell.

[0208] The effect on the sgRNA activity of the insulator sequence without the proUnit inactivation sequence is minimal (Figure 11). It was also shown that ribozyme activity is not sufficient to significantly inactivate sgRNA when the ribozyme is inserted without a stem or insulator sequence, i.e., inserted in such a way that there is no potential disruptive structural effect of the inserted sequence (Figure 14).

[0209] Example 4: Non-standard RNA Pol III terminator

[0210] In this predictive example, a non-standard terminator sequence such as that shown in Figure 12 is used in place of the polyU sequence to inactivate sgRNA activity. The non-target terminator sequence is targeted by Cas9 and a single nucleotide is inserted that disrupts the terminator sequence. To enhance the termination frequency, the hairpin position 10 nucleotides upstream of the terminator sequence is used.

[0211] Example 5: Multiple termination sequences

[0212] The purpose of examining multiple termination arrays is to invent a more effective transcription termination array for small RNAs transcribed by RNA Pol III. This concept is that there is even low-level read-through transcription by a 10-nt polyT tract, and extending the length of the tract results in a decrease in return because although the low-level read-through does not substantially decrease, the longer the polyT tract, the more functional problems it causes for plasmid DNA synthesis and stability. In contrast, having multiple (e.g., two) copies of the polyT tract can exhibit a multiplicative effect on transcription termination if each copy has the same probability of causing termination. As an experimental approach, it was to evaluate the importance of the sequence between multiple (e.g., two) polyT (e.g., 8-nt) tracts. Two different intervening sequences were evaluated. One contains DNA encoding 5S ribosomal RNA, and the other encodes a sequence predicted to have no secondary RNA structure (for example, for the non-polyT "linear sequence" placed between two polyT tracts, see SEQ ID NO: 36 in Table 1 and SEQ ID NO: 45 in Table 2).

[0213] Details of the experiment

[0214] Cells (e.g., HEK293 cells) having a genome-expressed transgene (e.g., EGFP) were transfected with a mixture of plasmid DNAs (e.g., those containing a combination of a Cas9-VPR expression plasmid, and a proGuide plasmid, an aGuide plasmid, and an sgRNA plasmid) to test the effect of multiple polyT tract configurations. Multiple proGuides (e.g., single polyT, linear multipolyT, 5S RNA multipolyT) were tested. All proGuide variants had the same spacer sequence targeting the disruption of the transgene (e.g., EGFP). The activity of the guide RNA was evaluated using the frequency of cells in which the signal (e.g., GFP fluorescence) disappeared.

[0215] Results

[0216] When juxtaposed and compared, proGuides containing multiple (e.g., two) 8-nt polyT tracts separated by a linear array exhibited background activity indistinguishable from negative control transfection (white bars; no sgRNA, no proGuide) (Figure 19). ProGuides containing polyT tracts (e.g., 5S RNA multipolyT) separated by a 5S RNA sequence showed detectable background activity and represented an inefficient method of inactivating guide RNAs compared to using linear multipolyT. By adding aGuide, proGuides with multiple polyT tracts were converted to the active matureGuide state at a frequency indistinguishable from the activity of sgRNAs (e.g., EGFP) that directly target genes.

[0217] Discussion

[0218] Adding a second polyT tract improved the transcription termination performance in proGuides. However, this effect was dependent on the sequence used to separate the two polyT tracts. By including a "linear" sequence between the polyT tracts, virtually no residual activity of the guide RNA was detected.

[0219] Example 6: Multi-step forward and reverse cascades

[0220] The systems and methods provided herein (e.g., based on polynucleotide sequences encoding activatable sgRNAs, which polynucleotide sequences include one or more polyT sequences) can be utilized to induce a multi-step cascade effect that is sequentially segmented, whereby the expression of an endogenous gene product can be activated at any stage of the cascade.

[0221] For example, the multi-step cascade effect may be a 10-step cascade effect such as a 10-step forward cascade or a 10-step reverse cascade.

[0222] Details of the experiment

[0223] In summary, the experiment begins by creating a mixture of plasmid DNAs encoding the components of the proGuide cascade, proceeds by introducing those DNAs into cells (e.g., HEK293 cells) via nucleofection, and concludes by using flow cytometry detection of cell surface gene products (e.g., CXCR4) to evaluate the effect on the activation of the target gene product at various time points.

