Crispr-associated transposon systems and components

Abstract

The present disclosure provides Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems, components thereof, and methods for nucleic acid modification using the systems or components.

Description

[0001]COLUM-43190.601 CRISPR-ASSOCIATED TRANSPOSON SYSTEMS AND COMPONENTS FIELD The present disclosure relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems and components thereof, for example, Cas proteins and transposon-associated proteins. SEQUENCELISTINGSTATEMENTThe content of the electronic sequence listing titled COLUM_43190_601_SequenceListing.xml (Size: 136,639 bytes; and Date of Creation: May 8, 2025) is herein incorporated by reference in its entirety. CROSSREFERENCETORELATEDAPPLICATIONSThis application claims the benefit of U.S. Provisional Application No. 63 / 644,890, filed May 9, 2024, the content of which is herein incorporated by reference in its entirety. STATEMENTREGARDINGFEDERALLYSPONSOREDRESEARCH ORDEVELOPMENTThis invention was made with government support under HG011650 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUNDIn bacteria and archaea, CRISPR / Cas systems provide immunity by incorporating fragments of invading phage, virus, and plasmid DNA into CRISPR loci and using corresponding CRISPR RNAs (“crRNAs”) to guide the degradation of homologous sequences. Transcription of a CRISPR locus produces a “pre-crRNA,” which is processed to yield crRNAs containing spacer-repeat fragments that guide effector nuclease complexes to cleave dsDNA sequences complementary to the spacer. Several different types of CRISPR systems are known, (e.g., type I, type II, or type III), and classified based on the Cas protein type and the use of a proto-spacer-adjacent motif (PAM) for selection of proto-spacers in invading DNA. Although RNA-guided targeting typically leads to endonucleolytic cleavage of the bound substrate, recent studies have uncovered a range of noncanonical pathways in which CRISPR protein-RNA effector complexes have been naturally repurposed for alternative functions. For example, some Type I (Cascade) and Type II (Cas9) systems leverage truncated guide RNAs to achieve potent transcriptional repression without cleavage and other Type I COLUM-43190.601 (Cascade) and Type V (Cas12) systems lie inside unusual bacterial Tn7-like transposons and lack nuclease components altogether. SUMMARYProvided herein are systems or kits for nucleic acid integration into a target nucleic acid sequence comprising: an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system or one or more nucleic acids encoding the engineered CAST system. In some embodiments, the CAST system comprises at least one or both of: a) one or more Cas proteins and b) one or more transposon-associated proteins. In some embodiments, at least one of the one or more Cas proteins and the one or more transposon- associated proteins are selected from SEQ ID NOs: 1-7. In some embodiments, the one or more Cas proteins and the one or more transposon-associated proteins comprise SEQ ID NOs: 1-7. In some embodiments, the engineered CAST system is a Type I-F system. In some embodiments, the engineered CAST system is a Type I-F3 system. In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or a combination thereof. In some embodiments, the one or more Cas proteins and the one or more transposon- associated proteins are encoded by different nucleic acids. In some embodiments, the one or more Cas proteins and the one or more transposon-associated proteins are encoded by a single nucleic acid. In some embodiments, the one or more Cas proteins comprises Cas5, Cas6, Cas7, Cas8, or a combination thereof. In some embodiments, the one or more Cas proteins comprise a Cas8-Cas5 fusion protein. In some embodiments, the one or more transposon-associated proteins comprise TnsA, TnsB, TnsC, TniQ, or a combination thereof. In some embodiments, the engineered CAST system comprises Cas5, Cas6, Cas7, Cas8, TnsA, TnsB, TnsC, and TniQ. In some embodiments, the engineered CAST system further comprises at least one gRNA complementary to at least a portion of the target nucleic acid sequence, or a nucleic acid encoding the at least one gRNA. In some embodiments, the at least one gRNA is encoded by a nucleic acid different from the nucleic acid(s) encoding the one or more Cas proteins and the one or more transposon-associated proteins. In some embodiments, the at least one gRNA is encoded COLUM-43190.601 by a nucleic acid also encoding the one or more Cas proteins and the one or more transposon- associated proteins. In some embodiments, the at least one gRNA is a non-naturally occurring gRNA. In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array. In some embodiments, the systems or kits further comprise a target nucleic acid sequence. In some embodiments, the systems or kits further comprise a donor nucleic acid flanked by at least one transposon end sequence. In some embodiments, the nucleic acid encoding the one or more Cas proteins, the one or more transposon-associated proteins, the at least one gRNA, or any combination thereof further comprises the donor nucleic acid. In some embodiments, the system or kits further comprise at least one unfoldase protein, or at least one nucleic acid encoding thereof. In some embodiments, the system is a cell-free system. Also provided herein are compositions and cells comprising the disclosed systems. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. Further provided herein are methods for DNA integration comprising contacting a target nucleic acid sequence with the system as disclosed herein, or a composition comprising thereof. In some embodiments, the target nucleic acid sequence is in a cell. In some embodiments, contacting a target nucleic acid sequence comprises introducing the system into the cell. In some embodiments, introducing the system into the cell comprises administering the system to a subject. In some embodiments, the administering comprises in vivo administration. In some embodiments, the administering comprises transplantation of ex vivo treated cells comprising the system. Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1D show the identification of PspA757, a hyperactive type I-F CAST in human cells. FIG. 1A is a schematic of a plasmid-based integration assay workflow. Cells are transfected with all necessary components and a crRNA that targets a plasmid-encoded target COLUM-43190.601 site. Transposition then mobilizes the miniTn from one plasmid (pDonor) to the target plasmid (pTarget). Integration efficiencies are then measured via next generation sequencing (NGS). FIG. 1B is a graph of plasmid integration efficiencies for PspA757, compared to Tn7016 (PseCAST). “NT” represents a transfection in which a crRNA is transfected that does not target pTarget, while “T” represents a transfection in which a crRNA is transfected that successfully targets pTarget. FIG. 1C is a schematic of genomic integration assay workflow. Cells are transfected with all necessary components (including ClpX, a bacterial accessory factor), and a crRNA that targets AAVS1. Transposition then mobilizes the miniTn from one plasmid (pDonor) to the genome. Integration efficiencies are then measured via NGS. FIG. 1D is a graph of genomic integration efficiencies for Tn7016 and PspA757, demonstrating markedly greater genomic integration efficiencies. FIGS. 2A-2B show subunit activity of PspA757. FIG. 2A is a schematic of TnsB reporter assay. Each TnsB is tested with its cognate right transposon end. FIG. 2B shows the transcriptional activation of Vch-, Pse-, and PspA757-derived TnsBs. PspA757 drives robust transcriptional activation compared to Pse and Vch, suggesting that PspA757 TnsB binds robustly to its cognate transposon end. FIG. 3 shows genomic integration efficiencies for Pse and PspA757, with various termini tagging permutations for PspA757. “All N-term” is a condition in which all components (except for TnsAB) encode an N-terminal bipartite NLS tag. Each component listed denotes the component in which the N-terminal bipartite NLS tag was re-coded onto the C-terminus. DETAILED DESCRIPTION Tn7-like and Tn5053-like transposons that encode nuclease-deficient CRISPR-Cas systems, also known as CRISPR-transposons (CRISPR-Tn) and CRISPR-associated transposons (CAST), catalyze the Insertion of Transposable Elements by Guide RNA-Assisted TargEting (sometimes referred to as INTEGRATE, or INTEGRATE technology). Described herein are engineered CAST systems tested in human cells. One system, hereafter referred to as PspA757, PspA757CAST, or PspCAST, performs targeted gene insertions in human cells at efficiencies greater than previously identified systems, including VchCAST (Klompe et al., Nature 571, 219– 225 (2019)) or PseCAST (Klompe et al., Mol Cell 82, 616-628.e5 (2022)). Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. COLUM-43190.601 Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. As used herein, comprising a certain sequence or a certain SEQ ID NO usually implies that at least one copy of said sequence is present in recited peptide or polynucleotide. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclature used in connection with, and techniques of cell and tissue culture, molecular biology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The term “contacting” as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination, such as, but not limited to, an organ, tissue, cell, or tumor, may occur by any means of administration known to the skilled artisan. The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide, or a precursor of any of the foregoing. The RNA or COLUM-43190.601 polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. Thus, a “gene” refers to a DNA or RNA, or portion thereof, that encodes a polypeptide or an RNA chain that has functional role to play in an organism. For the purpose of this disclosure, it may be considered that genes include regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. A cell has been “genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell. The presence of the exogenous DNA results in permanent or transient genetic change. