Nuclease resistant single stranded DNA product for non-viral delivery to a cell and methods of production thereof

Abstract

Exonuclease-resistant single stranded template DNA product and a method for producing the same are provided. Also provided are lipid compositions comprising exonuclease-resistant single stranded template DNA product and methods for inserting a custom DNA template into a cell.

Description

NUCLEASE RESISTANT SINGLE STRANDED DNA PRODUCT FOR NON-VIRAL DELIVERY TO A CELL AND METHODS OF PRODUCTION THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit of U.S. Provisional Application Nos. 63 / 583.524, filed September 18, 2023 and 63 / 610,960 filed December 15, 2023, which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The Sequence Listing filed with this application in XML format, which is entitled “5640-102PCT.xml,” was created on September 17, 2024 and is 8,192 bytes in size, is hereby incorporated by reference in its entirety.FIELD

[0003] The instant disclosure relates generally to methods for producing, delivering, and using a exonuclease-resistant single stranded template deoxyribonucleic acid (DNA) product, as well as exonuclease-resistant single stranded template DNA product comprising a custom DNA template.BACKGROUND

[0004] Polynucleotide-based therapeutics provide a means to treat numerous conditions. Among them, gene-editing strategies designed to ameliorate genetic disorders underlying many diseases. Gene-editing technologies use various enzy me-based systems to create single and double stranded breaks to alter targets sites. For example, inducing double stranded breaks (DSBs) followed by repair mechanisms including homology-directed repair (“HDR”) or homologous recombination (“HR’’) allows editing of the target sites using endogenous or exogenous template DNA. Efficient editing suitable for therapeutic applications requires the availability of the appropriate template DNA to mediate the repair. Therefore, stable storage and delivery of polynucleotides is critical for the development of numerous DNA-based therapeutic applications, particularly gene-editing techniques.

[0005] However, polynucleotides are susceptible to environmental degradation that occurs in a physiological setting and also during in vitro storage and handling. Exposure to nucleases can readily degrade single stranded polynucleotides, as well as, duplex oligonucleotides and plasmid DNA, which renders nuclease sensitive forms of DNA unsuitable for therapeutic applications.

[0006] Developing polynucleotide structures that are resistant to nucleases such as exonucleases is a critical aspect of DNA based therapeutic development. Single stranded polynucleotides tend to be less stable than double stranded DNA and pose a greater challenge for use in therapeutic applications. Indeed, it is known in the field that producing exonuclease-resistant single stranded template DNA product, e.g., exonuclease-resistant single stranded template DNA product greater than 1 kb or 2 kb is especially difficult. Moreover, efficient production of exonuclease-resistant, polynucleotides is difficult at scales needed for both clinical development and therapeutic applications. Production requires numerous processing steps to form a stabilized product. Therefore, there is an unmet need for exonuclease-resistant linear single stranded template DNA product that provides an extended half-life, and methods of producing such molecules, particularly long exonuclease-resistant linear single stranded template DNA products, efficiently (high speed, high yield, high purity, and low cost) at large scales.SUMMARY OF THE INVENTION

[0007] Provided herein are exonuclease-resistant single stranded template deoxyribonucleic acid (DNA) products and lipid compositions comprising an exonuclease-resistant single stranded template DNA product. In addition, the present disclosure includes methods for producing an exonuclease-resistant single stranded template DNA product and methods for inserting an exonuclease-resistant single stranded template DNA product into a cell.

[0008] In some aspects, the method comprises a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide configured to modify a second end of the template sequence, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonuclease-resistant single stranded template DNA product, e) inactivating the second exonuclease, and I performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture. In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both are resistant to exonuclease digestion.

[0009] In some aspects, the method comprises a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide comprising a hairpin and configured to modify a second end of the template sequence to produce a double stranded template DNA closed on one end, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonuclease-resistant single stranded template DNA product, e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture to obtain the exonuclease-resistant single stranded template DNA product,

[0010] In some aspects, one strand of the first capping oligonucleotide is resistant to exonuclease digestion. In some aspects, the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.

[0011] In some aspects, the capping reaction comprises adding the following components a) to c) to the cell-free reaction mixture in the specified order: a) the template DNA source molecule; b) an aqueous solvent; c) a buffer; d) the first capping oligonucleotide; e) the second capping oligonucleotide; f) at least one endonuclease; and g) the DNA ligase.In some aspects, the cell-free reaction mixture may be mixed after adding component c). In some aspects, components d) to g) may be added to the cell-free reaction mixture in any order. In some aspects, one or both of d) and e) are added (in any order) after c) is added. In some aspects, one or both of f) and g) are added (in any order) after d) or e) is added.

