Methods and compositions for generating polynucleotides

Abstract

Recombinant nucleic acids, compositions and methods for producing polynucleotides, such as donor sequences, as well as their use in a variety of applications including genome engineering.

Description

METHODS AND COMPOSITIONS FOR GENERATING POLYNUCLEOTIDESCROSS-REFERENCES TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 665,752, filed June 28, 2024, the disclosure of which is considered part of, and is incorporated by reference in the disclosure of this application.FIELD

[0002] The present disclosure relates to nucleic acid production, and more particularly to recombinant nucleic acids, compositions and methods for producing polynucleotides, as well as their use in a variety of applications.BACKGROUND

[0003] Single-stranded DNA (ssDNA) may be used in a variety of biotechnology applications including, for example, genome editing, gene synthesis, gene therapy, and drug delivery.

[0004] Recent advancements in genome editing technologies, such as CRISPR and TALEN, enable researchers to efficiently introduce double-stranded breaks (DSBs) in the human genome and subsequent insertion of a donor DNA to generate a gene-edited cell. When compared to the use of double-stranded donor DNA in genome editing applications, single-stranded donor DNA exhibits lower cell toxicity, lower off-targets, and higher integration efficiency. Thus, ssDNA is considered to be the gold standard for non-viral gene delivery. However, the synthesis of ssDNA is complex, expensive, and time-consuming.

[0005] There exists a substantial need for more efficient and less costly methods for synthesis of ssDNA for a variety of applications.SUMMARY

[0006] The technology set out herein is directed to improvements in polynucleotide production. The technology includes recombinant nucleic acids, compositions and methods for producing polynucleotides, such as donor sequences, as well as their use in a variety of applications, such as gene editing.

[0007] In various aspects, the disclosure provides methods for generating a DNA molecule, such as a ssDNA molecule, that can be either linear or circular.

[0008] In one aspect, the disclosure provides a method of generating a DNA molecule including: transforming a host cell with a phagemid as described herein; culturing the host cell under conditions suitable for producing an engineered phage particle which includes a ssDNA molecule having the donor sequence; and optionally isolating the ssDNA from the engineered phage particle. In some embodiments, the method includes transforming the host cell with a helper phage plasmid having a genome encoding proteins for producing the engineered phage particle.

[0009] In yet another aspect, the disclosure provides a method of generating a ssDNA molecule including: generating a circular dsDNA molecule having a first DNA strand with a nick or a gap; and contacting the dsDNA molecule with a thermostable exonuclease thereby degrading the first DNA strand and generating a circular ssDNA molecule having a donor sequence.

[0010] In still another aspect, the disclosure provides a method of generating a ssDNA molecule including: amplifying a recombinant nucleic acid using first and second oligonucleotide of a composition as described herein to generate a first single-stranded amplicon and a second singlestranded amplicon, the first amplicon comprising a terminal overhang sequence and a donor sequence, and a second amplicon comprising a nucleic acid sequence complementary to the donor sequence and a terminal overhang sequence; contacting the first and second amplicons with an RNase to remove the terminal overhang sequences, wherein the first and second amplicons have a different length; contacting the first and second amplicons with a ligase thereby forming a dsDNA molecule having a first DNA strand that is circular and second DNA strand having a gap devoid of a least one nucleotide; and denaturing the dsDNA molecule and contacting the denatured dsDNA molecule with a thermostable exonuclease to degrade the second DNA strand thereby generating a circular ssDNA molecule comprising the donor sequence. In some embodiments, the recombinant nucleic acid is the recombinant nucleic acid of the disclosure.

[0011] In various aspects, the present disclosure also provides a recombinant nucleic acid. In some embodiments, the nucleic acid is used to synthesize a nucleic acid molecule. In some embodiments, the recombinant nucleic acid is synthesized by a method of the disclosure.

[0012] In one aspect, the disclosure provides a recombinant nucleic acid including: a donor sequence having a 5’ homology arm, an insert sequence, and a 3’ homology arm; and a telomeraseoccupancy-site (TOS).

[0013] In another aspect, the disclosure provides a recombinant nucleic acid that is a hybrid RNA / DNA molecule. In some embodiments, the nucleic acid includes: a DNA sequence encoding a donor sequence having a 5’ homology arm, an insert sequence, and a 3’ homology arm; and an RNA sequence located 5’ to the donor sequence or located 3’ to the donor sequence.

[0014] In another aspect, the disclosure provides a phagemid including a recombinant nucleic of the disclosure additionally having an fl origin of replication sequence.

[0015] In another aspect, the disclosure provides a composition. The composition includes: a first nucleic acid strand having: i) a donor sequence including a 5’ homology arm, an insert sequence, and a 3’ homology arm, and ii) a terminal overhang sequence located at the 5’ end of the first nucleic acid strand, wherein the donor sequence is DNA and the terminal overhang sequence is RNA; and a second nucleic acid strand having: i) a sequence complementary to the donor sequence of the first nucleic acid strand, and ii) a terminal overhang sequence located at the 3 ’ end of the second nucleic acid strand, wherein the sequence complementary to the donor sequence is DNA and the terminal overhang sequence of the second nucleic acid strand is RNA.

[0016] In yet another aspect, the disclosure provides a composition including: a nucleic acid cutting entity; and a recombinant nucleic acid as described herein.

[0017] In still another aspect, the disclosure provides an expression vector including a recombinant nucleic acid as described herein.

[0018] In another aspect, the disclosure provides a host cell including a recombinant nucleic acid as described herein, an expression vector as described herein, or a phagemid as described herein.

[0019] In another aspect, the disclosure provides a kit including: a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminal overhang sequence, wherein the restriction endonuclease recognition sequence is DNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA. In some embodiments, the kit further includes a thermostable endonuclease and optionally a vector having a nucleic acid encoding a nicking endonuclease.

[0020] In another aspect, the disclosure provides a kit including: a thermostable endonuclease; and a vector having a nucleic acid encoding a nicking endonuclease. In some embodiments, the kit further includes a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminal overhang sequence, wherein the restriction endonuclease recognition sequence isDNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA.

[0021] A method of genetically modifying a cell is also provided herein. The method includes generating a DNA molecule having a donor sequence using a method of the disclosure; and delivering the DNA molecule and a nucleic acid cutting entity to a cell, wherein the delivering results in modification of a genome of the cell.

[0022] The disclosure further provides a gene-edited cell produced by the method of the disclosure.

[0023] A method of treating a disease or disorder in a subject in need thereof is also provided herein. The method includes administering to the subject a gene-edited cell as described herein.

[0024] A method of treating a genetic disorder or condition in a subject in need thereof is also provided herein. Such a method may include editing a genome of a cell of the subject according to a method set out herein to modify one or more mutations associated with the genetic disorder or condition in a target locus of the genome, thereby preventing or treating the genetic disorder or condition in the subject. In such a method, cells of the subject may be contacted with a recombinant nucleic acid as described herein along with a nucleic acid cutting entity to facilitate genome editing, either in vivo or ex vivo. Further, gene-edited cells generated using a method set out herein may be administered to a subject as part of a treatment regimen.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a diagram illustrating a donor sequence of a recombinant nucleic acid in embodiments of the disclosure.

[0026] Figure 2 is a diagram showing a recombinant nucleic acid used in a method described herein to generate a DNA molecule having a donor sequence in embodiments of the disclosure.

[0027] Figure 3 is a diagram showing a donor sequence of a recombinant nucleic acid of the disclosure being inserted into a vector for amplification of the donor sequence using a method described herein.

[0028] Figure 4 is a diagram illustrating a recombinant nucleic acid having a first and second single-stranded circular regions and a double-stranded hybridization region.

[0029] Figure 5 is a diagram illustrating aspects of a method described herein which includes amplification of a donor sequence using hybrid RNA / DNA oligonucleotide primers and amplification of the donor sequence via rolling circle amplification (RCA) with a strand-displacing DNA polymerase.

[0030] Figure 6 is a diagram illustrating aspects of a method described herein which includes amplification of a donor sequence using hybrid RNA / DNA oligonucleotide primers and amplification of the donor sequence via RCA with a strand-displacing DNA polymerase.

[0031] Figure 7 is a diagram illustrating aspects of a method described herein which includes amplification of a donor sequence to produce a recombinant nucleic acid molecule having the structure shown in Figure 4 which may subsequently be processed to isolate the donor sequence. Notably, the method includes treatment with a thermostable endonuclease to produce a circular ssDNA molecule including the donor sequence.

[0032] Figure 8 is a diagram illustrating aspects of a method described herein which includes amplification of a donor sequence to produce a recombinant nucleic acid molecule having the structure shown in Figure 4 which may subsequently be processed to isolate the donor sequence. Notably, the method includes treatment with a thermostable endonuclease to produce a circular ssDNA molecule including the donor sequence.

[0033] Figure 9 is a diagram illustrating aspects of a method described herein in which a recombinant nucleic acid molecule having the structure shown in Figure 4 is treated with multiple restriction endonucleases to produce a linear ssDNA molecule including the donor sequence.

[0034] Figure 10 is a diagram illustrating aspects of a method described herein in which a recombinant nucleic acid molecule having a tag moiety is used to facilitate isolation of the donor sequence.

[0035] Figure 11 is a diagram illustrating aspects of methods described herein to generate a recombinant nucleic acid molecule having a donor sequence.

[0036] Figure 12 is a diagram illustrating aspects of a method described herein which includes production of phage particles to generate a recombinant nucleic acid molecule having a donor sequence.

[0037] Figure 13 is a diagram illustrating aspects of a method described herein which includes production of phage particles to generate a recombinant nucleic acid molecule having a donor sequence.

[0038] Figure 14 is a diagram illustrating aspects of a method described herein which includes production of phage particles to generate a recombinant nucleic acid molecule having a donor sequence.DETAILED DESCRIPTION

[0039] Definitions

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the meanings provided herein.

[0041] The terminology used herein is for the purpose of describing particular instances only, and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0042] The term “naturally occurring” as used herein as applied to a nucleic acid, a protein, a cell, or an organism, refers to a nucleic acid, cell, protein, or organism that is found in nature.

[0043] As used herein the term “isolated” is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0044] ‘ ‘Recombinant”, as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, nucleotide sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant nucleotide sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interferewith manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms.

[0045] Thus, e.g., the term “recombinant” polynucleotide or “recombinant” nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such artificial combination can be carried out to join together nucleic acid segments of desired functions to generate a desired combination of functions.

[0046] Similarly, the term “recombinant” polypeptide refers to a polypeptide which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino acid sequence through human intervention. Thus, e.g., a polypeptide that comprises a heterologous amino acid sequence is recombinant.

[0047] “Heterologous”, as used herein, means a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, in the context of a nucleic acid, a recombinant nucleic acid may include a nucleotide sequence from a bacteriophage (e.g., a TOS sequence) and a donor sequence including an insert sequence from an organism other than a bacteriophage (e.g., a human or other mammal). As another example, relative to a CRISPR-Cas effector polypeptide, a heterologous polypeptide may include an amino acid sequence from a protein other than the CRISPR-Cas effector polypeptide. As another example, a CRISPR-Cas effector protein (e.g., a dead CRISPR-Cas effector protein) can be fused to an active domain from a non-CRISPR-Cas effector protein (e.g., a cytidine deaminase), and the sequence of the active domain could be considered a heterologous polypeptide (it is heterologous to the CRISPR-Cas effector protein). As another example, a CRISPR-Cas effector protein may be fused to a DNA- binding domain from a non-CRISPR-Cas effector protein (e.g., a transposase), resulting in a heterologous polypeptide.

[0048] As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide”, “nucleic acid sequence”, and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures,and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0049] The term “intracellular nucleic acid”, as used herein, refers to nucleic acid present within a cell that is intended for editing. Intracellular nucleic acid includes genomic nucleic acid, mitochondrial nucleic acid, chloroplast nucleic acid, and plasmid nucleic acid. Intracellular nucleic acid thus includes a vector, such as a plasmid, which is introduced into a cell that contains a target locus and also encodes one or more components of a nucleic acid cutting entity (e.g., a CRISPR- Cas effector polypeptide).