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that comprise a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and / or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793- 800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, COLUM-43190.601 and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA / RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and / or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and / or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double- stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T- Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3x, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951- 960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)). COLUM-43190.601 The terms “non-naturally occurring,” “engineered,” and “synthetic” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, engineered, or synthetic, or any combination thereof. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference. As used herein, the terms “providing,” “administering,” and “introducing,” are used interchangeably herein and refer to the placement of the systems of the disclosure into a cell, organism, or subject by a method or route which results in at least partial localization of the system to a desired site. The systems can be administered by any appropriate route which results in delivery to a desired location in the cell, organism, or subject. A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as COLUM-43190.601 described herein. Likewise, subject may include either adults or juveniles (e.g., children). Moreover, subject may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, guinea pigs, and the like. Examples of non- mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods and compositions provided herein, the mammal is a human. A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an “insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. Systems Disclosed herein are systems for DNA integration into a target nucleic acid sequence comprising: an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system or one or more nucleic acids encoding the engineered CAST system. The CAST system comprises at least one or both of: a) one or more Cas proteins and b) one or more transposon-associated proteins. In some embodiments, the system comprises two or more engineered CAST systems. Pairing of orthogonal systems with their orthogonal donor DNA substrates enables tandem insertion of multiple distinct payloads directly adjacent to each other without any risk of repressive effects from target immunity. For example, one, two, three, four, five, or more orthogonal CAST systems may be used to integrate large tandem arrays of payload DNA. In some embodiments, multiple orthogonal RNA-guided transposases and their transposon donor DNAs may be integrated into distal regions of a given chromosome or genome, such that the COLUM-43190.601 lack of sequence identity between the transposon ends of the distinct transposon DNA substrates prevents genetic instability and the risk of recombination. The system may be a cell free system. Also disclosed is a cell comprising the system described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a cell of a non- human primate or a human cell). CRISPR-Cas systems are currently grouped into two classes (1-2), six types (I-VI) and dozens of subtypes, depending on the signature and accessory genes that accompany the CRISPR array. In some embodiments, the engineered CAST system is a Type I-F system. In some embodiments, the engineered CAST system is a Type I-F3 system. In some embodiments, the engineered CAST system comprises Cas5, Cas6, Cas7, and Cas8. In some embodiments, the engineered CAST system comprises Cas8-Cas5 fusion protein. In certain embodiments, the Cas6 protein comprises an amino acid sequence having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more) similarity to that of SEQ ID NO: 7. In certain embodiments, the Cas6 protein comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the Cas7 protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 6. In certain embodiments, the Cas7 protein comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the Cas8-Cas5 fusion protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 5. In certain embodiments, the Cas8-Cas5 fusion protein comprises the amino acid sequence of SEQ ID NO: 5. A system of the present invention may comprise one or more transposon-associated proteins (e.g., transposases or other components of a transposon). The transposon-associated proteins may facilitate recognition or cleavage of the target nucleic acid and subsequent insertion of the donor nucleic acid into the target nucleic acid. In some embodiments, the one or more transposon-associated proteins comprise TnsA, TnsB, TnsC, TniQ, or combinations thereof. In certain embodiments, the TnsA protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 1. In certain embodiments, the TnsA protein comprises the amino acid sequence of SEQ ID NO: 1. COLUM-43190.601 In certain embodiments, the TnsB protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 2. In certain embodiments, the TnsB protein comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the TnsC protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 3. In certain embodiments, the TnsC protein comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the TniQ protein comprises an amino acid sequence having at least 70% similarity to that of SEQ ID NO: 4. In certain embodiments, the TniQ protein comprises the amino acid sequence of SEQ ID NO: 4. Any of the proteins described or referenced herein may comprise one or more amino acid substitutions as compared to the recited sequences. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (D or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg). The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups COLUM-43190.601 described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In some embodiments at least one of the one or more Cas proteins and the one or more transposon-associated proteins are provided as a fusion protein. For example, at least one of the one or more Cas proteins and the one or more transposon-associated proteins may be in a fusion protein with another Cas protein or transposon-associated protein. Alternatively, at least two of the disclosed modified Cas proteins or transposon-associated proteins may be linked in a fusion protein. In some embodiments, each of the one or more Cas proteins and the one or more transposon-associated proteins are provided as a single fusion protein. The term “fusion protein” as used herein refers to a polypeptide which comprises at least two different proteins or at least two protein domains from two different proteins. The fusion protein is not limited by orientation of the at least two different proteins. For example, the arrangement of the first protein in the fusion protein may be N-terminal or C-terminal to the second protein. In some embodiments, TnsA and TnsB are provided as a TnsA-TnsB fusion protein. TnsA and TnsB can be fused in any orientation: N-terminus to C-terminus; C-terminus to N- terminus; N-terminus to N-terminus; or C-terminus to C-terminus, respectively. Preferably the C-terminus of TnsA is fused to the N-terminus of TnsB. The fusion protein may comprise a linker polypeptide between the first amino acid sequence and the second amino acid sequence. The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a linker polypeptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. The linking peptides may have virtually any amino acid sequence, bearing in COLUM-43190.601 mind that the preferred linkers will have a sequence that results in a generally flexible peptide. Small amino acids, such as glycine and alanine, are useful in creating a flexible peptide linker. A variety of different linkers are considered suitable for use, including but not limited to, glycine- serine polymers, glycine-alanine polymers, and alanine-serine polymers. In the systems disclosed herein, at least one of the one or more Cas protein and the one or more transposon-associated protein comprise at least one nuclear localization sequence (NLS). The at least one nuclear localization sequence may be appended to at least one of the one or more Cas protein and the one or more transposon-associated protein at a N-terminus, a C-terminus, embedded in the protein (e.g., inserted internally within the open reading frame (ORF), in a linker of a fusion protein), or a combination thereof. In some embodiments, the linker of the TnsA-TnsB fusion protein comprises at least one NLS. In some embodiments, one or more or each of Cas6, Cas7, Cas8, TnsC, and TniQ comprise at least one NLS. In some embodiments, Cas6 and Cas7 comprise at least one NLS on the N-terminus. In some embodiments, TnsC, Cas8, and TniQ comprise at least one NLS on the N-terminus or C-terminus. The NLS may be embedded within a linker sequence, such that it is flanked by additional amino acids. In some embodiments, the NLS is flanked on each end by at least a portion of a flexible linker. In some embodiments, the NLS is flanked on each end by a glycine rich region of the linker. Suitable nuclear localization sequences for use with the disclosed system are described elsewhere herein and are applicable to use with the fusion proteins herein, e.g., TnsA-TnsB fusion protein. The nuclear localization sequence may comprise any amino acid sequence known in the art to functionally tag or direct a protein for import into a cell’s nucleus (e.g., for nuclear transport). Usually, a nuclear localization sequence comprises one or more positively charged amino acids, such as lysine and arginine. In some embodiments, the NLS is a monopartite sequence. A monopartite NLS comprises a single cluster of positively charged or basic amino acids. In some embodiments, the monopartite NLS comprises a sequence of K-K / R-X-K / R, wherein X can be any amino acid. Exemplary monopartite NLSs include, without limitation, those from the SV40 large T-antigen (PKKKRKVEDP; SEQ ID NO: 12), c-Myc (PAAKRVKLD; SEQ ID NO: 13), and TUS- proteins (Kaczmarczyk SJ et al. 2010). In select embodiments, the NLS comprises a c-Myc NLS. COLUM-43190.601 In some embodiments, the NLS is a bipartite sequence. Bipartite NLSs comprise two clusters of basic amino acids, separated by a spacer of about 9-12 amino acids. Exemplary bipartite NLSs include the NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 14), the NLS of EGL-13, MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 15), the bipartite SV40 NLS, KRTADGSEFESPKKKRKV (SEQ ID NO: 16). In some embodiments, the NLS comprises a bipartite SV40 NLS. In certain embodiments, the NLS comprises an amino acid sequence having at least 70% (e.g., having at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) similarity to KRTADGSEFESPKKKRKV (SEQ ID NO: 