[0012] In some aspects, the capping reaction is performed in a temperature range from about 30 °C to about 42 °C.

[0013] In one aspect, a lipid composition is provided comprising the exonuclease-resistant single stranded template DNA product prepared by the methods of any one of the aspects described herein.

[0014] In another aspect, the disclosure provides an exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification and the second modification are exonuclease resistant, and wherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15,000 nucleotides in length.

[0015] In another aspect, the disclosure provides an exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification, the second modification, or both the first and second modification comprises a linear sequence comprising one or more phosphorothioate bonds or an exonuclease resistant functional group, wherein the exonuclease resistant functional group does not comprise biotinylation.

[0016] In some aspects, the first modification, the second modification, or both the first and the second modification may be selected from 2'-O-methyl nucleotides. 2'-O-methoxy ethyl (MOE) nucleotides, phosphorothioate nucleotides, 2’-fluoro nucleotides, inverted nucleotides at a 3 '-end, at a 5 '-end, or both the 3' and the 5 '-ends, thiophosphate nucleotides, phosphoroselenoate nucleotides, selenophosphate nucleotides, locked nucleotides (e.g., containing a 2’-O, 4’-C methylene bridge), or reversed nucleotide bases. In some aspects, the first modification, the second modification, or both the first and the second modification may be selected from a G-quadruplex structure, for example G-4 quadruplex, an RNA aptamer, e.g., an MS2 RNA aptamer, or C spacer group. In some aspects, the C spacer is a C3 to C12 spacer group.

[0017] In some aspects, the first modification, the second modification, or both the first and the second modification may be one or more phosphorothioate bonds or a functional group comprising a polynucleotide secondary structure, wherein the second modification comprises a hairpin structure, wherein the second modification comprises one or more phosphorothioate bonds.

[0018] In some aspects, the lipid composition may comprise microspheres, liposomes, lipoplexes, or lipid nanoparticles. In some aspects, the lipid composition further encapsulates one or more peptides, polypeptides, one more cationic salts, one or more anionic salts, one or more additional polynucleotides.

[0019] In some aspects, the disclosure relates to a method for inserting a custom DNA template into a cell comprising introducing an exonuclease-resistant single stranded template DNA product into a cell comprising a) producing the exonuclease-resistant single stranded template DNA product by the method of any one of the aspects described herein; b) contacting the cell with the exonuclease-resistant single stranded template DNA product and a gene editing system; and c) inserting at least a part of the template sequence from the exonuclease-resistant single stranded template DNA product into DNA of the cell that has been cleaved by the gene editing system.

[0020] In some aspects, the gene editing system may comprise a CRISPR / Cas system; a Tth Argonaute (TtAgo) system; a zinc finger nuclease (ZFN) system; ARCUS nuclease system; megaTALs; or a transcription activator-like effector nuclease (TALEN) system.

[0021] In one aspect, the exonuclease-resistant single stranded template DNA product produced according to the methods of any one of the aspects described herein may be inserted into a cell by contacting the cell with the exonuclease-resistant single stranded template DNA product.

[0022] In an aspect, the lipid composition comprises (a) an ionizable lipid; (b) one or more helper lipids; (c) one or more neutral lipids; (d) optionally, a conjugated lipid; and (e) the exonuclease-resistant single stranded template DNA product prepared according to the methods of any one of the aspects described herein.

[0023] In some aspects at least a part of the template sequence from the exonuclease-resistant single stranded template DNA product may be inserted into DNA of the cell that has been cleaved by a gene editing system.

[0024] In certain aspects, the disclosure relates to any method of gene editing described herein, comprising administering a lipid composition comprising the exonuclease-resistant single stranded template DNA product to a cell or whole organism, for example a plant or an animal, particularly, for example, a human.

[0025] In some aspects, the disclosure relates to any method of gene editing described herein, wherein the gene editing results in a gene correction.

[0026] In certain aspects, the disclosure relates to any method of gene editing described herein, wherein the gene editing results in an insertion. In some aspects, the insertion is a gene insertion.

[0027] These and other aspects of the invention will be apparent upon reference to the following detailed description, claims, aspects, procedures, compounds, and / or compositionsand associated background information and references, which are hereby incorporated in their entirety.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1A shows a gel separation of single stranded template DNA product samples from mixtures containing unmodified LHP (left hairpin), mixture 1 , and modified LHP, mixture 2, following 5, 10, 15. 20 and 30 minutes of digestion. FIG. IB shows a gel separation of samples collected after the enzyme cutting step, after the T4 ligation step, and after the T7 digestion step.

[0029] FIG. 2 shows a gel separation of the double stranded template DNA precursor sample after Esp3I endonuclease digestion and ligation of the first and second capping oligonucleotides (lane 2) and after Exonuclease III and RecJF exonuclease digestion (lane 3).