[0050] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may, in some instances, be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0051] As used herein, “nucleic acid cutting entity” refers to one or more molecules, enzymes, or complex of molecules with nucleic acid cutting activity (e.g., double-stranded nucleic acid cutting activity). In most embodiments, nucleic acid cutting entity components will be either proteins or nucleic acids or a combination of the two, but they may be associated with cofactors and / or other molecules. The nucleic acid cutting entity will typically be selected based upon a number of factors, such as efficiency of DS break generation at target loci, the ability to generate DS break generation at suitable locations at or near target loci, low potential for DS break generation at undesired loci, low toxicity, and cost issues. A number of these factors will vary with the cell employed and target loci. A number of nucleic acid cutting entities are known in the art. By way of illustration, nucleic acid cutting entities include zinc finger nucleases, transcription activatorlike effector nucleases (TALENs), CRISPR-Cas effector polypeptides (e.g., CRISPR-Casnucleases), meganucleases, recombinases, integrases, transcriptases, endonucleases, transposases, CRISPR-Cas effector polypeptides fused to deaminase, CRISPR-Cas effector polypeptides fused to reverse transcriptase, and CRISPR-Cas effector polypeptides fused to DNA polymerase. The term also refers to variants, truncations, and fusion proteins of these exemplary entities. In instances, nucleic acid cutting entities will have an activity that allows them to be nuclear localized e.g., will contain nuclear localization signals (NLS)). In some embodiments, a ssDNA donor sequence could work with a nick or combination of nicks.

[0052] The term “cut”, as used herein when referring to nucleic acid, means a break in one or both strands of a nucleic acid molecule. The break may be single-stranded in that only one strand may be “broken” {e.g., the nucleic acid molecule is “nicked”) or the break may be double-stranded in that both strands are broken. Further, double-stranded cuts may generate blunt ends or staggered- / ” sticky” ends.

[0053] The term “editing”, as used herein, refers to the induction of a change in nucleotide sequence of a nucleic acid molecule. By way of example, the alteration of a single nucleotide in an intracellular nucleic acid molecule from A to T is an edit. Insertions, deletions, and substitutions of nucleic acid into or from intracellular nucleic acid molecules are also edits.

[0054] The terms “genome editing” or “gene editing” as provided herein refer to stepwise processes involving enzymes, such as polymerases, ligases, exonucleases, endonucleases or a combinations thereof. For example, gene editing may include processes where a nucleic acid molecule is cleaved, nucleotides at the cleavage site or in close vicinity thereto are excised, new nucleotides are newly synthesized and the cleaved strands are ligated.

[0055] As used herein, the term “donor nucleic acid”, “donor nucleic acid molecule”, “donor DNA”, “donor sequence” or “donor” refers to a nucleic acid molecule that is designed to be introduced into a nick or cleaved intracellular nucleic acid molecule at a target locus. A donor nucleic acid molecule will often have at least one region of sequence homology, referred to herein as a “homology arm”, to the target locus where the intracellular nucleic acid molecule is to be nicked or cleaved. In many instances, the donor nucleic acid molecule will have two regions of sequence homology to the locus {e.g., homology arms). These regions of homology may be at one or both termini flanking an insert sequence of the donor nucleic acid molecule, or may be internal to the donor nucleic acid molecule flanking the insert sequence. These regions of homology flanking the insert sequence of the donor nucleic acid molecule are referred to herein as a “5’homology arm”, which is upstream of the insert sequence, and a “3’ homology arm”, which is downstream of the insert sequence. A donor nucleic acid molecule may also include a nucleic acid cutting entity recognition sequence and its reverse complement both flanking the insert sequence. A donor nucleic acid molecule may also include a nucleic acid cutting entity recognition sequence located 5’ to a 5’ homology arm, and its reverse complement located 3’ to the 3’ homology arm, and an insert sequence is located between the homology arms.

[0056] The term “expression” of a polypeptide includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and the like).

[0057] The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0058] Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.

[0059] The term “plasmid” refers to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, gene and regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0060] A “vector” as used herein is a nucleic acid molecule that can be used as a vehicle to transfer genetic material into a cell. A vector can be a plasmid, a single or double-stranded mini circle that may be self-replicating, a virus or bacteriophage, a cosmid or an artificial chromosome, such as, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BAC) or othersequences which are able to replicate or be replicated in vitro or in a host cell, or to convey a desired nucleic acid segment to a desired location within a host cell. In some instances, a vector refers to a DNA molecule harboring at least one origin of replication, a multiple cloning site (MCS) and one or more selection markers. A vector is typically composed of a backbone region and at least one insert or transgene region or a region designed for insertion of a DNA fragment or transgene, such as an MCS. The backbone region often contains an origin of replication for propagation in at least one host and one or more selection markers. A vector can have one or more restriction endonuclease recognition sites (e.g., two, three, four, five, seven, ten, etc.) at which the sequences can be cut in a determinable fashion without loss of an essential biological function of the vector, and into which a nucleic acid fragment can be spliced in order to bring about its replication and cloning. Vectors can further provide primer sites (e.g., for PCR), transcriptional and / or translational initiation and / or regulation sites, recombinational signals, replicons, selectable markers, and the like. Clearly, methods of inserting a desired nucleic acid fragment which do not require the use of recombination, transpositions or restriction enzymes (such as, but not limited to, uracil N glycosylase (UDG) cloning of PCR fragments (U.S. Patent No. 5,334,575 and 5,888,795, both of which are entirely incorporated herein by reference), T : A cloning, and the like) can also be applied to clone a fragment into a cloning vector to be used according to the present technology. In instances, a vector contains additional features. Such additional features may include natural or synthetic promoters, unique restriction endonuclease recognition sequences to provide cut / cleavage sites (e.g., type II or type IIS) for removal, insertion or replacement of DNA fragments, genetic markers, antibiotic resistance cassettes or selection markers (e.g., toxins such as ccdB or tse2), epitopes or tag moieties for detection, manipulation or purification (e.g., V5 epitope, c-myc, hemagglutinin (HA), FLAG™, polyhistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP)), scaffold attachment regions (SARs) or reporter genes (e.g., green fluorescent protein (GFP), red fluorescence protein (RFP), luciferase, P-galactosidase, and the like). In instances, vectors are used to isolate, multiply or express inserted DNA fragments in a target host. A vector can for example be a cloning vector, an expression vector, a functional vector, a capture vector, a co-expression vector (for expression of more than one open reading frame), a viral vector or an episome (i.e., a nucleic acid capable of extrachromosomal replication), or the like.

[0061] A “cloning vector” as used herein includes any vector that can be used to delete, insert, replace or assemble one or more nucleic acid molecules. In embodiments a cloning vector may contain a counter selectable marker gene (such as, e.g., ccdB or tse2) that can be removed or replaced by another transgene or DNA fragment. In embodiments a cloning vector may be referred to as donor vector, entry vector, shuttle vector, destination vector, target vector, functional vector or capture vector. Cloning vectors typically contain a series of unique restriction enzyme cleavage sites (e.g., type II or type IIS) for removal, insertion or replacement of DNA fragments. Alternatively, DNA fragments can be replaced or inserted by TOPO® Cloning or recombination as, e.g., employed in the GATEWAY® Cloning System offered by Invitrogen / Life Technologies (Carlsbad, CA) and described in more detail elsewhere herein. A cloning vector that can be used for expression of a transgene in a target host may also be referred to as expression vector. In embodiments a cloning vector is engineered to obtain a TAL effector conjugate.

[0062] An “expression vector” is designed for expression of a transgene and generally harbors at least one promoter sequence that drives expression of the transgene. Expression as used herein refers to transcription of a transgene or transcription and translation of an open reading frame and can occur in a cell-free environment such as a cell-free expression system or in a host cell. In embodiments expression of an open reading frame or a gene results in the production of a polypeptide or protein. An expression vector is typically designed to contain one or more regulatory sequences such as enhancer, promoter and terminator regions that control expression of the inserted transgene. Suitable expression vectors include, without limitation, plasmids, single or double-stranded minicircles that may be self-replicating, and viral vectors. Vectors and expression systems for various applications are available from commercial suppliers such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Life Technologies Corp. (Carlsbad, CA). In embodiments an expression vector is engineered for expression of a gene of interest or portion thereof.

[0063] A “control” sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition, e.g., in the presence of a test compound (e.g., a first or second DNA-binding modulation-enhancing agent), and compared to samples from known conditions, e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control). A control can also represent an average value gathered from a number of tests or results.One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0064] A “guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a nucleoprotein, thereby forming ribonucleoprotein complex. Likewise, a “guide DNA” or “gDNA” as provided herein refers to a deoxyribonucleotide sequence capable of binding a nucleoprotein, thereby forming deoxyribonucleoprotein complex. In embodiments, the guide RNA includes one or more RNA molecules. In embodiments, the guide DNA includes one or more DNA molecules. In embodiments, the gRNA includes a nucleotide sequence complementary to a target site (e.g., a modulator binding sequence). In embodiments, the gDNA includes a nucleotide sequence complementary to a target site (e.g., a modulator binding sequence). The complementary nucleotide sequence may mediate binding of the ribonucleoprotein complex or the deoxyribonucleoprotein complex to said target site thereby providing the sequence specificity of the ribonucleoprotein complex or the deoxyribonucleoprotein complex. Thus, in embodiments, the guide RNA or the guide DNA is complementary to a target nucleic acid (e.g., a modulator binding sequence). In embodiments, the guide RNA binds a target nucleic acid sequence (e.g., a modulator binding sequence). In embodiments, the guide DNA binds a target nucleic acid sequence (e.g., a modulator binding sequence). In embodiments, the guide RNA is complementary to a CRISPR nucleic acid sequence. In embodiments, the complement of the guide RNA or guide DNA has a sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to a target nucleic acid (e.g., a modulator binding sequence). A target nucleic acid sequence as provided herein is a nucleic acid sequence expressed by a cell. In embodiments, the target nucleic acid sequence is an exogenous nucleic acid sequence. In embodiments, the target nucleic acid sequence is an endogenous nucleic acid sequence. In embodiments, the target nucleic acid sequence (e.g., a modulator binding sequence) forms part of a cellular gene. Thus, in embodiments, the guide RNA or guide DNA is complementary to a cellular gene or fragment thereof. In embodiments, the guide RNA or guide DNA is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to the target nucleic acid sequence (e.g., amodulator binding sequence). In embodiments, the guide RNA or guide DNA is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the sequence of a cellular gene. In embodiments, the guide RNA or the guide DNA binds a cellular gene sequence. The term “target nucleic acid sequence” refers to a modulator binding sequence as provided herein.

[0065] In embodiments, the guide RNA or guide DNA is a single-stranded ribonucleic acid. In embodiments, the guide RNA or guide DNA is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In embodiments, the guide RNA or guide DNA is from about 10 to about 30 nucleic acid residues in length. In embodiments, the guide RNA or guide DNA is about 20 nucleic acid residues in length. In embodiments, the length of the guide RNA or the guide DNA can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 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, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleic acid residues or sugar residues in length. In embodiments, the guide RNA or guide DNA is from 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, 95 to 100, or more residues in length. In embodiments, the guide RNA or guide DNA is from 10 to 15, 10 to 20, 10 to 30, 10 to 40, or 10 to 50 residues in length.

[0066] As used herein the term “homologous recombination system or “HR system” refers to components of systems set out herein that may be used to alter cells by homologous recombination. In particular, editing systems including a nucleic acid cutting entity, such as a CRISPR-Cas effector polypeptide fused to a DNA-binding domain of a transposase.

[0067] As used herein, “homology-directed repair” or “HDR” is a mechanism in cells to repair double-stranded breaks (DSBs) in DNA. In some embodiments, the HDR is greater than or equal to 10%, 25%, 50%, 75%, 90%, 95%, 98%, 99%, or 100%.