[0224] Essential components of a mixture of plasmid DNAs (e.g., Cas9-VPR expression plasmid and GFP expression plasmid) are used to identify transfected cells. To construct combinations of plasmids for activating endogenous genes at different stages in the proGuide cascade, the mixture of cascade plasmid DNAs was assumed to use the components described in Tables 1 and 2. The core cascade plasmid was gradually included in the transfection mixture, adding further stages to the cascade as follows. For example, the conditions for the first stage (e.g., stage 1) did not include proGuide but included sgRNAs, and the sgRNAs included spacer sequences targeting the 5' and 3' cleavage sites within the proGuide plasmid for the second stage (e.g., stage 2). The conditions for the second stage (e.g., stage 2) included all the plasmids of the conditions of the first stage (e.g., stage 1) and the proGuide plasmid described for the second stage (e.g., stage 2). The conditions for the third stage (e.g., stage 3) included all the plasmids of the conditions of the second stage (e.g., stage 2) and the proGuide etc. described for the third stage (e.g., stage 3). To keep the mass of each proGuide plasmid DNA constant and the total DNA mass of the whole transfection constant, a genetically inert plasmid DNA (e.g., pUC19) was used as a "filler" in conditions where there were fewer proGuide plasmids.

[0225] To activate the expression of an endogenous gene product (e.g., CXCR4), Cas9-VPR was targeted to the promoter region of the gene (e.g., CXCR4) using a 14nt spacer sequence. To activate in the first stage (e.g., stage 1), the activation of the gene (e.g., CXCR4) was stimulated by an sgRNA having a related spacer of the gene (e.g., 14nt CXCR4 spacer). For subsequent stages, a proGuide plasmid having a related spacer for the gene (e.g., 14nt CXCR4 spacer) was added to the plasmid DNA mixture. By matching the 5’ and 3’ cleavage sites at a particular stage of the cascade with the 5’ and 3’ cleavage sites of the gene (e.g., CXCR4) activation proGuide, the activation of the gene (e.g., CXCR4) was effectively programmed to occur at one particular stage of the cascade for each condition / mixture of plasmid DNA.

[0226] Using a standard procedure with a nucleofection system (e.g., Lonza 4D), a mixture of plasmid DNA was introduced into cells (e.g., HEK293 cells). Using standard mammalian tissue culture methods, the transfected cells were plated (e.g., in a multiwell tissue culture plate) and maintained. At specific time points after nucleofection (e.g., 12, 24, 36, 48, and 72 hours), the cells were processed to detect flow cytometry and cell surface expression of the gene product (e.g., CXCR4). For each condition, independent replicates (e.g., n = 4) (nucleofection) were examined by flow cytometry.

[0227] Results

[0228] As expected, the combination of Cas9-VPR and an sgRNA targeting the promoter region of an endogenous gene (e.g., CXCR4) activated the cell surface expression of the gene (e.g., CXCR4) (e.g., stage 1; FIGS. 15A-17D). In the first stage (e.g., stage 1), the maximum level of the gene (e.g., CXCR4) stimulated with the sgRNA increased within the first time point (e.g., 12 hours). In contrast, in each of the proGuide-mediated stages (e.g., stages 2-10), a delay in the activation of the gene (e.g., CXCR4) was shown compared to the sgRNA. Importantly, in the proGuide-mediated stages, a delay in activation was also shown compared to the previous proGuide-mediated stages. For example, the activation of the gene (e.g., CXCR4) programmed in the third stage (e.g., stage 3) was delayed compared to the activation of that programmed in the second stage (e.g., stage 2), and the activation in the fourth stage (e.g., stage 4) was delayed compared to the activation in the third stage (e.g., stage 3), etc. The delays programmed in the subsequent stages occurring after the previous stages were generally consistent in both the forward cascade (FIGS. 15A-15E, 17A-17B) and the reverse cascade (FIGS. 16A-16E, 17C-17D).

[0229] The activity levels gradually decreased slightly after each stage of the cascade. It seems that a plateau was reached by stage 7, and the activity at stages 7-10 was the same as after 72 hours (FIG. 16E). These cascades have been significantly improved compared to previous versions of the proGuide technology. As an example of the improvement, in a control comparison, the maximum activity in a 4-stage cascade using the previous technology was lower than the level in step 9 using the new technology (FIG. 18).