[0030] FIG. 3A shows a gel separation of the double stranded template DNA precursor sample after Bsal endonuclease digestion and ligation of the first and second capping oligonucleotides (lane 1) and after 60 minutes (lane 2), 40 minutes (lane 3), and 20 minutes (lane 4) of Exonuclease III digestion. FIG. 3B shows conversion of the sample in FIG. 3 A to single stranded DNA by the addition of T7 exonuclease for 10. 20. and 30 minutes following 60 minutes of digestion with Exonuclease III (lanes 1-3), the addition of T7 exonuclease for 30 minutes following 40 minutes of digestion with Exonuclease III (lane 4), and the addition of T7 exonuclease for 30 minutes following 20 minutes of digestion with Exonuclease III (lane 5).

[0031] FIG. 4 shows a gel separation of the double stranded template DNA precursor sample after Bsal digestion at 37 °C and ligation at 16 °C (“cut / ligate”), Exonuclease III digestion at 37 °C (“ExoIII”), and T7 digestion at 25 °C (“T7”) (Sample 1), Bsal digestion and ligation at 37 °C (“cut / ligate”), Exonuclease III digestion at 37 °C (“ExoIII”), heat inactivation at 70 °C, and T7 digestion at 25 °C (“T7”) (Samples 2-3).

[0032] FIG. 5 shows a schematic illustrating different configurations of a double stranded template DNA precursor resulting from the ligation of a phosphorothioate modified hairpin or a double stranded phosphorothioate modified prehybridized duplex oligonucleotide on each end of a double stranded template sequence excised from a source plasmid. The schematic also shows the final exonuclease-resistant single stranded template DNA product obtained after T7 exonuclease digestion of the double stranded template DNA precursor.

[0033] FIG. 6 shows a schematic illustrating the formation of a double stranded template DNA precursor ligation product from a source plasmid followed by T7 exonuclease digestion to produce exonuclease-resistant single stranded template DNA product. The leftover vectoris further digested using Exonuclease III prior to purification of the exonuclease-resistant single stranded template DNA product.

[0034] FIG. 7 shows a chromatogram of a HPLC purification, measured at 260 nm, of the cell-free reaction mixture prepared in Example 5 using the HPLC ramping method outlined in Table II.

[0035] FIG. 8 shows the results of atomic force microscopy, indicating that the exonucleaseresistant single stranded template DNA product was contained in the HPLC peak corresponding to the fraction collected at 21.227 minutes.

[0036] FIG. 9 shows a chromatogram of a HPLC purification, measured at 260 nm, of the cell-free reaction mixture prepared in Example 5 using the ramping method outlined in TableIII.

[0037] FIG. 10 shows a chromatogram of a HPLC purification, measured at 260 nm, of the cell-free reaction mixture prepared in Example 5 using the ramping method outlined in TableIV.

[0038] FIG. 11 shows an overlay of chromatograms of three HPLC purification runs of the cell-free reaction mixture prepared in Example 5 (one with 3000 pL and two with 2500 pL injection volume of the filtered reaction mixture), measured at 260 nm, using the ramping method outlined in Table V.

[0039] FIG. 12 shows gel electrophoresis of HPLC peaks obtained from the chromatograms shown in FIGS. 7, 9. and 10-11 after dialysis.

[0040] FIG. 13 shows gel electrophoresis of HPLC peaks obtained from the chromatograms shown in FIGS. 7, 9, and 10-11 after dialysis and lyophilization.

[0041] FIG. 14 show s a chromatogram of a HPLC purification, measured at 260 nm, of reaction mixture prepared in Example 5 using the ramping method outlined in Table VI.

[0042] FIGS. 15A-15B show the results of atomic force microscopy at different magnifications (1 pm inset, FIG. 15A and 200 nm inset, FIG. 15B), indicating that the exonuclease-resistant single stranded template DNA product was contained in the HPLC peak corresponding to the fraction collected at 17.645 minutes

[0043] FIG. 16 shows overlaid chromatograms of nine back-to-back injections of the reaction mixture measured at 260 nm using the stepwise method outlined in Table VII.

[0044] FIG. 17 show s gel electrophoresis of fractionated HPLC peaks obtained from the chromatogram shown in FIG. 16. The peak corresponding to the fraction collected between 5.7 minutes and 6.2 minutes from each of the nine chromatograms contains the exonucleaseresistant single stranded template DNA product.

[0045] FIG. 18 show s gel electrophoresis of HPLC purified exonuclease-resistant single stranded template DNA product before and after TFF.DETAILED DESCRIPTION

[0046] While aspects of the subject matter of the present disclosure may be embodied in a variety of forms, the following description is merely intended to disclose some of these forms as specific examples of the subject matter encompassed by the present disclosure.Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described.