[0068] A common form of HDR is “homologous recombination”, which refers to a mechanism of genetic recombination in which two DNA strands comprising similar nucleotide sequences exchange genetic material. Cells use homologous recombination during meiosis, where it servesto rearrange DNA to create an entirely unique set of haploid chromosomes, but also for the repair of damaged DNA, in particular, for the repair of double-stranded breaks. The mechanism of homologous recombination is well known to the skilled person and has been described, for example by Paques F., Haber J. E., Microbiol. Mol. Biol. Rev. 63:349-404 (1999). In some embodiments, homologous recombination is enabled by the presence of matched termini being placed upstream (5’) and downstream (3’), respectively, in a donor nucleic acid molecule, each of which are homologous to a continuous DNA sequence within the cleaved nucleic acid molecule.

[0069] As used herein the term “non-homologous end joining” (NHEJ) refers to cellular processes that join the two ends of double-strand breaks (DSBs) through a process largely independent of homology. Naturally occurring DSBs are generated spontaneously during DNA synthesis when the replication fork encounters a damaged template and during certain specialized cellular processes, including V(D)J recombination, class-switch recombination at the immunoglobulin heavy chain (IgH) locus and meiosis. In addition, exposure of cells to ionizing radiation (X-rays and gamma rays), UV light, topoisomerase poisons or radiomimetic drugs can produce DSBs. NHEJ (non-homologous end-joining) pathways join the two ends of a DSB through a process largely independent of homology. Depending on the specific sequences and chemical modifications generated at the DSB, NHEJ may be precise or mutagenic (Lieber M. R., Annu. Rev. Biochem. 79 : 181 -211).

[0070] As used herein, “nucleic acid cutting entity recognition sequence” refers to a nucleotide sequence within a nucleic acid molecule that is recognized and cleaved by a nucleic acid cutting entity at a target locus.

[0071] As used herein, “target locus” refers to a site within a nucleic acid molecule that is recognized and cleaved by a nucleic acid cutting entity. When, for example, a single CRISPR complex is designed to cleave double- stranded nucleic acid, then the target locus is the cut site and the surrounding region recognized by the CRISPR complex. When, for example, two CRISPR complexes are designed to nick double-stranded nucleic acid in close proximity to create a double-stranded break, then the region surrounding recognized by both CRISPR complexes and including the break point is referred to as the target locus.

[0072] As used herein, “telomerase-occupancy-site” refers to a nucleotide sequence, or its reverse complement, that when hybridized to form a double-stranded nucleic acid create a cut and resolution site for a Phage N15 protelomerase (TelN). TelN cleaves the hybridized double-strandedDNA molecule containing the cut and resolution site and rejoins the resulting linear DNA strand ends to form a closed-hairpin structure. Where a single-stranded circular DNA includes a TOS and its reverse complement, a double-stranded DNA region may be created at which TelN cleaves the double-stranded DNA region and rejoins the resulting linear DNA strand ends to form a circular ssDNA having a closed-hairpin structure.

[0073] As used herein, “restriction endonuclease recognition sequence” refers to a nucleotide sequence, or its reverse complement, that when hybridized to form a double-stranded nucleic acid create a cut site for a restriction endonuclease.

[0074] As used herein, “restriction endonuclease” refers to an enzyme that cleaves a doublestranded nucleic acid at a cut site. The cut site created by hybridization of a restriction endonuclease recognition sequence with its reverse complement can be a cut site for any of a variety of restriction endonuclease types. In some embodiments, the restriction endonuclease is a Type II restriction endonuclease that recognizes a cut site have asymmetric DNA sequences, such as a Type IIS restriction endonuclease. Examples of restriction endonucleases that can be used include, without limitation, I-Scel, PacI, Bsal, BsmBl, Lgul, SapI, FokI, EcoRI, EcoRII, BamHI, Hindlll, TaqI, Notl, Hinff, Sau3A, PovII, Smal, Haelll, Hgal, Alul, EcoRV, EcoP15I, Kpnl, PstI, Sad, Sall, Seal, SphI, Stul, Xbal, Aarl, Banll, BseGI, BspPI, CfrI, EcoNI, Hsp92II, NlalV, Rsal, Tail, AasI, BbsI, BseLI, BspTI, BspQI, Clal, EcoOl 091, 1-Ppol, NmuCI, RsrII, TaqaI, Aatll, Bbul, BseLI, BsrBI, Cpol, KasI, Acc65I, BbvCI, BseML BsrDI, Csp45I, Kpn2I, Nrul, SacII, TasI, AccB7I, Bbvl, BseMII, BsrFI, Csp6I, Ehel, Kpnl, Nsbl, Sall, Tati, AccI, BceAI, BseNI, BsrGI, CspI, Esp3I, KspAI, Nsil, SapI, and Taul restriction endonucleases.

[0075] As used herein, “nicking endonuclease” refers to an enzyme that cleaves a single strand of nucleic acid, which can either be single-stranded or double-stranded, at a nicking endonuclease recognition sequence contained within a nucleic acid molecule. Examples of nicking endonucleases that can be used include, without limitation, BbvCI, BsmI, BsrDI, BssSI, BtsI, Alwl, BbvCI, BsmAI, BstNBI, BspQI, CviPII, Mval269I and BpulOI nicking endonucleases, as well as modified forms thereof.

[0076] Recombinant Nucleic Acids

[0077] The present disclosure provides recombinant nucleic acids used for production of DNA (e. ., linear or circular ssDNA) via a method described herein, as well as those generated while conducting, or resulting from, a method described herein. In various embodiments, recombinantnucleic acids of the disclosure include a donor sequence, an illustrative embodiment of which is shown in Figure 1.

[0078] In some embodiments, the disclosure provides a recombinant nucleic acid having a donor sequence used for synthesis of DNA, or a recombinant nucleic acid having a donor sequence generated by a method described herein. In some embodiments, the nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; and a telomerase-occupancy-site (TOS).

[0079] In various embodiments, the recombinant nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; and a telomeraseoccupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the donor sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the donor sequence.

[0080] In various embodiments, a recombination nucleic acid also includes a nucleic acid cutting entity recognition sequence. In some embodiments, the nucleic acid includes: a donor sequence; a TOS and a nucleic acid cutting entity recognition sequence. In some embodiments, the nucleic acid includes: a donor sequence; a TOS and a nucleic acid cutting entity recognition sequence and a reverse complement of the nucleic acid cutting entity recognition sequence.

[0081] In various embodiments, the recombinant nucleic acid further includes one or more restriction endonuclease recognition sequences. In embodiments, a restriction endonuclease recognition sequence is located 5’ (e.g., upstream) to the donor sequence, and / or 3’ (e.g., downstream) to the donor sequence. In some embodiments, the nucleic acid includes a restriction endonuclease recognition sequence located 5’ to the donor sequence and a reverse complement of the sequence is located 3’ to the donor sequence. In some embodiments, the nucleic acid includes multiple restriction endonuclease recognition sequences that form cut sites specific for different restriction endonucleases. In some embodiments, the nucleic acid includes restriction endonuclease recognition sequences that form cut sites for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different restriction endonucleases. As such, it will be appreciated that one or more different restriction endonucleases may be used at different points in a method described herein to generate one or more specific intermediate or resultant DNA molecules, such as a linear or circular ssDNA molecule including a donor sequence as described herein.

[0082] In various embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a telomerase-occupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the donor sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the donor sequence; and one or more restriction endonuclease recognition sequences. In some embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a telomerase-occupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the donor sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the donor sequence; a restriction endonuclease recognition sequence, wherein the restriction endonuclease recognition sequence is located 5’ to a 5’ homology arm and a reverse complement of the restriction endonuclease recognition sequence is located 3’ to the 3’ homology arm.

[0083] In various embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a telomeraseoccupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the donor sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the donor sequence; and one or more restriction endonuclease recognition sequences. In some embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a telomerase-occupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the 5’ homology arm and a reverse complement of the TOS is located 3’ (e.g., downstream) to the 3’ homology arm; and a restriction endonuclease recognition sequence, wherein the restriction endonuclease recognition sequence is located 5’ to the TOS and a reverse complement of the restriction endonuclease recognition sequence is located 3’ to the reverse complement of the TOS.

[0084] In some embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a restriction endonuclease recognition sequence, wherein the restriction endonuclease recognition sequence is located 5’ to the 5’ homology arm and a reverse complement of the restriction endonuclease recognition sequence is located 3’ to the 3’ homology arm; and a telomerase-occupancy -site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the restriction endonuclease recognition sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the reverse complement of the restriction endonuclease recognition sequence.

[0085] In some embodiments, the recombination nucleic acid includes: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; a telomerase- occupancy-site (TOS), wherein the TOS is located 5’ (e.g., upstream) to the donor sequence and a reverse complement of the TOS is located 3’ (e.g., downstream) to the donor sequence; a first restriction endonuclease recognition sequence, wherein the first restriction endonuclease recognition sequence is located 5’ to the TOS and a reverse complement of the first restriction endonuclease recognition sequence is located 3’ to the reverse complement of the TOS; and a second restriction endonuclease recognition sequence, wherein the second restriction endonuclease recognition sequence is located between the 5’ homology arm and the TOS and a reverse complement of the second restriction endonuclease recognition sequence is located between the 3’ homology arm and the reverse complement of the TOS.

[0086] In various embodiments, a recombinant nucleic acid of the disclosure may also include a nucleotide sequence encoding a selectable marker. Examples of selectable markers include, without limitation, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes and chloramphenicol resistance genes) or other compounds. Additional examples of selectable markers include, without limitation, genes encoding proteins that enable cell growth in media deficient in an otherwise essential nutrient (auxotrophic markers).

[0087] In various embodiments, a recombinant nucleic acid of the disclosure may also include a tag moiety sequence for detection, manipulation, purification, and / or isolation of a nucleic acid molecule.

[0088] An insert sequence as used herein includes a nucleotide sequence optionally encoding a gene, e.g., a gene of interest (“GOI”) or portion thereof, which when expressed produces a polypeptide that may have a length of from about 250 amino acids to greater than about 3000 amino acids. For example, a therapeutic polypeptide encoded by an insert sequence of a donor sequence of the present disclosure may have a length of from about 250 amino acids to about 500 amino acids, from about 500 amino acids to about 1000 amino acids, from about 500 amino acids to about 750 amino acids, from about 750 amino acids to about 1500 amino acids, from about 750 amino acids to about 1000 amino acids, from about 1000 amino acids to about 1250 amino acids, from about 1000 amino acids to about 1500 amino acids, from about 1250 amino acids to about 1500 amino acids, from about 1250 amino acids to about 1750 amino acids, from about 1500amino acids to about 1750 amino acids, from about 1500 amino acids to about 2000 amino acids, from about 1500 amino acids to about 2500 amino acids, from about 2000 amino acids to about 2500 amino acids, from about 2000 amino acids to about 3000 amino acids, or from about 2500 amino acids to about 3000 amino acids.

[0089] Suitable therapeutic proteins include, but are not limited to, a chimeric antigen receptor (CAR), a T cell receptor (TCR), a natural killer cell receptor (NKR), a synNotch polypeptide, an antibody, a Modular Extracellular Sensor Architecture (MESA) receptor, and the like. In some embodiments, a therapeutic protein is a functional version of a protein, e.g., a cystic fibrosis transmembrane conductance (CFTR) protein, a globin polypeptide (e.g., 0-globin), and the like.

[0090] In some embodiments, the disclosure provides a donor nucleic acid. By a “donor nucleic acid” or “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence having an insert sequence as described herein, that is to be inserted at the site cleaved by a nucleic acid cutting entity, such as a CRISPR / Cas effector protein (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 10%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor polynucleotides can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0091] The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair or a non -disease-causing base pair). In some embodiments, the donor sequence comprises a non-homologous sequence flanked by two regions of homology, such thathomology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

[0092] The donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, and the like), which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some embodiments may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some embodiments, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.