[0230] It was unclear whether the array composition of the spacer region and the array composition of the cleavage site could affect each other's activity. For example, some spacer sequences might inhibit the conversion of proGuide or generate matureGuide with inferior activity. To test this possibility, the inventors reconfigured the composition of the spacer and the cleavage site within each individual proGuide to form two cascades. The order of events was changed from the forward cascade to the reverse cascade, such that the cleavage site sequence that was used to proceed from the first stage to the second stage (e.g., from stage 1 to 2) in the forward cascade was used to proceed from stage 9 to 10 in the reverse cascade, and the cleavage site sequence that was used for stage 2 to 3 in the forward cascade was used for stage 8 to 9 in the reverse cascade, etc. (Tables 1 and 2). When comparing the activation of a gene (e.g., CXCR4) via the forward cascade and the activation of the same gene (e.g., CXCR4) via the reverse cascade, only extremely minor differences in the kinetics and levels of activity were observed between these two (Figures 15A - 17D). These results are consistent with the progression of the cascade from one stage to the next, which is mainly governed by the effectiveness of the cleavage site sequence. Therefore, when using only highly efficient cleavage site sequences, at locations where the cleavage site sequences can be used to generate cascades of proGuides, they are likely to be almost interchangeable.

[0231] Discussion: Two important parameters in synthetic biology solutions that provide sequential genetic instructions are the efficiency of the system (e.g., the proportion of cells that have completed the intended instructions) and the sophistication of the system (e.g., the number of encodable stages). The latest development of proGuide technology brings about an efficiency and sophistication that substantially exceed those of other synthetic biology systems while maintaining the ability to activate essentially any combination of endogenous gene products.

[0232] The efficiency of the system is shown by comparing the activation of endogenous gene (e.g., CXCR4) expression in the first stage (e.g., stage 1) against the optimal criteria when the sgRNA activates the gene (e.g., CXCR4). In each successive stage of the cascade, more than 95% of the cells continue to be activated in the next stage of the cascade. The sophistication of the system is shown by completing a multi-stage (e.g., 10-stage) cascade. The number of stages of the continuous process is unprecedented and comparable to the conventional methods that use conditional gene activation methods to achieve two activation steps. The proGuide cascade system progresses autonomously when introduced into cells via transfection of plasmid DNA. Therefore, there is no need to apply conditional activation (e.g., doxycycline or cumate induction) by changing the culture conditions. Furthermore, since the entire proGuide cascade system is encoded by plasmid DNA, it does not involve gene editing or mutations of the host cell to perform epigenetic programming of the cell.

[0233]

Table 1

[0234]

Table 2

[0235] Example 7: Examination of the conversion to matureGuide RNA using DNA sequencing

[0236] The systems and methods herein may have one or more mechanism pathways. An important parameter in synthetic biology solutions is the efficiency of conversion at a particular stage. Optionally, the conversion can be from proGuide to matureGuide. Optionally, the structure of the proGuide can affect the efficiency of conversion to matureGuide.

[0237] To investigate the DNA repair processes required for the conversion from proGuide to matureGuide, the RNA sequences of matureGuideRNA transcripts were characterized in cells. Sequencing experiments were used to elucidate the potential causes underlying the increased efficiency seen in types 2 and 3 compared to type 1. Type 1 refers to the structure of the proGuide in Figures 1A - 1B (e.g., those with polyT having a length less than 7). The structures of types 2 and 3 are shown in Figures 22A and 22B, respectively. Examples of differences between type 1 and types 2 and 3 include the removal of components (insulators, restriction sites, ribozymes) from type 1 and the orientation of the cleavage sites from direct repeats in type 1 to inverted repeat orientation in types 2 and 3. Additionally, the length of polyT in type 1 proGuide (e.g., less than 7) is shorter than the length of polyT in type 2 or 3 proGuide (e.g., 7 or more, e.g., 8 or 9). In particular, type 3 incorporates multiple (e.g., two) polyT sequences within its structure. The experimental procedure for characterization involved the transfection of cells (e.g., HEK293 cells) having plasmid DNA encoding proGuides with different proGuide structures but the same cleavage site sequence. In each transfection, the proGuide was co - transfected with an expression plasmid (e.g., Cas9 - VPR) and an sgRNA (i.e., aGuide) that targets the cleavage site of the proGuide plasmid. After transfection, RNA was extracted at a specific time point (e.g., 36 hours), converted to cDNA, amplified using guide - RNA - specific primers, and thereby only the RNA molecules having the proGuide spacer and the complete scaffold (i.e., the tetraloop, hairpin 1, hairpin 2) were sequenced.