16). The protein components of the disclosed system (e.g., the Cas proteins or the transposon-associated proteins) may further comprise an epitope tag (e.g., 3xFLAG tag, an HA tag, a Myc tag, and the like). In some embodiments, the epitope tag may be adjacent, either upstream or downstream, to a nuclear localization sequence. The epitope tags may be at the N- terminus, a C-terminus, or a combination thereof of the corresponding protein. In some embodiments, the systems may further comprise a guide RNA (gRNA) or a nucleic acid encoding a gRNA, wherein the gRNA is complementary to at least a portion of a target nucleic acid sequence. In some embodiments, one or more of the at least one Cas protein are part of a ribonucleoprotein (RNP) complex with the gRNA. In some embodiments, one or more of the at least one Cas protein and one or more of the transposon-associated proteins are part of a ribonucleoprotein (RNP) complex with the gRNA. The gRNA may be a crRNA, crRNA / tracrRNA (or single guide RNA, sgRNA). The terms “gRNA,” “guide RNA,” “crRNA,” and “CRISPR guide sequence” may be used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the binding specificity of the CRISPR-Cas system. A gRNA hybridizes to (complementary to, partially or completely) a target nucleic acid sequence (e.g., the genome in a host cell). In some embodiments, the at least one gRNA is encoded in a CRISPR RNA (crRNA) array. The gRNA or portion thereof that hybridizes to the target nucleic acid (a target site) may be any length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. gRNAs or sgRNA(s) used in the present disclosure can be between about 5 and 100 nucleotides long, or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, COLUM-43190.601 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 5960, 61, 62, 63, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9192, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length, or longer). To facilitate gRNA design, many computational tools have been developed (See Prykhozhij et al. (PLoS ONE, 10(3): (2015)); Zhu et al. (PLoS ONE, 9(9) (2014)); Xiao et al. (Bioinformatics. Jan 21 (2014)); Heigwer et al. (Nat Methods, 11(2): 122–123 (2014)). Methods and tools for guide RNA design are discussed by Zhu (Frontiers in Biology, 10 (4) pp 289-296 (2015)), which is incorporated by reference herein. Additionally, there are many publicly available software tools that can be used to facilitate the design of sgRNA(s); including but not limited to, Genscript Interactive CRISPR gRNA Design Tool, WU-CRISPR, and Broad Institute GPP sgRNA Designer. There are also publicly available pre-designed gRNA sequences to target many genes and locations within the genomes of many species (human, mouse, rat, zebrafish, C. elegans), including but not limited to, IDT DNA Predesigned Alt-R CRISPR-Cas9 guide RNAs, Addgene Validated gRNA Target Sequences, and GenScript Genome-wide gRNA databases. In addition to a sequence that binds to a target nucleic acid, in some embodiments, the gRNA may also comprise a scaffold sequence (e.g., tracrRNA). In some embodiments, such a chimeric gRNA may be referred to as a single guide RNA (sgRNA). Exemplary scaffold sequences will be evident to one of skill in the art and can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, and Ran, et al. Nature Protocols (2013) 8:2281-2308, incorporated herein by reference in their entireties. In some embodiments, the gRNA sequence does not comprise a scaffold sequence and a scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further comprises an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence. The gRNA can comprise spacer sequence. The spacer sequence can be any length. In some embodiments, the spacer sequence is 30-40 nucleotides long (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to a target nucleic acid. In some embodiments, the gRNA sequence is at least 50%, 55%, 60%, 65%, 70%, COLUM-43190.601 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or at least 100% complementary to the 3’ end of the target nucleic acid (e.g., the last 5, 6, 7, 8, 9, or 10 nucleotides of the 3’ end of the target nucleic acid). The gRNA may be a non-naturally occurring gRNA. The system may further comprise a target nucleic acid. The terms “target sequence,” “target nucleic acid,” and “target site” (e.g., a “target genomic DNA sequence”) are used interchangeably herein to refer to a polynucleotide (nucleic acid, gene, chromosome, genome, etc.) to which a guide sequence (e.g., a synthetic guide RNA) is designed to have complementarity, wherein hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex, provided sufficient conditions for binding exist. The target sequence and guide sequence need not exhibit complete complementarity, provided that there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA. Suitable DNA / RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art. The target sequence may or may not be flanked by a protospacer adjacent motif (PAM) sequence. In certain embodiments, a nucleic acid-guided nuclease can only cleave a target sequence if an appropriate PAM is present, see, for example Doudna et al., Science, 2014, 346(6213): 1258096, incorporated herein by reference. A PAM can be 5' or 3' of a target sequence. A PAM can be upstream or downstream of a target sequence. In one embodiment, the target sequence is immediately flanked on the 3' end by a PAM sequence. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In certain embodiments, a PAM is between 2-6 nucleotides in length. The target sequence may or may not be located adjacent to a PAM sequence (e.g., PAM sequence located immediately 3' of the target sequence) (e.g., for Type I CRISPR / Cas systems). In some embodiments, e.g., Type I systems, the PAM is on the alternate side of the protospacer (the 5' end). Makarova et al. describes the nomenclature for all the classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). Guide structures and PAMs are described in by R. Barrangou (Genome Biol. 16:247 (2015)). COLUM-43190.601 Non-limiting examples of the PAM sequences include: CC, CA, AG, GT, TA, AC, CA, GC, CG, GG, CT, TG, GA, AGG, TGG, T-rich PAMs (such as TTT, TTG, TTC, etc.), NGG, NGA, NAG, NGGNG and NNAGAAW (W=A or T), NNNNGATT, NAAR (R=A or G), NNGRR (R=A or G), NNAGAA, and NAAAAC, where N is any nucleotide. In some embodiments, the PAM may comprise a sequence of CN, in which N is any nucleotide. In select embodiments, the PAM may comprise a sequence of CC. “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule, which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. There may be mismatches distal from the PAM. The system may further include a donor nucleic acid. The donor nucleic acid may be a part of a bacterial plasmid, bacteriophage, a virus, autonomously replicating extra chromosomal DNA element, linear plasmid, linear DNA, linear covalently closed DNA, mitochondrial or other organellar DNA, chromosomal DNA, and the like. In some embodiments, the donor nucleic acid comprises a cargo nucleic acid sequence. The donor nucleic acid may be flanked by at least one transposon end sequence. In some embodiments, the donor nucleic acid is flanked on the 5’ and the 3’ end with a transposon end sequence. The term “transposon end sequence” refers to any nucleic acid comprising a sequence capable of forming a complex with the transposase enzymes thus designating the nucleic acid between the two ends for rearrangement. Usually, these sequences contain inverted repeats and may be about 10-150 base pairs long, however the exact sequence requirements differ for the specific transposase enzymes. Transposon end sequences are well known in the art. Transposon ends sequences may or may not include additional sequences that promote or augment transposition. The transposon end sequences on either end may be the same or different. The transposon end sequence may be the endogenous CRISPR-transposon end sequences or may include deletions, substitutions, or insertions. The endogenous CRISPR-transposon end sequences may be truncated. In some embodiments, the transposon end sequence includes an about 40 base pair (bp) deletion relative to the endogenous CRISPR-transposon end sequence. In COLUM-43190.601 some embodiments, the transposon end sequence includes an about 100 base pair deletion relative to the endogenous CRISPR-transposon end sequence. The deletion may be in the form of a truncation at the distal (in relation to the cargo) end of the transposon end sequences. The donor nucleic acid, and by extension the cargo nucleic acid, may of any suitable length, including, for example, about 50-100 bp (base pairs), about 100-1000 bp, at least or about 10 bp, at least or about 20 bp, at least or about 25 bp, at least or about 30 bp, at least or about 35 bp, at least or about 40 bp, at least or about 45 bp, at least or about 50 bp, at least or about 55 bp, at least or about 60 bp, at least or about 65 bp, at least or about 70 bp, at least or about 75 bp, at least or about 80 bp, at least or about 85 bp, at least or about 90 bp, at least or about 95 bp, at least or about 100 bp, at least or about 200 bp, at least or about 300 bp, at least or about 400 bp, at least or about 500 bp, at least or about 600 bp, at least or about 700 bp, at least or about 800 bp, at least or about 900 bp, at least or about 1 kb (kilobase pair), at least or about 2 kb, at least or about 3 kb, at least or about 4 kb, at least or about 5 kb, at least or about 6 kb, at least or about 7 kb, at least or about 8 kb, at least or about 9 kb, at least or about 10 kb, or greater. The present systems may further include at least one unfoldase protein. Unfoldases are proteins that catalyze the unfolding of a native protein without affecting the primary structure. The unfoldase may be an NTP driven unfoldase. NTP driven unfoldases may include ATP- dependent proteases, including, but not limited to, ATPases, AAA proteases, or AAA+ enzymes (e.g., AAA+ enzyme). In some embodiments, the at least one unfoldase protein may comprise ClpX (caseinolytic mitochondrial matrix peptidase chaperone subunit X). In some embodiments, the at least one unfoldase protein may comprise a homolog of ClpX. ClpX homologs may be readily screened through systematic testing and optimization of a large panel of homologs, identified through bioinformatic search strategies such as BLASTp and psi-BLASTp. In some embodiments, the unfoldase protein (e.g., ClpX) is derived from the same host organism as that of the engineered CAST system. In some