[0093] In some embodiments, the donor sequence is provided to the cell as single-stranded DNA. In some embodiments, the donor sequence is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self- complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions of homology that can be degraded withoutimpacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.

[0094] The term “CRISPR” or “Clustered Regularly Interspaced Short Palindromic Repeats” is a general term that applies to three types of systems, and system sub-types. In general, the term CRISPR refers to the repetitive regions that encode CRISPR system components (e.g., encoded crRNAs).

[0095] As used herein, “CRISPR complex” refers to the CRISPR-Cas effector polypeptides and nucleic acid (e.g., RNA) that associate with each other to form an aggregate that has functional activity. An example of a CRISPR complex is a wild-type Cas9 (sometimes referred to as Csnl) protein that is bound to a guide RNA specific for a target locus.

[0096] As used herein, “CRISPR-Cas effector polypeptide” refers to a protein comprising a nucleic acid (e.g., RNA) binding domain nucleic acid and an effector domain (e.g., Cas9, such as Streptococcus pyogenes Cas9, or CPF1 (cleavage and polyadenylation factor 1)). The nucleic acid binding domains interact with a first nucleic acid molecule either having a region capable of hybridizing to a desired target nucleic acid (e.g., a guide RNA) or allows for the association with a second nucleic acid having a region capable of hybridizing to the desired target nucleic acid (e.g., a crRNA). CRISPR-Cas effector polypeptides also include a nuclease domains (i.e., DNase or RNase domains), additional DNA-binding domains, helicase domains, protein-protein interaction domains, dimerization domains, as well as other domains.

[0097] CRISPR-Cas effector polypeptide also refers to proteins that form a complex that binds the first nucleic acid molecule referred to above. Thus, one a CRISPR-Cas effector polypeptide may bind to, for example, a guide RNA and another protein may have endonuclease activity. These are all considered to be CRISPR-Cas effector polypeptides because they function as part of a complex that performs the same functions as a single protein, such as Cas9 or CPF1.

[0098] In some embodiments, a CRISPR-Cas effector polypeptide will contain nuclear localization signals (NLS) that allow them to be transported to the nucleus.

[0099] CRISPR-Cas effector polypeptides used in some embodiments may generate DS breaks or may have a combined action for the generation of DS breaks. For example, mutations may be introduced into CRISPR components that prevent CRISPR complexes from making DS breaks but still allow for these complexes to nick DNA. Mutations have been identified in Cas9 proteins thatallow for the preparation of Cas9 proteins that nick DNA rather than making double-stranded cuts. Thus, some embodiments include the use of Cas9 proteins that have mutations in RuvC and / or HNH domains that limit the nuclease activity of this protein to nicking activity.

[0100] CRISPR systems that may be used vary greatly. These systems will generally have the functional activities of being able to form a complex comprising a protein and a first nucleic acid where the complex recognizes a second nucleic acid. CRISPR systems can be of any type (e.g., type I-VI).

[0101] As discussed herein, a nucleic acid cutting entity may be a CRISPR-Cas effector polypeptide. The CRISPR-Cas effector polypeptide can be any of a variety of CRISPR-Cas effector polypeptides. Suitable CRISPR-Cas effector polypeptides are described in detail below. For example, in some embodiments, the CRISPR-Cas effector polypeptide is a type II CRISPR- Cas effector polypeptide. In some embodiments, the type II CRISPR-Cas effector polypeptide is a Cas9 polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a type V CRISPR-Cas effector polypeptide, e.g., a Casl2a, a Casl2b, a Casl2c, a Casl2d, or a Casl2e polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a type VI CRISPR- Cas effector polypeptide, e.g., a Casl3a polypeptide, a Casl3b polypeptide, a Casl3c polypeptide, or a Casl3d polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Casl4 polypeptide. In some embodiments, the CRISPR-Cas effector polypeptide is a Casl4a polypeptide, a Casl4b polypeptide, or a Casl4c polypeptide. Also suitable for use is a variant CRISPR-Cas effector polypeptide, where the variant CRISPR-Cas effector polypeptide has reduced nucleic acid cleavage activity. Also suitable for use is a CRISPR-Cas effector fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide is a variant that has reduced nucleic acid cleavage activity; and ii) a heterologous fusion polypeptide. In some embodiments, the heterologous fusion polypeptide is a protein modifying enzyme. In some embodiments, the heterologous fusion polypeptide is a nucleic acid modifying enzyme, such as cytidine deaminase, adenosine deaminase or a prime editor. In some embodiments, the heterologous fusion polypeptide is a transcription factor. In some embodiments, the heterologous fusion polypeptide is a transcription activator. In some embodiments, the heterologous fusion polypeptide is a transcription repressor. As described in more detail below, in some embodiments, the CRISPR-Cas effector polypeptide comprises one or more nuclear localization signals.

[0102] Examples of CRTSPR-Cas effector polypeptides are CRISPR-Cas endonucleases (e.g., class 2 CRISPR-Cas effector polypeptide such as a type II, type V, or type VI CRISPR-Cas effector polypeptide). Where a CRISPR-Cas effector polypeptide has endonuclease activity, the CRISPR- Cas effector polypeptide may also be referred to as a “CRISPR-Cas endonuclease.” A CRISPR- Cas effector polypeptide can also have reduced or undetectable endonuclease activity. A CRISPR- Cas effector polypeptide can also be a fusion CRISPR-Cas effector polypeptide comprising a heterologous fusion partner. In some embodiments, a suitable CRISPR-Cas effector polypeptide is a class 2 CRISPR-Cas effector polypeptide. In some embodiments, a suitable CRISPR-Cas effector polypeptide is a class 2 type II CRISPR-Cas effector polypeptide (e.g., a Cas9 protein). In some embodiments, a suitable CRISPR-Cas effector polypeptide is a class 2 type V CRISPR-Cas endonuclease e.g., a Cpfl protein, a C2cl protein, or a C2c3 protein). In some embodiments, a suitable CRISPR-Cas effector polypeptide is a class 2 type VI CRISPR-Cas effector polypeptide (e.g., a C2c2 protein; also referred to as a “Casl3a” protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein.

[0103] In some embodiments, a suitable CRISPR-Cas effector polypeptide is a fusion protein comprising a CRISPR-Cas effector polypeptide that is fused to a heterologous polypeptide (also referred to as a “fusion partner”). In some embodiments, a CRISPR-Cas effector polypeptide is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0104] A nucleic acid that binds to a class 2 CRISPR-Cas effector polypeptide (e.g., a Cas9 protein, a type V or type VI CRISPR-Cas protein, a Cpfl protein, and the like) and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “gRNA” or “CRISPR-Cas guide nucleic acid” or “CRISPR-Cas guide RNA.” A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.

[0105] In some embodiments, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some embodiments, the guide RNA is one molecule (e.g., for some class 2 CRISPR-Cas proteins, the corresponding guide RNA is asingle molecule; and in some embodiments, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA”, a “one-molecule guide RNA”, or simply “sgRNA.”

[0106] In some embodiments, a kit or composition of the present disclosure comprises a CRISPR- Cas effector polypeptide, or both a CRISPR-Cas effector polypeptide and a guide RNA. In some embodiments, e.g., where a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a retinal cell target nucleic acid), the CRISPR-Cas effector polypeptide / guide RNA complex, together with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence that encodes a functional copy of the protein encoded by the defective allele), can be used to correct the deleterious mutation, e.g., via homology-directed repair (HDR).

[0107] In some embodiments, a kit or composition of the present disclosure comprises: i) a CRISPR-Cas effector polypeptide; and ii) one guide RNA. In some embodiments, the guide RNA is a single guide RNA (an “sgRNA”). In some embodiments, the guide RNA is a dual guide RNA (“dgRNA”).

[0108] In some embodiments, a kit or composition of the present disclosure comprises: i) a CRISPR-Cas effector polypeptide; and ii) 2 or more gRNAs, where the two or more gRNAs provide for multiplexed gene knockout, e.g., each of the 2 or more guide RNAs is targeted to a different gene. In some embodiments, the guide RNAs are sgRNAs. In some embodiments, the guide RNAs are dgRNAs.

[0109] In some embodiments, a kit or composition of the present disclosure comprises: i) a CRISPR-Cas effector polypeptide; and ii) 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ). In some embodiments, the guide RNAs are sgRNAs. In some embodiments, the guide RNAs are dgRNAs.

[0110] In class 2 CRISPR systems, the functions of the effector complex (e.g., the cleavage of target DNA) are carried out by a single endonuclease (e.g., see Zetsche et al., Cell. 2015 Oct. 22; 163(3): 759-71 ; Makarova c / al., Nat Rev Microbiol. 2015 November; 13(11):722-36; Shmakov et al., Mol Cell. 2015 Nov. 5; 60(3):385-97); and Shmakov et al. (2017) Nature Reviews Microbiology 15: 169. As such, the term “class 2 CRISPR-Cas protein” is used herein to encompass the CRISPR-Cas effector polypeptide (e.g., the target nucleic acid cleaving protein) from class 2 CRISPR systems. Thus, the term “class 2 CRISPR-Cas effector polypeptide” as used hereinencompasses type II CRISPR-Cas effector polypeptides (e.g, Cas9); type V-A CRISPR-Cas effector polypeptides (e.g., Cpfl (also referred to a “Casl2a”)); type V-B CRISPR-Cas effector polypeptides (e.g., C2cl (also referred to as “Casl2b”)); type V-C CRISPR-Cas effector polypeptides (e.g., C2c3 (also referred to as “Casl2c”)); type V-Ul CRISPR-Cas effector polypeptides (e.g., C2c4); type V-U2 CRISPR-Cas effector polypeptides (e.g., C2c8); type V-U5 CRISPR-Cas effector polypeptides (e.g., C2c5); type V-U4 CRISPR-Cas proteins (e.g., C2c9); type V-U3 CRISPR-Cas effector polypeptides (e.g., C2cl0); type VI-A CRISPR-Cas effector polypeptides (e.g., C2c2 (also known as “Casl3a”)); type VI-B CRISPR-Cas effector polypeptides (e.g., Casl3b (also known as C2c4)); and type VI-C CRISPR-Cas effector polypeptides (e.g., Casl3c (also known as C2c7)). To date, class 2 CRISPR-Cas effector polypeptides encompass type II, type V, and type VI CRISPR-Cas effector polypeptides, but the term is also meant to encompass any class 2 CRISPR-Cas effector polypeptide suitable for binding to a corresponding guide RNA and forming an RNP complex.

[0111] Non-limiting examples of suitable CRISPR-Cas effector polypeptides include Casl, Cas2, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, Casl Od, CasF, CasG, CasH, Csyl , Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3,Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, Casl l, Casl2, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl 3, Casl3a, Casl3b, Casl3c, Casl3d, Casl4 Casl4a, Casl4b and Casl4c.

[0112] In some embodiments, a CRISPR-Cas effector polypeptide is a fusion polypeptide comprising: i) a CRISPR-Cas effector polypeptide; and ii) one or more heterologous fusion partners (one or more heterologous fusion polypeptides). In some embodiments, a fusion CRISPR- Cas effector polypeptide comprises one or more localization signal peptides. In some embodiments, a fusion CRISPR-Cas effector polypeptide comprises one or more localization signal peptides. Suitable localization signals (“subcellular localization signals”) include, e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES); a sequence to keep the fusion protein retained in the cytoplasm; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an endoplasmic reticulum (ER) retention signal; and ER export signal; and the like. In some embodiments, a fusion CRISPR-Caseffector polypeptide does not include a NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid is an RNA that is present in the cytosol).

[0113] In some embodiments, a fusion CRISPR-Cas effector polypeptide includes (is fused to) a nuclear localization signal (NLS) (e.g., in some embodiments 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some embodiments, a fusion polypeptide includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some embodiments, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or the C-terminus. In some embodiments, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some embodiments, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some embodiments, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some embodiments, an NLS is positioned at the N-terminus and an NLS is positioned at the C- terminus.