[0047] Unless otherwise defined, 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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0048] Other features and advantages of the invention will be apparent from the follow ing detailed description and figures, and from the claims.

[0049] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 0.01 to 2.0” should be interpreted to include not only the explicitly recited values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5. 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc.Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.

[0050] In understanding the scope of the present disclosure, the terms “including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms thatspecify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of,” as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. It is understood that reference to any one of these transition terms (i.e. “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of’ or “consisting of’ would find direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, the present disclosure supports including or excluding any element disclosed herein or incorporated by reference from the claims.

[0051] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group w ithout indications to the contrary.

[0052] The disclosure relates to novel methods for producing exonuclease-resistant single stranded template DNA product, with high efficiency, scalability, and reduced cost, as well as to lipid compositions for delivering the exonuclease-resistant single stranded template DNA product. The exonuclease-resistant single stranded template DNA product may be derived from template DNA source molecules having suitable restriction endonuclease sites to produce double stranded template DNA that is further processed to the exonuclease-resistant single stranded template DNA product. The exonuclease-resistant single stranded template DNA product of the present disclosure are capable of being inserted, at least in part, into the DNA of either a cycling or non-cycling cell that has been cleaved by a gene editing system. For example, exonuclease-resistant single stranded template DNA product may be insertedfollowing cleavage of target site DNA by non-homolog ous end joining repair (NHEJ) or homology-directed repair (HD).

[0053] Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0054] While the invention is described in conjunction with the illustrated embodiments, it is understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the invention as defined by the appended claims.

[0055] The summan' and detailed description, as well as the following examples, are exemplary and explanatory only and are not restrictive of the teachings. The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any literature incorporated by reference contradicts any term defined in this specification, this specification controls. All ranges given in the application encompass the endpoints unless stated otherwise.Definitions

[0056] The singular forms “a,” "an" and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0057] Furthermore, the term “about” as used herein when referring to a measurable value such as an amount of the length of a polynucleotide or polypeptide sequence, dose. time, temperature, and the like, is meant to encompass variations of the number in context. In certain aspects, variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% may be defined. Embodiments in the specification that recite “about” various values are also contemplated as encompassing “at” the recited values.

[0058] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0059] A “bioreactor” means any device or system that supports an environment in which a chemical process is carried out. In some aspects, the bioreactor may be a closed system, such that it is sealed or substantially sealed from exposure to the outside environment. In someaspects, the bioreactor may be an open system that is at least partially exposed to the outside environment. The volume, shape, and material of the bioreactor may be selected based on the desired application, for example, the size of the reaction process. In some aspects, the bioreactor may be suitable for batch, fed batch, or continuous (e.g. a continuous stirred-tank reactor model), such as a chemostat device. In some aspects, the bioreactor is able to support an environment capable of achieving an isothermal process according to any of the aspects disclosed herein. For example, the bioreactor material may be insulated or configured so as to minimize temperature variation suitable to achieve an isothermal process. In some aspects, the bioreactor may accommodate a reaction volume of 0.01 L to 50 L, 0.5 L to 25 L, 0.1 L to 12.5 L, 0.25 L to 6.25 L, 0.5 L to 3.5 L, or 0.75 L to 1.5 L.

[0060] A ‘"duplex structure” means a polynucleotide sequence forming a duplex, i.e., double stranded DNA, on at least a portion of the polynucleotide sequence. For example, the duplex structure may comprise a double stranded DNA polynucleotide with single strand overhangs on one or both ends of the polynucleotide sequence, e.g., overhangs of 2 nucleotides or more. In some aspects, a duplex structure may comprise a prehybridized duplex oligonucleotide or an oligonucleotide compnsing a hairpin structure. In some aspects, the prehybridized duplex oligonucleotide may comprise a hairpin structure on one or both strands.

[0061] As used herein, the term “endogenous sequence” refers to a sequence that is native to the cell. The term “exogenous sequence” refers to a sequence that is not native to a cell, or a sequence whose native location in the genome of the cell is in a different location.