[0238] Results and Discussion

[0239] Figure 20A shows the frequency of RNAs corresponding to the results of complete NHEJ repair of type 3 proGuide. The results of complete repair are defined as sequences where the Cas9 cleavage sites are ligated together without further nucleotide insertions or deletions. Figure 20B shows the DNA sequences seen from experiments on the type 3 proGuide also described in Figure 20A. The top sequence is an example of complete NHEJ repair....TA CCG TCG-----------CGA CGG Note that it is...TA (the PAM sequence is underlined here for reference). The sequencing results showed that the results of complete repair represented the majority of the matureGuide RNAs in the cells, and that single insertions of the next most frequent results, A or T (corresponding to U in the RNA), were rarely seen.

[0240] Significant improvements were shown by using DNA sequencing approaches to compare proGuides of different generations. Figures 21A - 21D show the size distributions of sequencing reads mapped to different proGuides. For example, in Figures 21A - 21D, by nomenclature, after the type of proGuide (e.g., type 1, type 2, or type 3), the nature of the cleavage site sequence within the proGuide for converting the proGuide to a matureGuide may be indicated. What is labeled as "Axin1" shared the same cleavage site sequence, where the type 1 cleavage sites were arranged in a direct repeat orientation and not in an inverted repeat orientation like type 2 and type 3. The RNA size distribution not only allowed for substantial read - through transcription and the presence of full - length proGuide RNA (triangles) due to the original structure, but also suggested that the results of complete NHEJ repair (arrows) were a minority compared to the results of repair that led to other - sized RNAs (Figure 21A). Type 2 (Figure 21B) and type 3 (Figure 21C) showed similar size distributions of matureGuide RNA, which mainly corresponded to the results of complete NHEJ repair (arrows). ProGuides with sub - optimal cleavage sites (e.g., type 3 APC) were repaired, and the frequency of the results of complete NHEJ repair was slightly lower than this (Figure 21D). Note that the sequencing assay does not have the ability to evaluate the activity of repair events and only has the ability to evaluate the results of repair events that result in full - length matureGuide RNA molecules.

[0241] Embodiment The following non - limiting embodiments provide exemplary examples of the present invention but do not limit the scope of the present invention.

[0242] Embodiment 1. A system for regulating the expression or activity of a target gene, the system comprising: A polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene for regulating the expression or activity of the target gene comprising The polynucleotide sequence comprises a domain that (i) corresponds to the tetraloop region of the guide nucleic acid molecule and (ii) contains a polyT sequence, the polyT sequence being sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene. Optionally, (1) the size of the polyT sequence is at least a threshold length, the threshold length being sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, Further optionally, (a) the polyT sequence contains at least 6 Ts and / or (b) the polyT sequence contains at least 7 Ts and / or (c) the polyT sequence contains at least 8 Ts and / or (d) the polyT sequence contains at least 9 Ts or at least 10 Ts and / or (e) the polyT sequence contains from 6 to 15 Ts and / or (2) the polyT sequence contains one or more additional nucleotides that are not Ts and / or (3) the polyT sequence is adjacent to a intervening sequence that is not a polyT sequence and / or (4) the polynucleotide sequence further comprises an insulator sequence, the insulator sequence being located adjacent to the polyT sequence, the insulator sequence containing a sequence that can be targeted by a gene editing moiety. Further optionally, (a) the insulator sequence is fully complementary and / or (b) the insulator sequence contains a non-complementary stem region, system.

[0243] Embodiment 2. A system for regulating the expression or activity of a target gene, the system comprising A polynucleotide sequence encoding a guide nucleic acid molecule, which (i) exhibits specific affinity for a target gene and (ii) has a size of at least about 12 nucleotides, for regulating the expression or activity of the target gene comprising The polynucleotide sequence comprises a polyX sequence having a length of 5 or more thresholds, whereby the polyX sequence is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, and the polyX sequence does not correspond to the terminal domain of the guide nucleic acid molecule Optionally (1) The polyX sequence comprises at least 6 Xs and / or (2) The polyX sequence comprises at least 7 Xs and / or (3) The polyX sequence comprises at least 8 Xs and / or (4) The polyX sequence comprises at least 9 Xs or at least 10 Xs and / or (5) The PolyX sequence comprises from 6 Xs to 15 Xs and / or (6) The PolyX sequence is a PolyT sequence and / or (7) The polyX sequence is located in a domain corresponding to the tetraloop region of the guide nucleic acid molecule and / or (8) The polyX sequence is located in a domain corresponding to the hairpin region of the guide nucleic acid molecule and / or (9) The guide nucleic acid molecule has a size of at most 300 nucleotides, a system