[0114] As used herein, “zinc finger nuclease” (ZFN) refers to a chimeric protein comprising a nuclease domain and a nucleic acid (e.g., DNA) binding domain that is stabilized by zinc. The individual DNA-binding domains are typically referred to as “fingers”, such that a zinc finger protein or polypeptide has at least one finger, more typically two fingers, or three fingers, or even four or five fingers, to at least six or more fingers. In some embodiments, ZFNs will contain three or four zinc fingers. Each finger typically binds from two to four base pairs of DNA. Each finger may comprise about 30 amino acids zinc-chelating, DNA-binding region (see, e.g., U.S. Pat. Publ. No. 2012 / 0329067 Al, the disclosure of which is incorporated herein by reference).

[0115] One example of a nuclease domain is the non-specific cleavage domain from the type Ils restriction endonuclease FokI (Kim, Y.G., et al., Proc. Natl. Acad. Sei. 93:1156-60 (1996)) typically separated by a linker sequence of 5-7 base pairs. A pair of the ok\ cleavage domain is generally required to allow for dimerization of the domain and cleavage of a non-palindromic target sequence from opposite strands. The DNA-binding domains of individual Cys2His2 ZFNs typically contain between 3 and 6 individual zinc-finger repeats and can each recognize between 9 and 18 base pairs.

[0116] As used herein, “transcription activator-like effectors” (TALEs) refer to proteins composed of more than one TAL repeat and is capable of binding to nucleic acid in a sequence specific manner. As used herein, “transcription activator-like effector nuclease” (TALEN) refer to a TALE protein fused to a nuclease.

[0117] TALEs represent a class of DNA-binding proteins secreted by plant-pathogenic bacteria of the species, such as Xanthomonas and Ralstonia, via their type III secretion system upon infection of plant cells. Natural TALEs specifically have been shown to bind to plant promoter sequences thereby modulating gene expression and activating effector-specific host genes to facilitate bacterial propagation (Romer, P., et al., Science 318:645-648 (2007); Boch, J., etal., Annu. Rev. Phytopathol. 45:419-436 (2010); Kay, S., et al. , Science 375:648-651 (2007); Kay, S., etal., Curr. Opin. Microbiol. 12:37-43 (2009)).

[0118] Natural TALEs are generally characterized by a central repeat domain and a carboxyl- terminal nuclear localization signal sequence (NLS) and a transcriptional activation domain (AD). The central repeat domain typically consists of a variable amount of between 1.5 and 33.5 amino acid repeats that are usually 33-35 residues in length except for a generally shorter carboxyl- terminal repeat referred to as half-repeat. The repeats are mostly identical but differ in certain hypervariable residues. DNA recognition specificity of TALEs is mediated by hypervariable residues typically at positions 12 and 13 of each repeat - the so-called repeat variable diresidue (RVD) wherein each RVD targets a specific nucleotide in a given DNA sequence. Thus, the sequential order of repeats in a TAL protein tends to correlate with a defined linear order of nucleotides in a given DNA sequence. The underlying RVD code of some naturally occurring TALEs has been identified, allowing prediction of the sequential repeat order required to bind to a given DNA sequence (Boch, J., et al., Science 326: 1509-1512 (2009); Moscou, M.J., et al., Science 326: 1501 (2009)). Further, TAL effectors generated with new repeat combinations have been shown to bind to target sequences predicted by this code. It has been shown that the target DNA sequence generally start with a 5’ thymine base to be recognized by the TAL protein.

[0119] The modular structure of TALs allows for combination of the DNA-binding domain with effector molecules such as nucleases. TALENs used in some embodiments may generate DS breaks or may have a combined action for the generation of DS breaks. For example, TAL-FoAl nuclease fusions can be designed to bind at or near a target locus and form double-stranded nucleic acid cutting activity by the association of two FokI domains.

[0120] In some embodiments, TALEs will contain greater than or equal to 6 (e.g.. greater than or equal to 8, 10, 12, 15, or 17, or from 6 to 25, 6 to 35, 8 to 25, 10 to 25, 12 to 25, 8 to 22, 10 to 22, 12 to 22, 6 to 20, 8 to 20, 10 to 22, 12 to 20, 6 to 18, 10 to 18, 12 to 18, etc.) TAL repeats. In some embodiments, a TALE may contain 18 or 24 or 17.5 or 23.5 TAL nucleic acid binding cassettes. In additional embodiments, a TALE may contain 15.5, 16.5, 18.5, 19.5, 20.5, 21.5, 22.5 or 24.5 TAL nucleic acid binding cassettes. TALEs will generally have at least one polypeptide region which flanks the region containing the TAL repeats. In many embodiments, flanking regions will be present at both the amino and carboxyl termini of the TAL repeats. Exemplary TALEs are set out in U.S. Pat. Publ. No. 2013 / 0274129 Al, the disclosure of which is incorporated herein by reference, and may be modified forms on naturally occurring proteins found in bacteria of the genera Burkholderia, Xanthamonas and Ralstonia.

[0121] In some embodiments, TALE proteins will contain nuclear localization signals (NLS) that allow them to be transported to the nucleus.

[0122] Compositions and Kits

[0123] The present disclosure provides kits and compositions, including pharmaceutical compositions, comprising a recombinant nucleic acid of the present disclosure optionally including a donor sequence.

[0124] Accordingly, in embodiments, the disclosure provides a composition produced in performing a method described herein for generating a DNA molecule having a donor sequence. The composition includes: a first nucleic acid strand having: i) a donor sequence including a 5’ homology arm, an insert sequence, and a 3’ homology arm, and ii) a terminal overhang sequence located at the 5’ end of the first nucleic acid strand, wherein the donor sequence is DNA and the terminal overhang sequence is RNA; and a second nucleic acid strand having: i) a sequence complementary to the donor sequence of the first nucleic acid strand, and ii) a terminal overhang sequence located at the 3’ end of the second nucleic acid strand, wherein the sequence complementary to the donor sequence is DNA and the terminal overhang sequence of the second nucleic acid strand is RNA.

[0125] In various embodiments, the disclosure further provides kits that may be used in performing a method described herein.

[0126] In embodiments, the kit is used for amplifying nucleic acid and includes: a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminaloverhang sequence, wherein the restriction endonuclease recognition sequence is DNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA. In some embodiments the kit further includes a thermostable endonuclease, and optionally a vector having a nucleic acid encoding a nicking endonuclease.

[0127] In embodiments, the kit includes: a thermostable endonuclease; and a vector having a nucleic acid encoding a nicking endonuclease. In some embodiments, the kit further includes: a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminal overhang sequence, wherein the restriction endonuclease recognition sequence is DNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA

[0128] The present disclosure also provides pharmaceutical compositions comprising a recombinant nucleic acid having a donor sequence of the present disclosure. The composition may comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, “Remington: The Science and Practice of Pharmacy”, 19th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.

[0129] In embodiments, a composition of the present disclosure may include: a nucleic acid cutting entity; and a recombinant nucleic acid having a donor sequence as described herein.

[0130] In embodiments, a composition of the present disclosure may include: a nucleic acid cutting entity; a recombinant nucleic acid having a donor sequence; and a pharmaceutical excipient.

[0131] In embodiments, a composition of the present disclosure may include: a nucleic acid cutting entity; a recombinant nucleic acid having a donor sequence; and a guide RNA.

[0132] In embodiments, a composition of the present disclosure may include: a nucleic acid cutting entity; a recombinant nucleic acid having a donor sequence; a guide RNA; and a pharmaceutical excipient.

[0133] As referred to herein, a “pharmaceutical excipient” includes, by way of example, one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative. Suitable buffers include, but are not limited to, (such as N,N-bis(2- hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyehtylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N'-bis(2-ethane- sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy- propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), tris(hydroxymethyl)-aminomethane (Tris), and the like). Suitable salts include, e.g., NaCl, MgCb, KC1, MgSO4, and the like.

[0134] In some embodiments, the composition is sterile and suitable for administration to a living subject, such as a human or other mammal.

[0135] Methods of Generating Polynucleotides Having a Donor Sequence

[0136] In some aspects, the disclosure provides methods of generating a ssDNA molecule, either linear or circular, having a donor sequence.

[0137] In various embodiments, the method includes: generating a circular dsDNA molecule having first and second DNA strands, wherein the first DNA strand has a nick or a gap and the second DNA strand is non-nicked and circular. The nick or gap may be generated by contacting the circular dsDNA molecule with a nicking endonuclease which cleaves the first DNA strand at a nicking endonuclease recognition sequence. Alternatively, the nick or gap may be generated during amplification of a double-stranded vector (e.g. , cloning vector, plasmid, and the like), using hybrid RNA / DNA oligonucleotide primers of different lengths as described herein.

[0138] In various embodiments, a recombinant nucleic acid of the disclosure is generated that includes a donor sequence, such as that illustrated in Figure 1. In some embodiments, the donor sequence may further include flanking restriction endonuclease recognition sequences as also shown in Figure 1.

[0139] In embodiments, a recombinant nucleic acid of the disclosure is generated, such as the circular dsDNA shown in Figure 2. It will be appreciated that the recombinant nucleic acid may be derived from a vector, such as a plasmid or minicircle having genetic elements for amplification of the nucleic acid (e.g., Ori element and the like) to amplify copies of the donor sequence and provide methodology to efficiently isolate the donor sequence. As discussed herein, amplified copies of the nucleic acid may be contacted with a nicking endonuclease to facilitate downstream manipulation and isolation of an amplified DNA product, such as a linear ssDNA molecule or a circular ssDNA molecule, having the donor sequence.

[0140] In embodiments, a recombinant nucleic acid molecule encoding a donor sequence is generated having restriction endonuclease recognition sequences flanking the donor sequence. The nucleic acid molecule may then be inserted into a vector (e.g., cloning vector, plasmid, minicircle and the like), at an MCS of the vector as illustrated in Figure 3. In some embodiments the vector includes binding sites flanking the inserted nucleic acid molecule for hybridization of hybrid RNA / DNA oligonucleotide primers of different lengths as described herein for use in amplifying the vector via PCR. Amplification of the vector produces two amplicons of differing lengths due to the different / asymmetric number of RNA nucleotides included in each of the hybrid primers. Once annealed, the hybridized product of the PCR reaction is treated with RNase to degrade the RNA overhang present on each strand of the product and subsequently treated with a DNA ligase to form a circular dsDNA molecule having a gap in one of the strands due to the differing lengths of the amplification products.

[0141] Accordingly, in some embodiments, the circular dsDNA molecule having a first strand gap is generated by amplification using a 5’ and 3’ oligonucleotide primer pair of hybrid RNA / DNA primers having different lengths of the RNA portion of each primer to amplify a vector including the donor sequence. As such, in some embodiments, amplification of a recombinant nucleic acid containing a donor sequence as described herein is performed using a composition of the disclosure including hybrid RNA / DNA primers having different lengths of RNA nucleotides. After amplification, treatment by an RNase degrades RNA overhangs of the hybridized first and second strand amplicons. The hybridized product may then be treated with a DNA ligase to produce the circular dsDNA having a first stand with a gap and a second strand that is devoid of a gap (e.g., fully contiguous circular DNA strand) as shown in the workflows depicted in Figures 5 and 6.

[0142] As discussed herein, a circular dsDNA molecule having a first strand with a gap or nick and a second strand that is devoid of a gap or nick is treated with an exonuclease to degrade the gapped or nicked strand. In embodiments, the circular dsDNA molecule is contacted with an exonuclease that is a thermostable exonuclease. Contacting the circular dsDNA molecule with the exonuclease degrades the first DNA strand to leave an intact circular ssDNA molecule (z.e., the second DNA strand having a donor sequence) which can be manipulated for downstream applications, as well as optionally amplified and subsequently isolated.