[0062] As used herein, a “nucleoside” consists of a nucleic acid base (e.g., the canonical nucleic acid bases: guanine (G), adenine (A), thymine (T), uracil (U), and cytosine (C)); or a modified nucleic acid base (e.g., 5 -methylcy tosine (m5C)), that is covalently linked to a pentose sugar (e.g., ribose or 2'-deoxyribose), whereas and a “nucleotide” consists of a nucleoside that is phosphorylated at one of the hydroxyl groups of the pentose sugar. Linear nucleic acid molecules are said to have a “5' terminus” (5' end) and a “3' terminus” (3' end) because, except with respect to capping or adenylation (e.g., adenylation by a ligase), mononucleotides are joined in one direction via a phosphodi ester linkage to make oligonucleotides or polynucleotides, in a manner such that a phosphate on the 5' carbon of one mononucleotide sugar moiety is joined to an oxygen on the 3' carbon of the sugar moiety of its neighboring mononucleotide. Therefore, an end of a linear single-stranded oligonucleotide or polynucleotide or an end of one strand of a linear double-stranded nucleic acid (RNA or DNA) is referred to as the “5' end” if its 5' phosphate is not joined or linked tothe oxygen of the 3' carbon of a mononucleotide sugar moiety, and as the “3' end” if its 3' oxygen is not joined to a 5' phosphate that is joined to a sugar of another mononucleotide.

[0063] As used herein, the term ‘‘exonuclease resistant moiety” means any molecular structure having an exonuclease resistant property and includes, for example, any one or more of an exonuclease resistant chemical modification, an exonuclease resistant secondary7structure, or exonuclease resistant functional group.

[0064] As used herein, the term “modified nucleotide”, “modified nucleoside” and “nucleotide analog” refer to a nucleotide or nucleoside that contains one or more chemical modifications (e.g. substitutions) in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U)), adenine (A) or guanine (G)). A nucleotide analog can contain further chemical modifications in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six- membered sugar analog, or open-chain sugar analog), or the phosphate. There are more than 96 naturally occurring modified nucleosides found on mammalian RNA. See, e.g., Limbach et al, Nucleic Acids Research, 22(12):2183-2196 (1994). A modified nucleobase species may include one or more substitutions that are not naturally occurring. The preparation of nucleotides and modified nucleotides and nucleosides are well-known in the art, e.g. from US Patent Nos 4373071, 4458066, 4500707, 4668777, 4973679, 5047524, 5132418, 5153319, 5262530, 5700642 all of which are incorporated by reference in their entirety herein.

[0065] As used herein, “nucleic acid” refers a nucleic acid molecule. According to the present disclosure, nucleic acids comprise genomic DNA, cDNA, RNA, mRNA, recombinantly prepared and chemically synthesized molecules. According to the present disclosure, a nucleic acid may be in the form of a single-stranded or double stranded and linear or covalently closed circular molecule. The nucleic acid of the present disclosure may also containing non-natural nucleotides and modified nucleotides. “Nucleic acid” also refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides.

[0066] As used herein, the term “genomic DNA” is referring to the heritable genetic information of a host organism. Said genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the other cellular organelles (e.g., mitochondria). In some aspects, the term genomic DNA refers to the chromosomal DNA of the nucleus.

[0067] As used herein, “custom DNA template” means a synthetic or an artificial sequence or a sequence that shares identity, or partial identity, with an endogenous nucleotide sequencefound in an organism. In certain aspects, the custom DNA template may comprise one or more nucleotide substitutions or deletions relative to an endogenous nucleotide sequence found in an organism. In certain aspects, the organism may be a mammal, such as a human.

[0068] As used herein, “capping oligonucleotide” means a polynucleotide configured to modify one or both ends of a template DNA sequence that is produced from a template DNA source molecule in order to form an exonuclease-resistant single stranded template DNA product. The capping oligonucleotide may be configured to modify an end of the template DNA sequence by a ligation reaction, e.g., by using a ligase to catalyze the formation of a phosphodi ester bond.

[0069] As used herein, a “diafiltration volume” is equal to a removed volume of permeate divided by a remaining volume of retentate. In some aspects, the permeate and retentate volumes may be automatically measured by a tangential flow filtration system.

[0070] “Isothermal” or “isothermally” means conducting a reaction at substantially constant temperature, i.e., without significant variation to the reaction temperature in which the reaction occurs. For example, an isothermal reaction may have a variation in temperature during the entirety of the reaction of no more than ± 2 °C. ± I °C. or ± 0.5 °C. In some aspects, one or more reactions may be conducted isothermally. For example, two reactions that are performed to produce a desired product (e.g., a exonuclease-resistant single stranded template DNA product) may be conducted isothermally, each at a different temperature, respectively.

[0071] “Molar excess” means a molar ratio being more than, i.e. above, 1 : 1. Likewise, a “mol excess” means a mole ratio being more than, i.e., above, 1 : 1. In some aspects, a mol excess or a molar excess may be 1: 1.1 or more, i.e., 1.1-fold or more.

[0072] “mRNA” refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’-methoxy ribose residues. In some aspects, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2-methoxy ribose residues, or a combination thereof. In general. mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). An mRNA can contain modified uridines at some or all of its uridine positions.