embodiments, the unfoldase protein (e.g., ClpX) is derived from a different host organism as that of the engineered CAST system. As such, the at least one unfoldase protein (e.g., ClpX) is not limited from which organism it is derived. In some embodiments, the unfoldase protein (e.g., ClpX) is derived from the E. coli genome. In other embodiments, the unfoldase protein (e.g., ClpX) from the cognate strain from which the engineered CAST system is derived. For example, unfoldase proteins from COLUM-43190.601 Pseudoalteromonas can be used alongside RNA-guided DNA integration machinery derived from PspA757. In some embodiments, the systems further comprise one or more additional genome engineering tools. For example, the systems may further comprise nucleases, such as zinc finger nucleases (ZFNs) and / or transcription activator like effector nucleases (TALENs); transcriptional activators, transcriptional repressors, histone-modifying proteins, integrases, and recombinases. In some embodiments, the system comprises components from or derived from different CAST systems. In some embodiments, at least one of the one or more Cas proteins and the one or more transposon-associated proteins may be derived from a homologous CAST system compared to the other protein components in the system. Examples of Cas proteins include, but are not limited to: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas 11, Cas12a (formerly Cpf1), Cas12b (formerly C2c1), Cas12c (formerly C2c3), Cas12d (formerly CasY), Cas12e (formerly CasX), Cas12k (formerly C2c5), Cas13a (formerly known as C2c2), Cas13b, Cas13c, Cas13d, homologs, orthologs, paralogs, modified versions, either engineered or naturally occurring, or active fragments thereof. Any Cas protein known in the art can be employed in the systems described herein, as appropriate. Cas proteins are described in detail in: U.S. Patent Nos. 8,697,359, 8,771,945, 8,945,839, 9,688,971, and 11,441,137; International Patent Publications: WO2016106239, WO2016205749, WO2017106657, WO2017070605, WO2017127807, WO2017184768, WO2017219027, WO2018170333, WO2019089796, WO2019089804, WO2019089820, WO2019104058, WO2020033601, WO2020181264, WO2020191102, WO2020257715, WO2021146641, WO2021216512, and WO2022159822; and Makarova et al., Nature Reviews Microbiology, 9(6): 467-477 (2011); Wiedenheft et al., Nature, 482: 331-338 (2012); Gasiunas et al., Proceedings of the National Academy of Sciences USA, 109(39): E2579-E2586 (2012); Jinek et al., Science, 337: 816-821 (2012); Carroll, Molecular Therapy, 20(9): 1658-1660 (2012); Al- Attar et al., Biol Chem., 392(4): 277-289 (2011); Hale et al., Molecular Cell, 45(3): 292-302 (2012), and Zhang Y., Pathog Dis. 2017;75(4):ftx036. doi:10.1093 / femspd / ftx036. Sequences of exemplary Cas proteins, transposon-associated proteins, gRNAs, and transposon ends can also be found in International Patent Publications WO 2020 / 181264, WO 2022 / 261122 and WO 2024 / 173573, incorporated herein by reference. However, the invention is COLUM-43190.601 not limited to the disclosed or referenced exemplary sequences. Indeed, genetic sequences can vary between different strains, and this natural scope of allelic variation is included within the scope of the invention. The one or more nucleic acids encoding the engineered CAST system may be any nucleic acid including DNA, RNA, or combinations thereof. In some embodiments, the one or more nucleic acids comprise one or more messenger RNAs, one or more vectors, or any combination thereof. The one or more Cas proteins, the one or more transposon-associated protein (e.g., TnsA, TnsB, TnsC, TnsD, and TniQ), the at least one gRNA, and the donor nucleic acid may be on the same or different nucleic acids (e.g., vector(s)). In some embodiments, the one or more Cas proteins are encoded by a single nucleic acid. In some embodiments, the one or more transposon-associated proteins are encoded by a single nucleic acid. In some embodiments, the nucleic acid encoding the one or more Cas proteins also encodes the one or more transposon- associated proteins. In some embodiments, the one or more Cas proteins are encoded by a different nucleic acid from the one or more transposon-associated proteins. In some embodiments, the at least one gRNA is encoded by a nucleic acid different from the nucleic acid(s) encoding the one or more Cas proteins and the one or more transposon- associated proteins. In some embodiments, the at least one gRNA is encoded by a nucleic acid also encoding at least one Cas protein, at least one transposon-associated protein, or both. In some embodiments, the one or more Cas proteins, the one or more transposon-associated proteins, and the at least one gRNA are encoded by a single nucleic acid. The gRNA may be encoded anywhere in the nucleic acid encoding the one or more Cas proteins or the one or more transposon-associated proteins. In some embodiments, the gRNA is encoded in the 3’ UTR of a protein coding nucleic acid. In some embodiments, the nucleic acid encoding the one or more Cas proteins, the one or more transposon-associated protein, the at least one gRNA, or any combination thereof further comprises the donor nucleic acid. Nucleic Acids and Delivery The present disclosure also provides for nucleic acids encoding the components of the disclosed systems, compositions comprising nucleic acids encoding the components of the disclosed systems, systems comprising nucleic acids encoding the components of the disclosed COLUM-43190.601 systems, and vectors containing or encoding these nucleic acids. The vectors may be used to propagate the nucleic acid in an appropriate cell and / or to allow expression from the nucleic acid (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence. The present disclosure further provides engineered, non-naturally occurring vectors and vector systems, which can encode one or more of the peptides or components of the present systems. The vector(s) can be introduced into a cell that is capable of expressing the polypeptide encoded thereby, including any suitable prokaryotic or eukaryotic cell. The vectors of the present disclosure may be delivered to a eukaryotic cell in a subject. Modification of the eukaryotic cells via the present system can take place in a cell culture, where the method comprises isolating the eukaryotic cell from a subject prior to the modification. In some embodiments, the method further comprises returning said eukaryotic cell and / or cells derived therefrom to the subject. Viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding the disclosed polypeptides or components of the present system into cells, tissues, or a subject. Such methods can be used to administer nucleic acids encoding the disclosed polypeptides or components of the present system to cells in culture, or in a host organism. Non- viral vector delivery systems include DNA plasmids, cosmids, RNA (e.g., a transcript of a vector described herein), a nucleic acid, and a nucleic acid complexed with a delivery vehicle. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Viral vectors include, for example, retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex viral vectors. In certain embodiments, plasmids that are non-replicative, or plasmids that can be cured by high temperature may be used, such that any or all of the necessary components of the system may be removed from the cells under certain conditions. For example, this may allow for DNA integration by transforming bacteria of interest, but then being left with engineered strains that have no memory of the plasmids or vectors used for the integration. Drug selection strategies may be adopted for positively selecting for cells. A donor nucleic acid may contain one or more drug-selectable markers within the cargo. Then presuming that the original donor plasmid is removed, drug selection may be used to enrich for integrated clones. Colony screenings may be used to isolate clonal events. COLUM-43190.601 A variety of viral constructs may be used to deliver the disclosed polypeptides or components of the present system (such as one or more Cas proteins and / or Tns proteins, gRNA(s), donor DNA, etc.) to the targeted cells and / or a subject. Nonlimiting examples of such recombinant viruses include recombinant adeno-associated virus (AAV), recombinant adenoviruses, recombinant lentiviruses, recombinant retroviruses, recombinant herpes simplex viruses, recombinant poxviruses, phages, etc. The present disclosure provides vectors capable of integration in the host genome, such as retrovirus or lentivirus. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989; Kay, M. A., et al., 2001 Nat. Medic. 7(1):33-40; and Walther W. and Stein U., 2000 Drugs, 60(2): 249-71, incorporated herein by reference. In one embodiment, a nucleic acid encoding the disclosed polypeptides or components of the present system is contained in a plasmid vector that allows expression of the disclosed polypeptides or components of the present system and subsequent isolation and purification of from the recombinant vector. Accordingly, the disclosed polypeptides or components of the present system disclosed herein can be purified following expression, obtained by chemical synthesis, or obtained by recombinant methods. To construct cells that express the disclosed polypeptides or components of the present system, expression vectors for stable or transient expression of the disclosed polypeptides or components of the present system may be constructed via conventional methods as described herein and introduced into host cells. For example, nucleic acids encoding the components of the disclosed polypeptides or components of the present system may be cloned into a suitable expression vector, such as a plasmid or a viral vector in operable linkage to a suitable promoter. The selection of expression vectors / plasmids / viral vectors should be suitable for integration and replication in eukaryotic cells. In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in prokaryotic cells. Promoters that may be used include T7 RNA polymerase promoters, constitutive E. coli promoters, and promoters that could be broadly recognized by transcriptional machinery in a wide range of bacterial organisms. The system may be used with various bacterial hosts. In certain embodiments, vectors of the present disclosure can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of COLUM-43190.601 mammalian expression vectors include pCDM8 (Seed, Nature (1987) 329:840, incorporated herein by reference) and pMT2PC (Kaufman, et al., EMBO J. (1987) 6:187, incorporated herein by reference). When used in mammalian cells, the expression vector's control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd eds., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference. Vectors of the present disclosure can comprise any of a number of promoters known to the art, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue- specific, or species specific. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns). Many promoter / regulatory sequences useful for driving constitutive expression of a gene are available in the art and include, but are not limited to, for example, CMV (cytomegalovirus promoter), EF1a (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta-actin promoter, rodent beta-actin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta actin promoter, and rabbit beta- globin splice acceptor), TRE (Tetracycline response element promoter), H1 (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Additional promoters that can be used for expression of the components of the present system, include, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Maloney murine leukemia virus (MMLV) LTR, myeoloproliferative sarcoma virus (MPSV) LTR, spleen focus-forming virus (SFFV) LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter, elongation factor 1- alpha (EF1-α) promoter with or without the EF1-α intron. Additional promoters include any constitutively active promoter. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within a cell. COLUM-43190.601 Moreover, inducible and tissue specific expression of a RNA, transmembrane proteins, or other proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissue specific or inducible promoter / regulatory sequences which are useful for this purpose include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Various commercially available ubiquitous as well as tissue-specific promoters and tumor-specific are available, for example from InvivoGen. In addition, promoters which are well known in the art can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated for use with the invention. Thus, it will be appreciated that the present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto. The vectors of the present disclosure may direct expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Such regulatory elements include promoters that may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; 5’-and 3’-untranslated regions for mRNA stability and COLUM-43190.601 translation efficiency from highly-expressed genes like α-globin or β-globin; SV40 polyoma origins of replication and ColE1 for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the chimeric receptor. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Selectable markers also include chloramphenicol resistance, tetracycline resistance, spectinomycin resistance, streptomycin resistance, erythromycin resistance, rifampicin resistance, bleomycin resistance, thermally adapted kanamycin resistance, gentamycin resistance, hygromycin resistance, trimethoprim resistance, dihydrofolate reductase (DHFR), GPT; the URA3, HIS4, LEU2, and TRP1 genes of S. cerevisiae. When introduced into the cell, the vectors may be maintained as an autonomously replicating sequence or extrachromosomal element or may be integrated into host DNA. In one embodiment, the donor DNA may be delivered using the same gene transfer system as used to deliver the Cas protein, and / or transposon-associated proteins (included on the same vector) or may be delivered using a different delivery system. In another embodiment, the donor DNA may be delivered using the same transfer system as used to deliver gRNA(s). In one embodiment, the present disclosure comprises integration of exogenous DNA into the endogenous gene. Alternatively, an exogenous DNA is not integrated into the endogenous gene. The DNA may be packaged into an extrachromosomal or episomal vector (such as AAV vector), which persists in the nucleus in an extrachromosomal state, and offers donor-template delivery and expression without integration into the host genome. Use of extrachromosomal gene vector technologies has been discussed in detail by Wade-Martins R (Methods Mol Biol. 2011; 738:1-17, incorporated herein by reference). The disclosed polypeptides or components of the present system (e.g., proteins, polynucleotides encoding these proteins, donor polynucleotides and compositions comprising the proteins and / or polynucleotides described herein) may be delivered by any suitable means. In certain embodiments, the polypeptides or system is delivered in vivo. In other embodiments, the polypeptides or system is delivered to isolated / cultured cells (e.g., autologous iPS cells) in vitro COLUM-43190.601 to provide modified cells useful for in vivo delivery to patients afflicted with a disease or condition. Vectors according to the present disclosure can be transformed, transfected, or otherwise introduced into a wide variety of cells. Transfection refers to the taking up of a vector by a cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral infection, and other methods known in the art. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of sequences delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome. Any of the vectors comprising a nucleic acid sequence that encodes the disclosed polypeptides or components of the present system is also within the scope of the present disclosure. Such a vector may be delivered into host cells by a suitable method. Methods of delivering vectors to cells are well known in the art and may include DNA or RNA electroporation, transfection reagents such as liposomes or nanoparticles to delivery DNA or RNA; delivery of DNA, RNA, or protein by mechanical deformation (see, e.g., Sharei et al. Proc. Natl. Acad. Sci. USA (2013) 110(6): 2082-2087, incorporated herein by reference); or viral transduction. In some embodiments, the vectors are delivered to host cells by viral transduction. Nucleic acids can be delivered as part of a larger construct, such as a plasmid or viral vector, or directly, e.g., by electroporation, lipid vesicles, viral transporters, microinjection, and biolistics (high-speed particle bombardment). Similarly, constructs containing the one or more transgenes can be delivered by any method appropriate for introducing nucleic acids into a cell. In some embodiments, constructs or nucleic acids encoding the disclosed polypeptides or components of the present system are DNA. In some embodiments, the nucleic acid encoding the disclosed polypeptides or components of the present system is a DNA vector and may be electroporated to cells. In some embodiments, the nucleic acid encoding the disclosed polypeptides or components of the present system is an RNA molecule, which may be electroporated to cells. Additionally, delivery vehicles such as nanoparticle- and lipid-based mRNA or protein delivery systems can be used. Further examples of delivery vehicles include lentiviral vectors, COLUM-43190.601 ribonucleoprotein (RNP) complexes, lipid-based delivery system, gene gun, hydrodynamic, electroporation or nucleofection microinjection, and biolistics. Various gene delivery methods are discussed in detail by Nayerossadat et al. (Adv Biomed Res. 2012; 1: 27) and Ibraheem et al. (Int J Pharm. 2014 Jan 1; 459(1-2):70-83), incorporated herein by reference. In some embodiments, the system is delivered as a ribonucleoprotein (RNP) complex comprising any or all of the one or more Cas proteins and one or more transposon-associated proteins and a gRNA. Methods of Use Also disclosed herein are methods for nucleic acid modification or integration utilizing the disclosed systems. The methods may comprise contacting a target nucleic acid sequence with a system or component thereof disclosed herein. The descriptions and embodiments provided above for the systems are applicable to the methods described herein. The phrase “modifying a nucleic acid sequence” or “nucleic acid modification” as used herein, refers to modifying at least one physical feature of a nucleic acid sequence of interest. Nucleic acid modifications include, for example, single or double strand breaks, deletion, or insertion of one or more nucleotides, and other modifications that affect the structural integrity or nucleotide sequence of the nucleic acid sequence. The target nucleic acid sequence may be in a cell. In some embodiments, contacting a target nucleic acid sequence comprises introducing the system into the cell. As described above the system may be introduced into eukaryotic or prokaryotic cells by methods known in the art. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the target nucleic acid is a nucleic acid endogenous to a target cell. In some embodiments, the target nucleic acid is a genomic DNA sequence. The term “genomic,” as used herein, refers to a nucleic acid sequence (e.g., a gene or locus) that is located on a chromosome in a cell. In some embodiments, the target nucleic acid encodes a gene or gene product. The term “gene product,” as used herein, refers to any biochemical product resulting from expression of a gene. Gene products may be RNA or protein. RNA gene products include non-coding RNA, such as tRNA, rRNA, microRNA (miRNA), and small interfering RNA (siRNA), and coding RNA, such as messenger RNA (mRNA). In some embodiments, the target nucleic acid sequence encodes a protein or polypeptide. COLUM-43190.601 Polynucleotides containing the target nucleic acid sequence may include, but is not limited to, purified chromosomal DNA, total cDNA, cDNA fractionated according to tissue or expression state (e.g., after heat shock or after cytokine treatment other treatment) or expression time (after any such treatment) or developmental stage, plasmid, cosmid, BAC, YAC, phage library, etc. Polynucleotides containing the target site may include DNA from organisms such as Homo sapiens, Mus domesticus, Mus spretus, Canis domesticus, Bos, Caenorhabditis elegans, Plasmodium falciparum, Plasmodium vivax, Onchocerca volvulus, Brugia malayi, Dirofilaria immitis, Leishmania, Zea maize, Arabidopsis thaliana, Glycine max, Drosophila melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus pneumonia, Mycobacterium tuberculosis, Aquifex, Thermus aquaticus, Pyrococcus furiosus, Thermus littoralis, Methanobacterium thermoautotrophicum, Sulfolobus caldoaceticus, and others. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells, an effective amount of the described system or components thereof. In some embodiments, the vector(s) is delivered to the tissue of interest by, for example, an intramuscular, intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. The components of the present system, or ex vivo treated cells may be administered with a pharmaceutically acceptable carrier or excipient as a pharmaceutical composition. In some embodiments, the components of the present system may be mixed, individually or in any combination, with a pharmaceutically acceptable carrier to form pharmaceutical compositions, which are also within the scope of the present disclosure. In some embodiments, an effective amount of the components of the present system, or compositions as described herein can be administered. As used herein the term “effective amount” may be used interchangeably with the term “therapeutically effective amount” and refers to that quantity that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “effective amount” refers to that quantity of the components of the system such that successful DNA integration is achieved. When utilized as a method of treatment, the effective amount may depend on the particular condition being treated, the severity of the condition, the individual patient parameters COLUM-43190.601 including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In the context of the present disclosure insofar as it relates to any of the disease conditions recited herein, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition. Within the meaning of the present disclosure, the term “treat” also denotes to arrest, delay the onset (e.g., the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease. For example, in connection with cancer the term “treat” may mean eliminate or reduce a patient's tumor burden, or prevent, delay, or inhibit metastasis, etc. The phrase “pharmaceutically acceptable,” as used in connection with compositions and / or cells of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a subject (e.g., a mammal, a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions and / or cells to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions. Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., COLUM-43190.601 Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. The methods may be used for a variety of purposes. For example, the methods may include, but are not limited to, inactivation of a microbial gene, RNA-guided DNA integration in a plant or animal cell, methods of treating a subject suffering from a disease or disorder (e.g., cancer, Duchenne muscular dystrophy (DMD), sickle cell disease (SCD), β-thalassemia, and hereditary tyrosinemia type I (HT1)), and methods of treating a diseased cell (e.g., a cell deficient in a gene which causes cancer). The disclosed methods may modify a target DNA sequence in a cell so as to modulate expression of the target DNA sequence, e.g., expression of the target DNA sequence is increased, decreased, or completely eliminated (e.g., via deletion of a gene). The modifications of the target sequence may lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, gene knock- down, protein tagging, etc. In some embodiments, the methods described herein may be used to correct one or more defects or mutations in a gene (referred to as “gene correction”). In such cases, the target sequence encodes a defective version of a gene, and the disclosed compositions and systems further comprise a donor nucleic acid molecule which encodes a wild-type or corrected version of the gene. Accordingly, in some embodiments, the methods described herein may be used to insert a gene or fragment thereof into a cell. In another embodiment, the method of modifying a target sequence can be used to delete nucleic acids from a target sequence in a host cell by cleaving the target sequence and allowing the host cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule. Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research. In some embodiments, the methods described herein may be used to genetically modify a plant or plant cell. As used herein, genetically modified plants include a plant into which has been introduced an exogenous polynucleotide. Genetically modified plants also include a plant that has been genetically manipulated such that endogenous nucleotides have COLUM-43190.601 been altered to include a mutation, such as a deletion, an insertion, a transition, a transversion, or a combination thereof. For instance, an endogenous coding region could be deleted. Such mutations may result in a polypeptide having a different amino acid sequence than was encoded by the endogenous polynucleotide. Another example of a genetically modified plant is one having an altered regulatory sequence, such as a promoter, to result in increased or decreased expression of an operably linked endogenous coding region. The genetically modified plant may promote a desired phenotypic or genotypic plant trait. Genetically modified plants can potentially have improved crop yields, enhanced nutritional value, and increased shelf life. They can also be resistant to unfavorable environmental conditions, insects, and pesticides. The present systems and methods have broad applications in gene discovery and validation, mutational and cisgenic breeding, and hybrid breeding. The present methods may facilitate the production of a new generation of genetically modified crops with various improved agronomic traits such as herbicide resistance, herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, disease (e.g. bacterial, fungal, and viral) resistance, high yield, and superior quality. The present methods may also facilitate the production of a new generation of genetically modified crops with optimized fragrance, nutritional value, shelf-life, pigmentations (e.g., lycopene content), starch content (e.g., low- gluten wheat), toxin levels, propagation and / or breeding and growth time. See, for example, CRISPR / Cas Genome Editing and Precision Plant Breeding in Agriculture (Chen et al., Annu Rev Plant Biol. 2019 Apr 29;70:667-69), incorporated herein by reference. The present method may confer one or more of the following traits to the plant cell: herbicide tolerance, drought tolerance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, resistance to bacterial disease, resistance to fungal disease, and resistance to viral disease. The present disclosure provides for a modified plant cell produced by the present method, a plant comprising the plant cell, and a seed, fruit, plant part, or propagation material of the plant. Transformed or genetically modified plant cells of the present disclosure may be as populations of cells, or as a tissue, seed, whole plant, stem, fruit, leaf, root, flower, stem, tuber, COLUM-43190.601 grain, animal feed, a field of plants, and the like. The present disclosure provides a transgenic plant. The transgenic plant may be homozygous or heterozygous for the genetic modification. Also provided by the present disclosure are transformed or genetically modified plant cells, tissues, plants, and products that contain the transformed or genetically modified plant cells. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants. The present system and method may be used to modify a plant stem cell. The present disclosure further provides progeny of a genetically modified cell, where the progeny can comprise the same genetic modification as the genetically modified cell from which it was derived. The present disclosure further provides a composition comprising a genetically modified cell. In one embodiment, the transformed or genetically modified cells, and tissues and products comprise a nucleic acid integrated into the genome, and production by plant cells of a gene product due to the transformation or genetic modification. Methods of introducing exogenous nucleic acids into plant cells are well known in the art. Such plant cells are considered “transformed.” DNA constructs can be introduced into plant cells by various methods, including, but not limited to PEG- or electroporation-mediated protoplast transformation, tissue culture or plant tissue transformation by biolistic bombardment, or the Agrobacterium-mediated transient and stable transformation. The transformation can be transient or stable transformation. Suitable methods also include viral infection (such as double stranded DNA viruses), transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, silicon carbide whiskers technology, Agrobacterium-mediated transformation, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). Transformation methods based upon the soil bacterium Agrobacterium tumefaciens are useful for introducing an exogenous nucleic acid molecule into a vascular plant. The wild-type form of Agrobacterium contains a Ti (tumor-inducing) plasmid that directs production of tumorigenic crown gall growth on host plants. Transfer of the tumor-inducing T-DNA region of the Ti plasmid to a plant genome requires the Ti plasmid-encoded virulence genes as well as T-DNA borders, which are a set of direct DNA repeats that delineate the region to be transferred. An Agrobacterium-based vector is COLUM-43190.601 a modified form of a Ti plasmid, in which the tumor inducing functions are replaced by the nucleic acid sequence of interest to be introduced into the plant host. Agrobacterium-mediated transformation generally employs cointegrate vectors or binary vector systems, in which the components of the Ti plasmid are divided between a helper vector, which resides permanently in the Agrobacterium host and carries the virulence genes, and a shuttle vector, which contains the gene of interest bounded by T-DNA sequences. A variety of binary vectors are well known in the art and are commercially available, for example, from Clontech (Palo Alto, Calif.). Methods of coculturing Agrobacterium with cultured plant cells or wounded tissue such as leaf tissue, root explants, hypocotyledons, stem pieces or tubers, for example, also are well known in the art. See., e.g., Glick and Thompson, (eds.), Methods in Plant Molecular Biology and Biotechnology, Boca Raton, Fla.: CRC Press (1993), incorporated herein by reference. Microprojectile-mediated transformation also can be used to produce a transgenic plant. This method, first described by Klein et al. (Nature 327:70-73 (1987), incorporated herein by reference), relies on microprojectiles such as gold or tungsten that are coated with the desired nucleic acid molecule by precipitation with calcium chloride, spermidine, or polyethylene glycol. The microprojectile particles