[0244] Embodiment 3. The system further comprises a gene editing part configured to perform at least one editing on the polyT sequence or the polyX sequence, and the at least one editing achieves the transcription of the guide nucleic acid molecule Optionally (1) The at least one editing is an insertion and / or (2) The at least one editing is a deletion and / or (3) At least one edit is the excision of the polyX sequence and / or (4) The excision of the polyX sequence is achieved using two cleavage sites adjacent to the polyX sequence and / or (5) At least one edit includes microhomology-mediated end joining (MMEJ) repair and / or (6) At least one edit enhances the expression of the guide nucleic acid molecule from the polynucleotide sequence as compared to that without a gene editing moiety and / or (7) The gene editing moiety includes a Cas protein and / or (8) The polyX sequence includes one or more additional nucleotides other than X and / or (9) The polyX sequence is adjacent to an intervening sequence that is not the polyX sequence, the system of embodiment 1 or embodiment 2.

[0245] Embodiment 4. Optionally, (1) The polynucleotide sequence includes (i) a first region encoding a guide nucleic acid molecule and (ii) a second region encoding an endonuclease recognition site, and the second region is disposed adjacent to the first region and / or (2) The polyT sequence or polyX sequence is at least 80 nucleotides away from the 3’ end of the polynucleotide sequence and / or (3) The polyT sequence or polyX sequence is at least 14 nucleotides away from the 5’ end of the polynucleotide sequence and / or (4) The polynucleotide sequence further includes at least one filler sequence adjacent to the polyT sequence or polyX sequence, Optionally further, (i) At least one filler sequence includes a first filler sequence and a second filler sequence, and the polyT sequence or polyX sequence is adjacent to the first filler sequence and the second filler sequence and / or (5) The system further includes an endonuclease capable of forming a complex with a guide nucleic acid molecule, and the complex achieves regulation of the expression or activity of a target gene. Optionally further, (i) The endonuclease includes a Cas protein and / or (6) The guide nucleic acid molecule does not include a ribozyme and / or (7) The polynucleotide sequence has the structure: T a NT b including wherein (i) T a is a first polyT sequence, (ii) T b is a second polyT sequence, (iii) a and b are integers of 4 or more, and (iv) N is an intervening sequence containing at least one nucleobase other than T. Optionally further, a and b are integers of 7 or more and / or (8) The polynucleotide sequence has the structure: M-T-M’ including wherein (i) T is a polyT sequence, (ii) M and M’ are polynucleotide sequences that are at least partially complementary to each other, and (iii) “-” is a polynucleotide linker or does not exist and / or (9) The polynucleotide sequences M and the additional polynucleotide sequence M' are pairs of polynucleotide sequences each selected from the group consisting of (1) SEQ ID NO: 17 and SEQ ID NO: 54, (2) SEQ ID NO: 18 and SEQ ID NO: 55, (3) SEQ ID NO: 19 and SEQ ID NO: 56, (4) SEQ ID NO: 20 and SEQ ID NO: 57, (5) SEQ ID NO: 21 and SEQ ID NO: 58, (6) SEQ ID NO: 22 and SEQ ID NO: 59, (7) SEQ ID NO: 23 and SEQ ID NO: 60, (8) SEQ ID NO: 24 and SEQ ID NO: 61, (9) SEQ ID NO: 26 and SEQ ID NO: 62, (10) SEQ ID NO: 27 and SEQ ID NO: 63, (11) SEQ ID NO: 28 and SEQ ID NO: 64, (12) SEQ ID NO: 29 and SEQ ID NO: 65, (13) SEQ ID NO: 30 and SEQ ID NO: 66, (14) SEQ ID NO: 31 and SEQ ID NO: 67, (15) SEQ ID NO: 32 and SEQ ID NO: 68, (16) SEQ ID NO: 33 and SEQ ID NO: 69, (17) SEQ ID NO: 34 and SEQ ID NO: 70, and (18) SEQ ID NO: 35 and SEQ ID NO: 71, and pairs of their complementary sequences show at least about 50% sequence identity, Optionally, (i) The polynucleotide sequences M and the additional polynucleotide sequence M' show at least about 60% sequence identity with pairs of polynucleotide sequences each selected from the group consisting of (1) - (18), and / or (ii) A system according to any one of Embodiments 1 - 3, wherein the polynucleotide sequences M and the additional polynucleotide sequence M' show at least about 80% sequence identity with pairs of polynucleotide sequences each selected from the group consisting of (1) - (18).