[0143] Figures 5 and 6 are diagrams illustrating aspects of a method described herein which includes amplification of a donor sequence using hybrid RNA / DNA oligonucleotide primers and amplification of the donor sequence via rolling circle amplification (RCA) with a strand-displacing DNA polymerase which can optionally include use of a thermostable exonuclease.

[0144] In embodiments, after contacting the circular dsDNA molecule having a nicked or gapped strand with the thermostable exonuclease, the molecule may be annealed to form a DNA molecule having a region where the DNA hybridizes to form a DNA molecule have a first circular ssDNA region including nucleic acids that encode regulatory elements and amplification elements of the DNA molecule before amplification (e.g., regulatory and amplification elements, bacterial elements and the like) and a second circular ssDNA region including the donor sequence, such that the first and second circular ssDNA regions are separated by a region of hybridized DNA formed by hybridization regions of the ssDNA molecule as shown in Figure 4. In embodiments, the region of hybridized DNA includes restriction endonuclease recognition sequences that form a cut site adjacent the first circular ssDNA region and a TOS site for being catalyzed by TelN adjacent the second circular DNA region including the donor sequence as shown in Figure 4.

[0145] The DNA molecule may then be contacted with TelN which produces two circular ssDNA molecules having hairpin regions, a first DNA molecule including the first circular ssDNA region including the restriction endonuclease cut site in the hairpin region and a second DNA molecule including the second circular ssDNA region having the donor sequence. The mixture is then contacted with a restriction endonuclease that cleaves the first DNA molecule at the cut site and an exonuclease that degrades the first DNA molecule. The second DNA molecule including the donor sequence remains intact preserving the donor sequence for isolation. In some embodiments, the second DNA molecule may be isolated as a circular ssDNA having a hairpin turn or furtherprocessed to generate a linear ssDNA molecule having the donor sequence by treatment with a second unique restriction endonuclease or unique nicking endonuclease.

[0146] In alternative embodiments, a recombinant nucleic acid of the disclosure is generated, such as the circular dsDNA shown in Figure 1 , the donor sequence is amplified by PCR after the nucleic acid is treated with a restriction endonuclease with a cut site positioned to excise the donor sequence such that the donor sequence is amplified. The donor sequence may then be introduced into a minicircle in which a circular dsDNA is generated such that the circular dsDNA includes a first strand having a gap or nick produced by the methodology described herein. The circular dsDNA having the gapped or nicked strand may then be contacted with an exonuclease, such as a thermostable exonuclease to degrade the first strand which has a nick or gap to produce a circular ssDNA having the donor sequence which may be processed and isolated.

[0147] In various embodiments, depending on the options for downstream processing to isolate the donor sequence, the circular ssDNA molecule may include nucleic acid elements that allow for rolling circle amplification (RCA) of the molecule as described herein. In such instances, the donor sequence may be amplified and processed to isolate the donor sequence.

[0148] It will be appreciated that the recombinant nucleic acid may be derived from a plasmid or minicircle having genetic elements for amplification of the nucleic acid (e.g., Ori element and the like) to amplify copies of the donor sequence for subsequent isolation of the donor sequence. As discussed herein, amplified copies of the nucleic acid may be contacted with a nicking endonuclease to facilitate downstream manipulation and isolation of an amplified DNA product having a donor sequence.

[0149] In embodiments, the disclosure provides a method of generating a ssDNA molecule as illustrated in the truncated workflow shown in Figure 5. The method includes: amplifying a recombinant nucleic acid using first and second hybrid RNA / DNA oligonucleotides of a composition described herein to generate a first single-stranded amplicon and a second singlestranded amplicon, the first amplicon comprising a terminal overhang sequence and a donor sequence, and a second amplicon comprising a nucleic acid sequence complementary to the donor sequence and a terminal overhang sequence; contacting the first and second amplicons with an RNase to remove the terminal overhang sequences, wherein the first and second amplicons have a different length; contacting the first and second amplicons with a ligase thereby forming a dsDNA molecule having a first DNA strand that is circular and second DNA strand having a gap devoidof a least one nucleotide; and denaturing the dsDNA molecule and contacting the denatured dsDNA molecule with a thermostable exonuclease to degrade the second DNA strand thereby generating a circular ssDNA molecule comprising the donor sequence.

[0150] As discussed previously, Figure 6 shows an illustrative workflow in which the donor sequence of a recombinant nucleic acid of the disclosure is amplified and isolated using a composition of the disclosure having hybrid RNA / DNA oligonucleotide primers to amplify the donor sequence for isolation. Notably, a circular dsDNA molecule is generated and then amplified by RCA and the donor sequence isolated.

[0151] As used herein, “thermostable exonuclease” refers to a thermostable exonuclease, wherein the thermostable exonuclease has 3’ to 5’ exonuclease activity or 5’ to 3’ exonuclease activity and retains such activity at temperatures greater than about 75, 80, 85, 90, 95°C. Examples of thermostable exonucleases include naturally occurring, as well as modified forms of those found in thermophile or hypothemiophile microorganisms of the genera Bacillus, Clostridium, Pyrococcus, Thermus, Thermotaga and Aquifex. Illustrative examples of thermostable exonucleases include naturally occurring, as well as modified forms of PfuExo I of Pyrococcus furiosus, PhoExo I of Pyrococcus horikoshii OT3 and TK1646 of Thermococcus kodakarensis .

[0152] In a related embodiment, the disclosure provides a method of generating a DNA molecule using a phagemid. The method includes: transforming a host cell with a phagemid including a recombinant nucleic acid of the disclosure having a donor sequence; culturing the host cell under conditions suitable for producing an engineered phage particle which includes a ssDNA molecule having the donor sequence; and optionally isolating the ssDNA from the engineered phage particle. In some embodiments, the method includes transforming the host cell with a helper phage plasmid having a genome encoding proteins for producing the engineered phage particle. In some embodiments, the helper phage plasmid includes a genome that is devoid of any viral packaging sequences. In some embodiments, the generated recombinant nucleic acid may have a structure as shown in Figure 4 and further processed to isolate the donor sequence as described herein. Figures 12-14 illustrate methods that include use of phage particles to produce a donor sequence, such as a linear ssDNA molecule including a donor sequence.

[0153] In various embodiments, a recombinant nucleic acid of the disclosure, e.g., including a DNA molecule having a donor sequence or bacterial backbone sequence, is immobilized on a solid support using a tag moiety which facilitates isolating the donor sequence or removing the bacterialbackbone sequence. In some embodiments, the recombinant nucleic acid having a donor sequence is a recombinant nucleic acid having a tag moiety, and the recombinant nucleic acid is immobilized on a solid support to facilitate isolation of the donor sequence. In some embodiments, the recombinant nucleic acid having a bacterial replication origin and / or an antibiotic selection marker is a recombinant nucleic acid having a tag moiety, and the recombinant nucleic acid is immobilized on a solid support to facilitate removal of the bacterial genetic element. The immobilization of the recombinant nucleic acid reduces contamination of the DNA byproduct, e.g., residual DNA that is produced by exonuclease and endonuclease digestion of DNA. In addition, immobilization makes the use of donor DNA more accessible and easily isolated for downstream processes and uses. Figures 10 and 14 illustrate methodology utilizing a tag moiety to facilitate immobilization of a recombinant nucleic acid to a solid support to assist in isolating the donor sequence.

[0154] In some embodiments, enzymes used in a method of synthesizing recombinant nucleic acid molecules are immobilized on a solid support. Enzyme immobilization methods are known in the art via interaction of a tag moiety within the DNA and a corresponding hybridizing or capture entity immobilized on a solid support. In some embodiments, an enzyme is used to catalyze a tag moiety of the DNA and the enzyme is labeled with a moiety that may be immobilized on a solid support, such as an HUH endonuclease. In some embodiments, an inactive form of the enzyme or DNA binding domain is immobilized on a solid support for affinity capture. In some embodiments, a non-enzyme based capture entity, such as biotinylated DNA oligonucleotide that is complementary to part of the recombinant nucleic acid, is immobilized on a solid support via binding to streptavidin.

[0155] As used herein, the term "solid support" refers to each undissolved support that is capable of immobilizing a DNA molecule or enzyme or other reaction component on its surface. The solid support material is non-degradable and can be selected from, for example, agarose, modified agarose, sepharose, thiopropyl-sepharose, Sephadex™, polystyrene, latex, cellulose and ferro- or ferrimagnetic particles, acrylamide, nitrocellulose, glass, gold, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, polysilicates, polycarbonates, Teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polyactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, or any combination thereof.

[0156] A solid support may have different shapes, such as a slide, a membrane, a matrix, a plate, a chip, a resin or a bead. A solid support may further have holes or depressions to perform reactions at defined locations in an arrayed format on or within the solid support. Reactions on a solid support may be carried out in the presence of one or more additives. Such additives may help to keep beads in suspension or otherwise increase the fidelity of enzymes acting in close proximity to the solid support. The additive may be a chemical compound, a polymer, a polysaccharide, a protein, a chaperon, or any mixture thereof.

[0157] Methods and strategies for choosing appropriate solid supports and for coupling DNA molecules to said solid supports are known in the art (see, e.g., Arndt-Jovin et al. 1975. EUR. J. Biochem. 54 (2): 41 1-8; Kerrigan et al., 2001. Current Protocols in Molecular Biology. 24: 12.10.1-12.10.18; WO 1995 / 08626).

[0158] In various embodiments, the solid support is a bead, wherein the bead is of any convenient dimension and is constructed from any number of known materials. In some instances, the bead may be monodisperse. Examples of such materials include inorganic materials, natural polymers, and synthetic polymers. Specific examples of these materials include cellulose, cellulose derivatives, acrylic resins, glass; silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene cross-linked with divinylbenzene, polyacrylamide, latex gel, polystyrene dextran, rubber, silicon, plastic, nitrocellulose, cellulose, natural sponge, silica gel, glass, metal, plastic, cellulose, methacrylate beads, cross-linked dextran (e.g., Sephadex™) and agarose gels (Sepharose). In a specific embodiment, the solid support is a bead, wherein the bead is a magnetic bead or a sepharose bead.

[0159] In some embodiments, magnetic beads may include microparticles or nanoparticles. In some examples, the magnetic beads may contain iron oxide. For example, magnetic nanoclusters as described in patent application No. GB2210796.5 which is hereby incorporated by reference may be used. For example, the magnetic beads may be selected from Dynabeads™ MyOne™ Silane, Dynabeads™ MyOne™ Carboxylic Acid, Dynabeads™ M-270™ Carboxylic Acid, Dynabeads™, Oligo(dT)25 magnetic beads (all available from Thermo Fisher Scientific), SpeedBead™ (General Electric, Boston, MA), BioMagPlus COOH™ and ProMag 1 COOH™ (both Bangs Laboratories, INC Fishers), 4.4 pm fluorescent ferromagnetic beads or 2.0 pm ferromagnetic beads (both available from Spherotech INC Lake Forest, IL), 2 pm beads designated WHM-S001™ or 2 pm beads designated WHM-S002™ (both available from Creative31Diagnostics, New York, NY), Silicon Hydroxyl Magnetic Microspheres or Carboxyl Magnetic Microspheres or Oligo(dT) Magnetic Microspheres (available at different nm or pm sizes from VDO Biotech, Suzhou, China), Carboxyl Adembeads (available at 100 nm, 200 nm, 300 nm or 500 nm from Ademtech, France) etc.

[0160] In some embodiments, a recombinant nucleic acid is immobilized on nanoparticles. Nanoparticles include, but are not limited to, metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS coated with ZnS) and magnetic (e.g., ferromagnetic) colloidal materials. Methods to attach recombinant nucleic acids to the nanoparticles are known in the art. In another embodiment, nanoparticles are attached to a substrate. Nanoparticles with or without immobilized recombinant nucleic acid can be attached to substrates as described in, e.g., Grabar et aL, Analyt. Chem., 67, 73-743 (1995); Bethell et al., J. Electroanal. Chem., 409, 137 (1996); Bar et al., Langmuir, 12, 1172 (1996); Colvin et al., J. Am. Chem. Soc., 114, 5221 (1992). Naked nanoparticles may be first attached to the substrate and recombinant nucleic acids can be attached to the immobilized nanoparticles.