[0073] “Guide RNA” or “gRNA” are used herein interchangeably to refer to a cognate guide nucleic acid for an RNA-guided DNA-binding agent. Guide RNAs can include modified RNAs as described herein. A gRNA may be, for example, either a single guide RNA, or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (as a single guide RNA, sgRNA) or, for example, in two separate RNA strands (dual guide RNA, dgRNA). In some systems a gRNA may be a crRNA (also known as a CRISPR RNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.

[0074] “Hairpin structure” means a polynucleotide sequence comprising tw o regions of the same strand that base-pair to form a double helix that ends in unpaired or non-Watson-Crick- paired nucleotides. In certain aspects, the hairpin structure may comprise a hairpin loop, stem loop, or a secondary structure comprising multiple hairpin loops or stem loops.

[0075] “Nuclease-resistant nucleotide” encompasses any type of nucleotide that provides or enhances resistance to nuclease digestion, for example exonuclease digestion. For example the nuclease resistant nucleotides may comprise modified nucleotides that provide or increase resistance to nucleases (e.g. including, but not limited to, exonucleases).

[0076] “Double stranded template DNA precursor” means a template sequence that has been modified on one or both ends by an exonuclease resistant capping oligonucleotide. In some aspects, the double stranded template DNA precursor may be formed by modifying a template DNA source molecule comprising a template sequence so that one or both ends of the template sequence may be ligated to an exonuclease resistant capping oligonucleotide. In certain aspects, the double stranded template DNA precursor is formed by excising a template sequence (e.g., using an endonuclease) from a template DNA source molecule and then modifying one or both ends of the template sequence with an exonuclease resistant capping oligonucleotide (e.g., by ligating a capping oligonucleotide to one end, or ligating two capping oligonucleotides, i.e., ligating one to each end of the template sequence, using a ligase enzyme). In some aspects, the double stranded template DNA precursor is modified with a first capping oligonucleotide having a prehybridized duplex oligonucleotide that has exonuclease resistant nucleotides on only one strand such that the other strand is susceptible to degradation by an exonuclease, and a second capping oligonucleotide that is resistant to exonuclease digestion. In some aspects, the double stranded template DNA precursor is modified with a first and second capping oligonucleotide having a prehybridized duplex oligonucleotide that has exonuclease resistant nucleotides on only one strand of theprehybridized duplex oligonucleotide such that only one strand of the double stranded template DNA precursor has exonuclease resistant nucleotides on both the 5’ and 3‘ end.

[0077] The double stranded template DNA precursor may be further processed by performing one or more exonuclease digestion reactions to produce an exonuclease-resistant single stranded template DNA product.

[0078] An “exonuclease-resistant single stranded template DNA product” means a template DNA that has a modification on each end of the sequence that confers resistance to exonuclease degradation and is single stranded over at least a portion of the sequence forming the exonuclease-resistant single stranded template DNA product. In certain aspects, the exonuclease-resistant single stranded template DNA product may be partially double stranded, for example, by hybridizing to itself and, in some aspects, forming one or more secondary structures. In some aspects, the exonuclease-resistant single stranded template DNA product may be uniformly single stranded across the entire sequence. In aspects, the exonuclease-resistant single stranded template DNA product may be single stranded across about 40% or more, about 50% or more, about 60% or more, about 75% or more, 80% or more, about 85% or more, about 90% or more, or about 95% or more of the entire sequence.

[0079] The term “PEG” as used herein means any polyethylene glycol or other polyalkylene ether polymer, such as an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain aspects, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer such as PEG- polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Polyethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety may be a PEG homopolymer.

[0080] "Phosphorothioated nucleotide" means a nucleotide that has an altered phosphate backbone, wherein, the sugar moieties are linked by a phosphorothioate bond. In the phosphate backbone of an oligonucleotide sequence, the phosphorothioate bond contains a sulphur atom as a substitute for a non-bridging oxygen atom. This intemucleotide linkage modification confers resistance to exonuclease degradation. In some aspects, resistance to exonuclease degradation is achieved by including 4 or more phosphorothioate bonded nucleotides.

[0081] “Purify,” “purifying,” or “purification” are used interchangeably and mean the physical separation of a product or substance of interest from one or more foreign or contaminating substances. In contrast, "impurities" or "impurity" refers to the one or moreforeign or contaminating substances, other than product or substance of interest, that are undesirable.

[0082] An HPLC “i socratic phase,” “isocratic mode,” or “isocratic elution” means the composition of the mobile phase is substantially the same over the run time or over a particular phase.

[0083] An HPLC “ramping gradient,” “ramping method,” “ramping mode,” or “ramping phase” means the buffer composition of the mobile phase changes gradually over a set time or volume. As used herein, “ramp” and “ramping” may be used interchangeably.