[0246] Embodiment 5. A method for regulating the expression or activity of a target gene in a cell, the method comprising: contacting a cell with a polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene, in order to regulate the expression or activity of the target gene comprising, The polynucleotide sequence comprises a domain that (i) corresponds to the tetraloop region of the guide nucleic acid molecule and (ii) contains a polyT sequence, the polyT sequence being sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene. Optionally, (1) the size of the polyT sequence is at least a threshold length, the threshold length being sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence in a cell, and / or (2) the polyT sequence contains at least 6 Ts, and / or (3) the polyT sequence contains at least 7 Ts, and / or (4) the polyT sequence contains at least 8 Ts, and / or (5) the polyT sequence contains at least 9 Ts or at least 10 Ts, and / or (6) the polyT sequence contains from 6 Ts to 15 Ts, and / or (7) the polyT sequence contains one or more additional nucleotides that are not T, and / or (8) the polyT sequence is adjacent to a intervening sequence that is not a polyT sequence, and / or (9) the polynucleotide sequence further comprises an insulator sequence, the insulator sequence being located adjacent to the polyT sequence, the insulator sequence containing a sequence that can be targeted by a gene editing moiety. Optionally further, (a) the insulator sequence is fully complementary, and / or (b) the insulator sequence contains a non-complementary stem region, method.

[0247] Embodiment 6. A method for regulating the expression or activity of a target gene in a cell, the method comprising A step of providing a polynucleotide sequence encoding a guide nucleic acid molecule to a cell, the guide nucleic acid molecule being characterized in that: (i) it shows specific affinity for a target gene and (ii) has a size of at least about 12 nucleotides comprising The polynucleotide sequence comprises a polyX sequence having a length of 5 or more threshold values, whereby the polyX sequence is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, and the polyX sequence does not correspond to the terminal domain of the guide nucleic acid molecule, Optionally (1) the polyX sequence comprises at least 6 Xs and / or (2) the polyX sequence comprises at least 7 Xs and / or (3) the polyX sequence comprises at least 8 Xs and / or (4) the polyX sequence comprises at least 9 Xs or at least 10 Xs and / or (5) The PolyX sequence comprises 6 to 15 Xs and / or (6) The PolyX sequence is a PolyT sequence and / or (7) the polyX sequence is located in a domain corresponding to the tetraloop region of the guide nucleic acid molecule and / or (8) the polyX sequence is located in a domain corresponding to the hairpin region of the guide nucleic acid molecule and / or (9) the polyX sequence comprises one or more additional nucleotides that are not X and / or (10) the polyX sequence is adjacent to an intervening sequence that is not a polyX sequence, method.

[0248] Embodiment 7. Optionally, a step of modifying the polyT sequence or polyX sequence in the polynucleotide sequence to change the expression level of the guide nucleic acid molecule from the polynucleotide sequence, thereby achieving regulation of the expression or activity of the target gene in the cell, further comprising the step Optionally (1) The modifying step comprises generating at least one edit to the polyT sequence or the polyX sequence, and optionally, (a) at least one edit comprises microhomology-mediated end joining (MMEJ) repair and / or (b) at least one edit enhances the expression of a guide nucleic acid molecule from a polynucleotide sequence and / or (2) at least one edit is an insertion and / or (3) at least one edit is a deletion and / or (4) at least one edit is excision of the polyX sequence, and optionally, (a) excision of the polyX sequence is achieved using two cleavage sites flanking the polyX sequence and / or (5) the modifying step reduces the size of the polyX sequence below a threshold length and / or (6) the modifying step comprises contacting the polynucleotide sequence with a gene editing moiety, a method of embodiment 6 or embodiment 7.