are accelerated at high speed into an angiosperm tissue using a device such as the BIOLISTIC PD-1000 (Biorad; Hercules Calif.). In one embodiment, the present methods may be adapted to use in plants. The vectors may be optimized for transient expression of the present system in plant protoplasts, or for stable integration and expression in intact plants via the Agrobacterium-mediated transformation. In certain embodiments, the present methods use a monocot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a monocot plant. In certain embodiments, the present methods use a dicot promoter to drive the expression of one or more components of the present systems (e.g., gRNA) in a dicot plant. The present methods may be used with various microbial species, including human pathogens that are medically important, and bacterial pests that are key targets within the agricultural industry, as well as antibiotic resistant versions thereof. The method may be designed to target any gene or any set of genes, such as virulence or metabolic genes, for clinical and industrial applications in other embodiments. For example, the present methods may be used to target and eliminate virulence genes from the population, to perform in situ gene knockouts, or COLUM-43190.601 to stably introduce new genetic elements to the metagenomic pool of a microbiome. The present systems and methods may be used to treat a multi-drug resistance bacterial infection in a subject. The present systems and methods may be used for genomic engineering within complex bacterial consortia. The present systems and methods may be used to inactivate microbial genes. In some embodiments, the gene is an antibiotic resistance gene. For example, the coding sequence of bacterial resistance genes may be disrupted in vivo by insertion of a DNA sequence, leading to non-selective re-sensitization to drug treatment. The methods described here also provide for treating a disease or condition in a subject. The method may comprise administering to the subject, in vivo, or by transplantation of ex vivo treated cells (e.g., disclosed T cells), a therapeutically effective amount of the present system, polypeptides, or components thereof. In some embodiments, the methods are used to treat a pathogen or parasite on or in a subject by altering the pathogen or parasite. In some embodiments, the methods target a “disease-associated” gene. The term “disease-associated gene,” refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease. A disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and / or progression of the disease. A disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. Examples of genes responsible for such “single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, α-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), β-hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin 1 (NF1), polycystic kidney disease 1 (PKD1), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate-regulating endopeptidase homologue, X-linked (PHEX), methyl-CpG-binding protein 2 (MECP2), and ubiquitin-specific peptidase 9Y, Y-linked (USP9Y). Other single gene or monogenic diseases are known in the art and described in, e.g., Chial, H. Rare Genetic Disorders: Learning About Genetic Disease COLUM-43190.601 Through Gene Mapping, SNPs, and Microarray Data, Nature Education 1(1):192 (2008); Online Mendelian Inheritance in Man (OMIM); and the Human Gene Mutation Database (HGMD). In another embodiment, the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes. Diseases caused by the contribution of multiple genes which lack simple (i.e., Mendelian) inheritance patterns are referred to in the art as a “multifactorial” or “polygenic” disease. Examples of multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia. Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip / palate, congenital heart defects, and neural tube defects. In another embodiment, the target DNA sequence can comprise a cancer oncogene. The present disclosure provides for gene editing methods that can ablate a disease-associated gene (e.g., a cancer oncogene), which in turn can be used for in vivo gene therapy for patients. In some embodiments, the gene editing methods include donor nucleic acids comprising therapeutic genes. Kits Also within the scope of the present disclosure are kits that include components of the present system. The kit may include instructions for use in any of the methods described herein. The instructions can comprise a description of administration to a subject to achieve the intended effect. The instructions generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. The packaging may be unit doses, bulk packages (e.g., multi-dose packages) or sub- unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical COLUM-43190.601 compositions are used for treating, delaying the onset, and / or alleviating a disease or disorder in a subject. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. The kit may further comprise a device for holding or administering the present system, polypeptides, or composition. The device may include an infusion device, an intravenous solution bag, a hypodermic needle, a vial, and / or a syringe. The present disclosure also provides for kits for performing nucleic acid modification and integration in vitro. Optional components of the kit include one or more of the following: buffer constituents, control plasmid, sequencing primers, cells. Examples The following are examples of the present invention and are not to be construed as limiting. Example 1 CAST systems in human cells The following nomenclature details for the text that follows: (1) Tn7016 encodes a naturally occurring Cas8-Cas5 fusion protein, as part of the Type I-F CRISPR-Cas system, which we refer to it simply as Cas8, for simplicity; (2) the Type I-F CRISPR-Cas system encoded within Tn7-like transposons may be more specifically referred to as Type I-F3, however Type I-F is used for simplicity; (3) the complex known as TniQ-Cascade, or QCascade (for simplicity), comprises crRNA (one copy), Cas8 (one copy), Cas7 (six copies), Cas6 (one copy), and TniQ (two copies); (4) in some contexts, QCascade subunits have been referred to with other gene and protein naming schemes, e.g. Csy1 or Csy2 or Cas8f instead of Cas8; Csy3 or Cas7f Cas7; Csy4 or Cas6f instead of Cas6; (5) the mini-transposon, also known as a mini-Tn, refers to the mobilizable DNA containing a cargo / payload sequence flanked by conserved left (L) and right (R) ends of the transposon; the mini-Tn may be encoded within a larger donor DNA molecule, for example a plasmid-based donor, or pDonor. Finally, CAST systems may also be referred to as INTEGRATE systems; CRISPR-transposon systems; CRISPR-Tn systems; RNA- guided transposase systems; RNA-guided DNA integration system; or a similar set of COLUM-43190.601 synonymous terms to refer to the core technology as molecular machinery. RNA-guided DNA integration by CAST systems involve a diverse array of targeting proteins, which include Cascade from Type I-B, Type I-D, and Type I-F CRISPR-Cas systems, and Cas12k from Type V-K CRISPR-Cas systems. A wide range of type I-F CASTs that captured the diversity of the set of identified CAST systems were screened. The selected systems varied greatly in sequence diversity within both the QCascade and TnsABC operons. PspA757 was identified and RNA-guided transposition was performed on a transfected, plasmid, based substrate (FIG. 1A) and genomic integration (FIG. 1C). PspA757 performed detectable RNA-guided transposition (FIG. 1B) and genomically targeted RNA-guided integration at efficiencies approximately 200-fold greater than PseCAST, e.g., Tn7016 (FIG. 1D). Example 2 Optimization of PspA757 and mechanistic dissection An additional subunit assay was performed to understand the origins of the PspA757 system’s improved performance in human cells. A previously described transcriptional activation assay was used. In the transcriptional activation assay a transcriptional activator is fused to the TnsB subunit of a CAST system and binding of the TnsB subunit to its cognate transposon end drives transcription of a fluorescent reporter (Table 2), which can be analyzed via flow cytometry (FIG. 2A). When comparing VchCAST (also referred to as VchINTEGRAT or Tn6677), PseCAST (also referred to as PseINTEGRATE or Tn7016), and PspA757, robust transcriptional activation was observed when PspA757 is expressed, demonstrating that PspA757 exhibits robust binding to its cognate transposon ends in human cells (FIG. 2B). This data suggests that TnsB•transposon end affinity may be a defining feature to identify active variants in human cells. Diverse combinations of NLS-tags were tested on each subunit of PspA757, as N- and C-terminal appendages can often disrupt the functions of type I-F CASTs. N-terminal NLS tag were individually changed to the C-terminus in all components except for TnsAB, as this rarely tolerates alternative tagging (FIG. 3, Table 2). Cas6 and Cas7 did not tolerate the C-terminal NLS tag, while TnsC, Cas8, and TniQ tolerated the C-terminal tag at varied efficiencies. In some COLUM-43190.601 embodiments, this may enable polycistronic expression vectors in which many components are linked via 2A viral peptides, or effector domains are fused to these subunits at tolerant termini. Table 1 ID PspA757 Genus Pseudoalteromonas Y V V K D E I L L D S V L M L L R C E V S K S A N L L F L L COLUM-43190.601 Cas7 MKLPRQLSYTRSLSPGKAVFFYKTTESDFEPLQIERQKIRGQKSGFAEAYKNEVTPKE LAPQDLAFGNPHTIELCYVPPTVQQLYCRFSLRVEANSLEPNVCGEPKVSYWLTRFM STYKQHGGFGELAKRYAKNILMCEWLWRNKTSPNVDLEIIGEGFEPISISKANRLRW A F Q A G G ab e Plasmid Description DNA SEQ Protein Protein Sequence ID NO Encoded P L G F K E Q L TI A R D L V I G E D D V Y COLUM-43190.601 EFEGRICRYTPDFLINHNEKPQKYIEVKPY SKIANPEFRARFAQKQLVAKEQLGIELILV TDKQIRVYPALDNLKLLHRYSGFQSLTEL W G E L H P T I L Y Q Q P D L S Q A : A L Y S G I Q L L K Y R T P R P G R E V Q S COLUM-43190.601 PNVDLEIIGEGFEPISISKANRLRWDGKWR EAEDALHTLTEVIQTGLEDPYSFCFLEVT AKLDTYFGQEIYPSQSFAENDDVARTYA S D T G A I F A H R A K T E L P L V A R L C G D S M V L L K L N L R N L V A G L G I COLUM-43190.601 KLANALAGNAVAKLVGLMAWFSHWRAI SCDDHDFSELFVNFFSNWPSGFADELSNI AELAKVKQLRPFNHTPFNEVFGSLLKDSK K R R Y L T S G T A F K G L S V Q N C A K L S R ES P Y F L S D I VI N E I Q L G D S COLUM-43190.601 pSL7453 PspA757_TnsC_Cterm 30 TnsC MGMLTEAKKAKLNQFKEVFIEYTIPKTIIN DFDKLRLHHDLAGEKPCMMLSGDTGCG KSALIKHYYDNNPPHFADGRRKVPVLLSR K L S L I M T and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