[0084] An HPLC “step-wise method,” “step mode,” or “step phase” means the buffer composition of the mobile phase changes in one or more steps. In some aspects, one or more steps in the step-wise method may comprise an isocratic phase. In some aspects, one or more steps in the step-wise method may comprise an isocratic phase before changing to the next buffer composition and then an isocratic phase again at that step. In certain aspects, a ramping phase may be performed between one or more steps.

[0085] As used herein, an “RNA-guided DNA-binding agent” means a polypeptide or complex of polypeptides having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, wherein the DNA-binding activity is sequence-specific and depends on the sequence of the RNA.

[0086] As used herein, “thermocycling” or “thermocy cle” means a set of conditions comprising repetitions of two or more temperatures, wherein the two or more temperatures are repeated.

[0087] As used herein, “transfection” refers to the introduction of a species (e.g., an RNA or DNA) into a cell. Various methods may be employed in order to introduce nucleic acids into cells in vitro or in vivo. Such methods include transfection of nucleic acid-CaPCh precipitates, transfection of nucleic acids associated with DEAE, transfection of infection with viruses carrying the nucleic acids of interest, liposome mediated transfection, lipid nanoparticle (LNP) mediated transfection, lipofectamine and the like.

[0088] As used herein a “prehybridized duplex oligonucleotide” means a double stranded oligonucleotide formed from two independent complementary oligonucleotide strands, wherein one strand is resistant to exonuclease digestion and the other strand can be digested by an exonuclease. In certain aspects, the top strand of the prehybridized duplex oligonucleotide has one or more exonuclease resistant nucleotides, e.g., four or more phosphorothioate nucleotides, or an exonuclease resistant functional group, and the bottom strand of the prehybridized duplex oligonucleotide is susceptible to exonuclease digestion,including, but not limited to digestion by a T7 exonuclease. The prehybridized duplex oligonucleotide is provided under conditions sufficient to promote hybridization, thereby- forming a duplex oligonucleotide. In some aspects, conditions sufficient to promote hybridization may comprise combining two complementary oligonucleotide strands in molecular biology grade water. In some aspects, the molecular biology grade water may- further include magnesium chloride. The complementary oligonucleotides strands may be combined in a ratio of 1 : 1. In some aspects, the ratio of a first complementary oligonucleotide to a second complementary oligonucleotide to a IM magnesium chloride solution may be 1: 1 :0.02. In some aspects, the complementary strands are incubated at 95 °C and cooled to a temperature of about 20°C to about 30°C, about 22°C to about 28°C, or about 23°C to about 25°C. In some aspects, the complementary strands are incubated at 95 °C for 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more before cooling.

[0089] In some aspects, the complementary strands are incubated at about 19 °C to about 25 °C or about 20 °C to about 23 °C for about 6 to 12 hours or about 8 to 10 hours.

[0090] As used herein, 'yield'’ (also referred herein as “% yield”, "percent yield” or “% of theoretical maximum yield”) is calculated as the molar amount of the exonuclease-resistant single stranded template DNA product obtained after purification (e g., by measuring the amount of product using Nanodrop or other spectral absorbance methods), as a percentage of the theoretical maximum molar amount of the exonuclease-resistant single stranded template DNA product produced from the template DNA source molecule. The theoretical maximum molar amount is based on the limiting reactant. For example, when 50 nanomoles of the template DNA source molecule is incubated with a molar excess of capping oligonucleotides following cleavage with at least one endonuclease, and processing with one or more exonucleases to produce the exonuclease-resistant single stranded template DNA product according to any of the aspects described herein, obtaining 50 nanomoles of exonucleaseresistant single stranded template DNA product, after purification, would equal a 100% yield.

[0091] In some aspects, the method for producing an exonuclease-resistant single stranded template DNA product comprises a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide configured to modify a second end of the template sequence, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-freereaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonuclease-resistant single stranded template DNA product, e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonucleaseresistant single stranded template DNA product from the cell-free reaction mixture,

[0092] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both are resistant to exonuclease digestion.

[0093] In some aspects, the method for producing an exonuclease-resistant single stranded template DNA product for non-viral delivery to a cell, comprises a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide comprising a hairpin and configured to modify a second end of the template sequence to produce a double stranded template DNA closed on one end. b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonuclease-resistant single stranded template DNA product, e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture to obtain the exonuclease-resistant single stranded template DNA product,

[0094] In some aspects, one strand of the first capping oligonucleotide is resistant to exonuclease digestion. In some aspects, the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.

[0095] In some aspects, the exonuclease-resistant single stranded template DNA product may be produced isothermally in steps a) to e). In some aspects, steps a) to e) are performed at a temperature from about 30°C to about 42°C. In some aspects, steps a) to e) are performed at about 37°C.