[0161] Methods of Delivering Compositions

[0162] The present disclosure provides methods of delivering a composition (e.g., pharmaceutical composition) of the disclosure to a cell, such as a eukaryotic cell. The methods generally involve contacting the cell with a composition including a donor sequence and a nucleic acid cutting entity. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo and the method comprises administering the composition to an individual.

[0163] A cell that serves as a recipient for a composition of the present disclosure can be any of a variety of eukaryotic cells which may be in vitro cells, in vivo cells, or ex vivo cells. In some embodiments, the target cell is in vitro. In some embodiments, cells are removed from an individual, contacted with a composition of the present disclosure in vitro, such that the cells are modified to produce a therapeutic protein of a donor nucleic acid as described herein; and returning the modified cells to the individual from whom the cells were obtained. In some embodiments, cells are removed from an individual, contacted with a composition of the present disclosure in vitro, such that the cells are modified to produce a therapeutic protein; and administering the modified cells to an individual other than the individual from whom the cells were obtained.

[0164] Suitable cells include a stem cell (e.g., an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, and the like),a somatic cell, e.g., a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, and the like.

[0165] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and post-natal stem cells.

[0166] In some embodiments, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some embodiments, the immune cell is a cytotoxic T cell. In some embodiments, the immune cell is a helper T cell. In some embodiments, the immune cell is a regulatory T cell ( reg).

[0167] In some embodiments, the cell is as adult stem cell, also referred to as somatic stem cells. Adult stem cells are resident in differentiated tissue, but retain the properties of self-renewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells, hematopoietic stem cells, epithelial stem cells, neural stem cells, mesenchymal stem cells, mammary stem cells, intestinal stem cells, mesodermal stem cells, endothelial stem cells, olfactory stem cells, neural crest stem cells, and the like.

[0168] Stem cells include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, and the like. In some embodiments, the stem cell is a human stem cell. In some embodiments, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some embodiments, the stem cell is a non-human primate stem cell.

[0169] In some embodiments, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver andyolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0170] In other embodiments, the stem cell is a neural stem cell (NSC). NSCs are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g. , cells committed to become one or more types of neurons and glial cells respectively.

[0171] In other embodiments, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon.

[0172] Therapeutic Methods

[0173] Also disclosed herein are methods for treating a genetic disorder or condition in a subject in need thereof using the processes and compositions described herein. In some instances, a target locus having a nucleic acid sequence associated with a genetic disorder or condition is modified by a donor nucleic acid as described herein to treat the genetic disorder or condition. For example, using the processes and compositions described herein, a donor nucleic acid is delivered to a cell of the subject to correct a mutation (e.g., insertion, deletion, substitution) associated with the genetic disorder or condition. In some instances, genome editing of a cell from a subject is perfomied in vivo by contacting the cell with a composition described herein, for example a composition including a donor sequence of the disclosure. In some instances, genome editing of a cell from a subject is performed ex vivo and the genome edited cell is administered to the subject.

[0174] The terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, murines, simians, humans, and veterinary and companion animals.

[0175] As used herein, “treatment”, “treating” and “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to atherapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0176] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0177] The methods and compositions described herein may be used to treat a variety of genetic disorders and conditions. In instances, treatment is achieved by replacing a fragment of a mal functioning or mutated gene that is associated with the genetic disorder or condition. A genetic disorder or condition as used herein refers to a disease caused by an abnormality in the DNA sequence of an individual. Typically, abnormalities are small mutations in a single gene. A genetic disorder or condition may be a heritable disorder and as such may be present from before birth. Other genetic disorders or conditions are caused by new mutations or changes to the DNA. In various instances, genetic disorders and conditions include, but are not limited to, proliferative disorders, such as cancer, monogenic genetic diseases, autosomal dominant disorders, autosomal recessive disorders, X-linked dominant disorders, Y-linked disorders, and hereditary diseases.

[0178] Therapeutic methods may involve ex vivo or in vivo genome modification. With ex vivo genome modification, cells will typically be removed from an individual, the genomes of these cells will be altered, then the cells will be introduced into the same individual (e.g., an allogeneic therapy) or different individual (e.g. an autologous therapy). Thus, provided herein are compositions and methods for the modification of genomes of cell, as well as the modified cells themselves.

[0179] Provided herein are compositions and methods for the alteration of genomes for therapeutic applications, including SNP alterations. Two genetic afflictions resulting from SNP alterations are set out below for purposes of illustration.

[0180] One example of an autologous, ex vivo genome modification application is where the single-nucleotide polymorphism (SNP) causing sickle cell anemia is corrected. The most common SNP associated with sickle cell anemia is rs334, which results in the alteration of a change of a single codon from GAG to GTG. This change results in the replacement of a glutamic acid residue with a valine residue. Compositions and methods set out herein are suited for altering this SNP from GTG to GAG, especially in individual homozygous for SNP rs334.

[0181] An exemplary ex vivo workflow for altering SNP rs334 in a patient would include the removal of bone marrow tissue from the patient, alteration of SNP rs334, followed by reintroduction of the editing cells back into the patient.

[0182] Of course, allogeneic workflows may also be performed. While such workflows can involve the modification of cells to render these cells allogeneic, allogeneic cells may be used as a starting point. The genomes of such allogenic cells may have been previously modified to render the cells allogeneic or they may be “naturally” allogeneic or a combination of both. One example of cells with allogeneic character are adipose derived stem cells.

[0183] One of the most common genome alterations associated with cystic fibrosis is based upon a three base pair deletion (SNP rsl99826652) in the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in the deletion of the amino acid phenylalanine at position 508.

[0184] An in vivo workflow for altering SNP rs 199826652 in a patient would include delivery of donor DNA molecules to airway cells of the patient, under conditions where a three base pair insertion would occur to correct SNP rsl99826652.

[0185] In addition to SNP correction, regions of nucleic acid may be added to or deleted from intracellular nucleic acid as part of therapeutic methods. By way of example, nucleic acid encoding a chimeric antigen receptor may be inserted into intracellular nucleic acid of T cells, resulting what are generally referred to as CAR-T cells. In many such instance, a donor nucleic acid molecule would be inserted into intracellular nucleic acid. Such donor nucleic acid molecules may comprise a promoter or may be inserted into a location where the chimeric antigen receptor (CAR) is operably linked to a promoter present in the intracellular nucleic acid (either naturally present in or previously introduced into the intracellular nucleic acid).

[0186] CAR-T cells may be activated, expanded, then introduced into patients (e.g., cancer patients). Methods for the activation and expansion of T cells can be found in Almasbak el al., PCT Publication WO 2017 / 072251.

[0187] Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description and the preferred versions contained within this specification. Various aspects of the present invention will be illustrated with reference to the following non-limiting examples.EXAMPLES

[0188] Example 1: Generation of circular ssDNA and dsDNA having a donor sequence.

[0189] Current methods for synthesis of long ssDNA often give rise to low yield and low purity as well as poor reproducibility. Chemical synthesis is limited to small ssDNA fragment. Methods using asymmetric PCR often give rise to unwanted PCR byproducts, while production of ssDNA with exonucleases often leads to incomplete or over digestion. In this example, a method is described for synthesis of either linear ssDNA or circular ssDNA and dsDNA based on a Ml 3 phage production system. The Ml 3 phage system is easy to scale up through fermentation and has the capability to produce micrograms to milligrams of ssDNA at low cost. In addition, the yield and quality of ssDNA that are produced by M13 phage system are higher than other methods.

[0190] As discussed herein a conventional phagemid is engineered to insert a pair of telomeraseoccupancy-sites (tos) flanking the donor DNA sequence, which includes of a 5’ homology arm, insert sequence (e.g., gene of interest), and 3’ homology arm. ssDNA, which is derived from a phagemid, is produced using an M13 phage production system and then purified using anion exchange column. The resulting ssDNA is denatured and re-annealed, was then digested with TelN to generate two lollipop-shaped circular DNA molecules, one containing the bacterial origin and antibiotic selection marker and one containing the donor sequence. The DNA molecule containing the bacterial origin and antibiotic selection marker is removed by selective digestion with restriction enzymes and exonucleases resulting in isolation of the DNA molecule harboring the donor sequence. Figures 12-13 show aspects of the general workflow utilized in this example.

[0191] Cloning a donor sequence into a phagemid.

[0192] The B2M EmGFP donor template containing 200nt left and right homology arms is cloned into the EcoRl site of a modified pTZ19R phagemid. The modified pTZ19R phagemid contains a pair of inverted repeats of I-Scel restriction recognition sequences and Tos sites. The pTZ19R_200nt HA-B2M -EmGFP plasmid was verified by Sanger sequencing.

[0193] Production of ssDNA using an Ml 3 phage system.

[0194] The pTZ19R_200nt HA-B2M-EmGFP plasmid is transformed into the F’ E. coll strain DH12S by electroporation and then selected with ampicillin. The colonies are inoculated into 40 ml of TBG medium containing 1.2% Tryptone, 2.4% yeast extract, 0.4% Glycerol, 17 mM KH2PO4, 55 mM K2HPO4, 20 mM glucose and ampicillin antibiotic, followed by addition of 200 ul M13 helper phage. The bacterial culture is incubated at 37oC for 2 hours with a shaking speed of 275 rpm, followed by the addition of kanamycin antibiotic. The culture is grown for approximately 18 hours and then centrifuged at 4000 rpm for 20 minutes to harvest the supernatant. To the supernatant, 0.2 volume of 2.5 M NaCl / 40% PEG-8000 solution is added, followed by rotating at 4oC for 1 hour with a Hula mixer. The phage particles are then precipitated by centrifugation at 4000 rpm for 20 minutes. The ssDNA is isolated using the PureLink™ HiPure Plasmid Miniprep Kit. The concentration of ssDNA is measured by NanoDrop™.

[0195] Purification of DNA molecule containing donor sequence.

[0196] The ssDNA isolated from phage particles were heat denatured and annealed, followed by digestion with TelN enzyme at room temperature for 1 hour. 3 ul of TelN enzyme is used to digest approximately 1 ug ssDNA. The digested DNA is purified by PureLink™ PCR Purification Kit, followed by double digestion with LScel and PacI restriction enzymes at 37oC for 1 hour. The digested DNA was purified by PureLink™ PCR Purification Kit, followed by treatment with Exonuclease III and Exonuclease I for 1 hour at 37°C. The DNA molecules containing donor sequence was further purified by PureLink™ PCR Purification Kit. The concentration of purified DNA was measured by NanoDrop™.

[0197] After reading this description it will become apparent to one skilled in the art how to implement the disclosed technology in various alternative instances and alternative applications. However, all the various instances of the present technology will not be described herein. It will be understood that the instances presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative instances should not be construed to limit the scope or breadth of the present technology as set forth below.

[0198] The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

Claims

1. WHAT IS CLAIMED IS:

1. A recombinant nucleic acid comprising: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm; and a telomerase-occupancy-site (TOS).

2. The recombinant nucleic acid of claim 1, wherein the donor sequence further comprises a nucleic acid cutting entity recognition sequence.

3. The recombinant nucleic acid of claim 2, wherein the nucleic acid cutting entity recognition sequence is located 5’ to the donor sequence and a reverse complement of the nucleic acid cutting entity recognition sequence is located 3’ to the donor sequence.

4. The recombinant nucleic acid of claim 2, wherein the nucleic acid cutting entity is a CRISPR-Cas effector polypeptide, zinc finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease, recombinase, integrase, transcriptase, endonuclease, transposase, or variant, mutant or truncation thereof.