[0249] Embodiment 8. Optionally, (1) the polynucleotide sequence comprises (i) a first region encoding a guide nucleic acid molecule and (ii) a second region encoding an endonuclease recognition site, the second region being disposed adjacent to the first region and / or (2) the polyT sequence or the polyX sequence is at least 80 nucleotides away from the 3' end of the polynucleotide sequence and / or (3) the polyT sequence or the polyX sequence is at least 14 nucleotides away from the 5' end of the polynucleotide sequence and / or (4) the polynucleotide sequence further comprises at least one filler sequence adjacent to the polyT sequence or the polyX sequence, and optionally, (a) At least one filler array includes a first filler array and a second filler array, and the polyT array or polyX array is adjacent to the first filler array and the second filler array, and / or (5) The guide nucleic acid molecule further includes an endonuclease recognition site, and / or (6) The cell is a mammalian cell, and / or (7) The method further includes a step of forming a complex with the guide nucleic acid molecule and the endonuclease, and the complex can regulate the expression or activity of the target gene in the cell, Optionally further, (a) The endonuclease is a Cas protein, and / or (8) The guide nucleic acid molecule does not contain a ribozyme, and / or (9) The polynucleotide sequence has the structure: T a NT b including, wherein (i) T a is the first polyT array, (ii) T b is the second polyT array, (iii) a and b are integers of 4 or more, (iv) N is an intervening sequence containing at least one nucleobase other than T, Optionally further, a and b are integers of 7 or more, and / or (10) The polynucleotide sequence has the structure: M-T-M’ including, wherein (i) T is a polyT array, (ii) M and M’ are polynucleotide sequences that are at least partially complementary to each other, (iii) “-” is a polynucleotide linker or does not exist, and / or (11) M, which is a polynucleotide sequence, and M', which is a further polynucleotide sequence, are pairs of polynucleotide sequences each selected from the group consisting of (1) SEQ ID NO: 17 and SEQ ID NO: 54, (2) SEQ ID NO: 18 and SEQ ID NO: 55, (3) SEQ ID NO: 19 and SEQ ID NO: 56, (4) SEQ ID NO: 20 and SEQ ID NO: 57, (5) SEQ ID NO: 21 and SEQ ID NO: 58, (6) SEQ ID NO: 22 and SEQ ID NO: 59, (7) SEQ ID NO: 23 and SEQ ID NO: 60, (8) SEQ ID NO: 24 and SEQ ID NO: 61, (9) SEQ ID NO: 26 and SEQ ID NO: 62, (10) SEQ ID NO: 27 and SEQ ID NO: 63, (11) SEQ ID NO: 28 and SEQ ID NO: 64, (12) SEQ ID NO: 29 and SEQ ID NO: 65, (13) SEQ ID NO: 30 and SEQ ID NO: 66, (14) SEQ ID NO: 31 and SEQ ID NO: 67, (15) SEQ ID NO: 32 and SEQ ID NO: 68, (16) SEQ ID NO: 33 and SEQ ID NO: 69, (17) SEQ ID NO: 34 and SEQ ID NO: 70, and (18) SEQ ID NO: 35 and SEQ ID NO: 71, and show at least about 50% sequence identity with pairs of their complementary sequences, Optionally, (i) M, which is a polynucleotide sequence, and M', which is a further polynucleotide sequence, show at least about 60% sequence identity with pairs of polynucleotide sequences each selected from the group consisting of (1) to (18), and / or (ii) A method according to any one of embodiments 5 to 7, wherein M, which is a polynucleotide sequence, and M', which is a further polynucleotide sequence, show at least about 80% sequence identity with pairs of polynucleotide sequences each selected from the group consisting of (1) to (18).

[0250] Further details regarding non-identical genetic circuits (HGCs) and their use are provided in International Application PCT / US2018 / 052211, entitled "CRISPR / CAS SYSTEM AND METHOD FOR GENOME EDITING AND MODULATING TRANSCRIPTION", International Application PCT / US2023 / 013240, entitled "SYSTEMS FOR CELL PROGRAMMING AND METHODS THEREOF", and Clarke et al., Molecular Cell, Vol. 81, pp. 226-238, 2021, entitled "Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities", each of which is hereby incorporated by reference in its entirety.

[0251] It will be understood that the various aspects of the invention can be highly regarded individually, collectively, or in combination with each other. The various embodiments of the invention described herein may be applied to any of the specific uses disclosed herein. A composition of matter comprising a compound of any of the formulas in the composition of the compositions of the disclosure herein may be utilized in the methods section, including the methods of use and manufacture disclosed herein, and vice versa.