COLUM-43190.601 CLAIMS What is claimed is:

1. A system for nucleic acid integration into a target nucleic acid sequence comprising: an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)- associated transposon (CAST) system or one or more nucleic acids encoding the engineered CAST system, wherein the CAST system comprises at least one or both of: a) one or more Cas proteins; and b) one or more transposon-associated proteins, wherein at least one of the one or more Cas proteins and the one or more transposon-associated proteins are selected from SEQ ID NOs: 1-7.

2. The system of claim 1, wherein the engineered CAST system is a Type I-F system.

3. The system of claim 1 or claim 2, wherein the engineered CAST system is a Type I-F3 system.

4. The system of any of claims 1-3, wherein the one or more Cas proteins comprises Cas5, Cas6, Cas7, Cas8, or a combination thereof.

5. The system of any of claims 1-4, wherein the one or more Cas proteins comprises Cas8-Cas5 fusion protein.

6. The system of any of claims 1-5, wherein the one or more transposon-associated proteins comprises TnsA, TnsB, TnsC, TniQ, or a combination thereof.

7. The system of any of claims 1-6, wherein the engineered CAST system comprises Cas5, Cas6, Cas7, Cas8, TnsA, TnsB, TnsC, and TniQ.

8. The system of any of claims 1-7, wherein the one or more nucleic acids comprises one or more messenger RNAs, one or more vectors, or a combination thereof.

9. The system of any of claims 1-8, wherein the engineered CAST system further comprises a gRNA complementary to at least a portion of the target nucleic acid sequence, or a nucleic acid encoding the at least one gRNA.COLUM-43190.601 10. The system of any of claim 1-9, further comprising a target nucleic acid sequence, a donor nucleic acid flanked by at least one transposon end sequence, and / or at least one unfoldase protein, or at least one nucleic acid encoding thereof.

11. The system of any of claims 1-10, wherein the system is a cell-free system.

12. A method for DNA integration comprising contacting a target nucleic acid sequence with the system of any of claims 1-11 or a composition comprising thereof.

13. The method of claim 12, wherein the target nucleic acid sequence is in a cell and wherein the method comprises introducing the system into the cell.

14. The method of claim 13, wherein the introducing the system into the cell comprises administering the system to a subject.

15. The method of claim 14, wherein the administering comprises in vivo administration or transplantation of ex vivo treated cells comprising the system.

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