5. The recombinant nucleic acid of claim 4, wherein the nucleic acid cutting entity is a CRISPR-Cas effector polypeptide.

6. The recombinant nucleic acid of claim 5, wherein the CRISPR-Cas effector polypeptide is Cas9.

7. The recombinant nucleic acid of claim 1, further comprising a restriction endonuclease recognition sequence located 5’ to the donor sequence or 3’ to the donor sequence.

8. The recombinant nucleic acid of claim 7, wherein the restriction endonuclease recognition sequence is located 5’ to the donor sequence and a reverse complement of the restriction endonuclease recognition sequence is located 3’ to the donor sequence.

9. The recombinant nucleic acid of claim 1, wherein the TOS is located 5’ to the donor sequence and a reverse complement of the TOS is located 3’ to the donor sequence.

10. The recombinant nucleic acid of claim 1, further comprising a nucleic acid sequence encoding a marker.

11. The recombinant nucleic acid of claim 1, wherein the marker is an antibiotic resistance marker.

12. The recombinant nucleic acid of claim 1, further comprising a tag moiety sequence.

13. A phagemid comprising: a recombinant nucleic acid of any preceding claim having an fl origin of replication sequence.

14. A method of generating a DNA molecule comprising: transforming a host cell with the phagemid of claim 13; culturing the host cell under conditions suitable for producing an engineered phage particle comprising a single-stranded DNA (ssDNA) molecule having the donor sequence; and optionally isolating the ssDNA from the engineered phage particle.

15. The method of claim 14, further comprising transforming the host cell with a helper phage plasmid comprising a genome encoding proteins for producing the engineered phage particle.

16. The method of claim 15, wherein the genome is devoid of a viral packaging sequence.

17. The method of claim 14, wherein the host cell comprises a genome encoding proteins for producing the engineered phage particle.

18. The method of claim 14, wherein the ssDNA molecule is circular and comprises a first and a second hybridization region wherein the first and second hybridization regions are capable of hybridizing to form a dsDNA region, first circular ssDNA region and a second circular ssDNA region having the donor sequence, the dsDNA region being interposed between the first and second circular ssDNA regions.

19. The method of claim 18, wherein the dsDNA region comprises a telomerase-occupancy- site (TOS) and one or more restriction endonuclease recognition sequences.

20. The method of claim 19, further comprising contacting the ssDNA molecule with a first restriction endonuclease that specifically cleaves one or more restriction endonuclease recognition sequences located adjacent the first circular ssDNA region, and a phage N15 protelomerase (TelN).

21. The method of claim 20, further comprising contacting the ssDNA molecule with an exonuclease.

22. The method of claim 21, wherein isolating comprises isolating a circular DNA molecule having: i) a hairpin region, and ii) the second circular ssDNA region having the donor sequence.

23. The method of claim 22, further comprising contacting the circular DNA molecule with a second restriction endonuclease that specifically cleaves one or more restriction endonuclease sequences located adjacent the donor DNA thereby producing a linear ssDNA molecule comprising the donor sequence.

24. The method of claim 19, further comprising immobilizing the ssDNA molecule to a solid support.

25. The method of claim 24, further comprising contacting the immobilized ssDNA molecule with a phage N15 protelomerase (TelN) thereby generating a circular DNA molecule having: i) a hairpin region, and ii) a circular ssDNA region having the donor sequence.

26. The method of claim 25, further comprising contacting the circular DNA molecule with a restriction endonuclease thereby generating a linear ssDNA molecule comprising the donor sequence.

27. The method of claim 24, further comprising contacting the ssDNA molecule with a restriction endonuclease thereby generating a linear ssDNA molecule comprising the donor sequence.

28. A recombinant nucleic acid comprising: a donor sequence comprising a 5’ homology arm, an insert sequence, and a 3’ homology arm, wherein the donor sequence is DNA; and a terminal overhang sequence located at the 5’ end of the donor sequence or the 3’ end of the donor sequence, wherein the terminal overhang sequence is RNA.

29. The recombinant nucleic acid of claim 28, wherein the terminal overhang sequence is located at the 5’ end of the donor sequence.

30. The recombinant nucleic acid of claim 28, wherein the terminal overhang sequence is located at the 3’ end of the donor sequence.

31. The recombinant nucleic acid of claim 28, further comprising a restriction endonuclease recognition sequence.

32. A composition comprising: a first nucleic acid strand having: i) a donor sequence including a 5’ homology arm, an insert sequence, and a 3’ homology arm, and ii) a terminal overhang sequence located at the 5’ end of the first nucleic acid strand, wherein the donor sequence is DNA and the terminal overhang sequence is RNA; and a second nucleic acid strand having: i) a sequence complementary to the donor sequence of the first nucleic acid strand, and ii) a terminal overhang sequence located at the 3’ end of thesecond nucleic acid strand, wherein the sequence complementary to the donor sequence is DNA and the terminal overhang sequence of the second nucleic acid strand is RNA.

33. The composition of claim 32, wherein the terminal overhang sequence of the first nucleic acid strand and the terminal overhang sequence of the second nucleic acid strand include a different number of ribonucleotides.

34. The composition of claim 32, wherein the terminal overhang sequence of the first nucleic acid strand includes about 1-40 ribonucleotides, and the terminal overhang sequence of the second nucleic acid strand includes about 1-45 ribonucleotides.

35. A kit compri si ng : a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminal overhang sequence, wherein the restriction endonuclease recognition sequence is DNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA.

36. The kit of claim 35, wherein the DNA sequence of the second oligonucleotide comprises a reverse complement of the restriction endonuclease recognition sequence.

37. The kit of claim 35, further comprising a thermostable endonuclease.

38. The kit of any one of claims 35-37, further comprising a vector having a nucleic acid encoding a nicking endonuclease.

39. A kit compri sing : a thermostable endonuclease; and a vector having a nucleic acid encoding a nicking endonuclease.

40. The kit of claim 39, further comprising:a first oligonucleotide having a restriction endonuclease recognition sequence and a first terminal overhang sequence, wherein the restriction endonuclease recognition sequence is DNA and the first terminal overhang sequence is RNA; and a second oligonucleotide having a DNA sequence and a second terminal overhang sequence, wherein the second terminal overhang sequence is RNA.

41. The kit of claim 40, wherein the DNA sequence of the second oligonucleotide comprises a reverse complement of the restriction endonuclease recognition sequence.

42. A method of generating a single-stranded DNA (ssDNA) molecule comprising: generating a circular double-stranded DNA (dsDNA) molecule having a first DNA strand with a nick or a gap; and contacting the dsDNA molecule with a thermostable exonuclease thereby degrading the first DNA strand and generating a circular single-strand DNA (ssDNA) molecule having a donor sequence.

43. The method of claim 42, wherein the circular dsDNA molecule generated from a DNA plasmid.

44. The method of claim 43, wherein the DNA plasmid comprises the recombinant nucleic acid of any preceding claim.

45. The method of claim 42, wherein the DNA plasmid comprises one or more nicking endonuclease recognition sequences.

46. The method of claim 45, further comprising contacting with the DNA plasmid with one or more nicking endonucleases to generate the circular dsDNA.

47. The method of claim 42, further comprising contacting the circular dsDNA with a thermostable exonuclease thereby generating the circular ssDNA molecule.

48. The method of claim 47, further comprising amplifying the circular ssDNA molecule via rolling circle amplification thereby generating a ssDNA amplification product comprising the donor sequence.

49. The method of claim 42, wherein the circular dsDNA molecule is generated from a DNA minicircle.

50. The method of claim 49, wherein the DNA minicircle comprises the recombinant nucleic acid of any preceding claim.

51. The method of claim 49, wherein generating the circular dsDNA molecule comprises amplifying the DNA minicircle followed by enzymatic treatment to generate sticky ends.

52. The method of claim 49, wherein the nick is generated by a nicking endonuclease.

53. The method of claim 49, wherein the nick is generated via amplification using an oligonucleotide comprising DNA and RNA and contacting an amplicon generated via the amplification with uracil DNA glycosylase or RNase to introduce the nick.

54. The method of any preceding claim, wherein the thermostable exonuclease comprises 3’ to 5’ exonuclease activity or 5’ to 3’ exonuclease activity.

55. The method of claim 54, wherein the thermostable exonuclease is from a thermophile or hypothermophile microorganism of a genus selected from Bacillus, Clostridium, Pyrococcus, Thermus, Thermotaga and Aquifex.

56. The method of claim 54, wherein the thermostable exonuclease is selected from Pyrococcus furiosus PfuExo I, Pyrococcus horikoshii 01’3 PhoExo I and Thermococcus kodakarensis TK1646.

57. The method of any preceding claim, wherein the nicking endonuclease is selected from BbvCI, BsmI, BsrDI, BssSI, BtsI, Alwl, BbvCI, BsmAI, BstNBI, BspQI, CviPII, Mval269I, BpulOI and modified forms thereof.

58. A method of generating a single-stranded DNA (ssDNA) molecule comprising: amplifying a recombinant nucleic acid using the first and the second oligonucleotides of the kit of claim 32 thereby generating a first single-stranded amplicon and a second singlestranded amplicon, the first amplicon comprising a terminal overhang sequence and a donor sequence, and a second amplicon comprising a nucleic acid sequence complementary to the donor sequence and a terminal overhang sequence; contacting the first and second amplicons with an RNase to remove the terminal overhang sequences, wherein the first and second amplicons have a different length; contacting the first and second amplicons with a ligase thereby forming a double-stranded DNA (dsDNA) molecule having a first DNA strand that is circular and second DNA strand having a gap devoid of a least one nucleotide; denaturing the dsDNA molecule and contacting the denatured dsDNA molecule with a thermostable exonuclease to degrade the second DNA strand thereby generating a circular ssDNA molecule comprising the donor sequence.

59. The method of claim 59, wherein the recombinant nucleic acid is the recombinant nucleic acid of any preceding claim.

60. The method of claim 59, further comprising contacting the circular ssDNA molecule with a restriction endonuclease thereby generating a linear ssDNA molecule comprising the donor sequence.

61. The method of claim 59, further comprising amplifying the circular DNA molecule via rolling circle amplification thereby generating a ssDNA amplification product comprising the donor sequence.

62. The method of claim 61, further comprising contacting the ssDNA amplification product with a restriction endonuclease thereby generating a linear ssDNA molecule comprising the donor sequence.

63. The method of any preceding claim, wherein the thermostable exonuclease comprises 3’ to 5’ exonuclease activity or 5’ to 3’ exonuclease activity.

64. The method of claim 63, wherein the thermophile or hypothermophile is of a genus selected from Bacillus, Clostridium, Pyrococcus, Thermus, Thermotaga and Aquifex.

65. The method of claim 63, wherein the thermostable exonuclease is selected from Pyrococcus furiosus PfuExo I, Pyrococcus horikoshii OT3 PhoExo I and Thermococcus kodakarensis TK1646.

66. An expression vector comprising the recombinant nucleic acid of any preceding claim.

67. A composition comprising: a nucleic acid cutting entity; and a recombinant nucleic acid of any preceding claim.

68. A host cell comprising the phagemid of any preceding claim, the expression vector of any preceding claim, the composition of any preceding claim, or the recombinant nucleic acid of any preceding claim.

69. A method of genetically modifying a cell comprising: generating a DNA molecule having a donor sequence using any one of the methods of claims 14-27 or 42-65; and delivering the DNA molecule and a nucleic acid cutting entity to a cell, wherein the delivering results in modification of a genome of the cell.

70. A gene-edited cell produced by the method of claim 69.

71. A method of treating a disease or disorder in a subject, the method comprising: administering to the subject the gene-edited cell of claim 70.

72. A method of treating a genetic disorder or condition in a subject in need thereof comprising editing a genome of a cell of the subject according to the method of claim 71 to modify a mutation associated with the genetic disorder or condition in a target locus of the genome, thereby preventing or treating the genetic disorder or condition in the subject.

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