[0252] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. The present invention has been described with reference to the foregoing specification, but the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative ratios set forth herein, depending on various conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein may be employed in the practice of the present invention. Therefore, the present invention is also intended to encompass any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that the methods and structures within these claims, as well as their equivalents, be covered by these claims.

Claims

1. A system for regulating the expression or activity of a target gene, wherein the system is A polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity to the target gene in order to regulate the expression or activity of the target gene. Includes, A system comprising a polynucleotide sequence that (i) corresponds to the tetraloop region of the guide nucleic acid molecule and (ii) includes a domain containing a polyT sequence, wherein the polyT sequence does not correspond to the terminal domain of the guide nucleic acid molecule, and the polyT sequence is sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene.

2. The system according to claim 1, wherein the size of the polyT sequence is greater than or equal to a threshold length, and the threshold length is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence.

3. The system according to claim 1, wherein the polyT sequence comprises at least seven Ts.

4. The system according to claim 1, wherein the polyT sequence includes at least eight Ts.

5. The system according to claim 1, wherein the polyT sequence includes at least nine Ts.

6. The system according to claim 1, wherein the polyT sequence comprises at least 10 T cells.

7. The system according to claim 1, wherein the polyT sequence comprises 6 to 15 T.

8. The system according to claim 1, wherein the domain of the polynucleotide sequence further comprises at least one insulator region comprising (i) a target polynucleotide sequence located adjacent to the polyT sequence and (ii) a target polynucleotide sequence that can be targeted by a gene editing moiety, and the targeting by the gene editing moiety induces at least one edit in the at least one insulator region.

9. The system according to claim 8, wherein the at least one editing achieves the excision of the polyT sequence from the polynucleotide sequence in order to enhance the expression of the guide nucleic acid molecule from the polynucleotide sequence.

10. The system according to claim 8, wherein the gene editing portion is a Cas endonuclease.

11. The system according to claim 10, wherein the Cas endonuclease is Cas9 endonuclease.

12. The system according to claim 8, wherein the at least one insulator region includes a first insulator region and a second insulator region adjacent to the polyT array.

13. The system according to claim 12, wherein the first insulator region and the second insulator region include the target polynucleotide sequence.

14. The system according to claim 12, wherein (i) the first insulator region comprises a first insulator array, and (ii) the second insulator region comprises a second insulator array, and the first insulator array and the second insulator array are complementary to each other.

15. The system according to claim 1, wherein the polyT sequence is adjacent to an intervening sequence that is not the polyT sequence.

16. The polynucleotide sequence has the following structure: T a NT b (1) Includes, The system according to claim 15, wherein (i) T a is a first polyT sequence, (ii) T b is a second polyT sequence, (iii) a and b are integers of 4 or more, and (iv) N is an intervening sequence comprising at least one nucleic acid base that is not T.

17. The system according to claim 16, wherein a and b are integers of 7 or more.

18. A system used in a method for regulating the expression or activity of a target gene in a cell, the method comprising the step of contacting the cell with a polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene, in order to regulate the expression or activity of the target gene, according to any one of claims 1 to 17.

19. A method for regulating the expression or activity of a target gene in a cell, wherein the method is: A step of contacting the cells with a polynucleotide sequence encoding a guide nucleic acid molecule that exhibits specific affinity for the target gene in order to regulate the expression or activity of the target gene. Includes, A method comprising a polynucleotide sequence comprising a domain that (i) corresponds to a tetraloop region of the guide nucleic acid molecule and (ii) includes a polyT sequence, wherein the polyT sequence is sufficient to reduce the expression of the guide nucleic acid molecule, thereby regulating the expression or activity of the target gene.

20. A method for regulating the expression or activity of a target gene in a cell, wherein the method is: (i) Providing the cells with a polynucleotide sequence that encodes a guide nucleic acid molecule characterized by exhibiting specific affinity to the target gene and (ii) having a size of at least about 12 nucleotides, in order to regulate the expression or activity of the target gene. Includes, A method wherein the polynucleotide sequence includes a polyX sequence having a threshold length of 7 or more, wherein the polyX sequence is sufficient to reduce the expression of the guide nucleic acid molecule from the polynucleotide sequence, and the polyX sequence does not correspond to the terminal domain of the guide nucleic acid molecule.

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