[0096] In some aspects, the capping reaction may be performed for about 1 hour to about 72 hours. In some aspects, the capping reaction may be performed for about 1 hour to about 10 hours, about 2 hours to about 8 hours, about 3 hours to about 6 hours, or about 4 hours.

[0097] In some aspects, the capping reaction comprises adding the following components a) to c) to the cell-free reaction mixture in the specified order: a) the template DNA source molecule: b) an aqueous solvent; c) a buffer; d) the first capping oligonucleotide; e) the second capping oligonucleotide; f) the at least one endonuclease; and g) the DNA ligase.In some aspects, the cell-free reaction mixture may be mixed after adding component c). In some aspects, components d) to g) may be added to the cell-free reaction mixture in any order.

[0098] In some aspects, the capping reaction comprises: i) incubating the template DNA source molecule comprising the at least one endonuclease recognition sequence and the template sequence in a cell-free reaction mixture comprising a first endonuclease, a first DNA ligase, and the first capping oligonucleotide to modify the first end of the template sequence; optionally inactivating the first endonuclease prior to step ii); and ii) incubating the template sequence modified on the first end with the second capping oligonucleotide to modify the second end of the template sequence, and optionally adding a second endonuclease, a second DNA ligase, or both after step i). In some aspects, the first and second endonuclease may be the same or different. In some aspects, step i) and ii) may be performed sequentially in the same reaction. In some aspects, step i) and ii) may be performed as separate reactions. In some aspects, step i) and ii) may be performed in the same reaction vessel. In some aspects, step i) and ii) may be performed in separate reaction vessels, optionally the template sequence modified on the first end is purified prior to step ii). In some aspects, step i) and ii) may be performed at the same or different temperatures. In some aspects, step i) and ii) are each independently performed at one or more temperatures.

[0099] In some aspects, the capping reaction may be performed in a temperature range from about from about 10 °C to about 42 °C, about 14 °C to about 40 °C, about 15 °C to about 39 °C, or about 16 °C to about 37°C. In some aspects, the capping reaction may be performed isothermally at a temperature of about 37 °C. In some aspects, the capping reaction may be at least partially performed at a first temperature and optionally at a second temperature that is different than the first temperature. In some aspects, the first temperature and the secondtemperature may be each independently selected from a temperature in a range of about 10 °C to about 42 °C, about 14 °C to about 40 °C, about 15 °C to about 39 °C. or about 16 °C to about 37°C.

[0100] In some aspects, the capping reaction may be performed at a first temperature after adding the at least one endonuclease and a second temperature after adding the ligase. In some aspects, the first temperature and the second temperature may each independently selected from a temperature in a range of about 10°C to about 42°C, about 14°C to about 40°C, about 15°C to about 39°C, about 16°C to about 37°C, or about 37°C. In some aspects, the capping reaction may be conducted at the first temperature for 1 hour or more, 2 hours or more, 3 hours or more, or 4 hours or more. In some aspects, the capping reaction may be conducted at the first temperature for about 1 hour to about 72 hours. In some aspects, the capping reaction may be conducted at the first temperature for about 1 hour to about 10 hours, about 2 hours to about 8 hours, about 3 hours to about 6 hours, or about 4 hours. In some aspects, the capping reaction may be conducted at the second temperature for 1 hour or more, 2 hours or more. 3 hours or more, or 4 hours or more. In some aspects, the capping reaction may be conducted at the second temperature for about 1 hour to about 72 hours. In some aspects, the capping reaction may be conducted at the second temperature for about 1 hour to about 10 hours, about 2 hours to about 8 hours, about 3 hours to about 6 hours, or about 4 hours.

[0101] In some aspects, the capping reaction is performed at a single temperature. In some aspects, the capping reaction is performed at about 37 °C.

[0102] The capping reaction may be performed for a longer duration than the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both. In some aspects, the capping reaction is performed for about 10 minutes to 20 hours, about 0.5 to about 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 1 hour to about 2 hours. In some aspects, the capping reaction may be performed for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7, hours, 8 hours, 9 hours, or 10 hours. In some aspects, the capping reaction is performed for 2 hours or less or 1 hour or less.

[0103] In some aspects, the template DNA source molecule may comprise a first endonuclease recognition sequence at the first end of the template sequence and a second endonuclease recognition sequence at the second end of the template sequence. In some aspects, the first end and the second end of the template sequence comprise a different overhang end following digestion of the template DNA source molecule by two different endonucleases.

[0104] The methods according to the present disclosure may comprise one or more or two or more exonuclease digestion reactions to produce the exonuclease-resistant single stranded template DNA product. In some aspects, a first exonuclease digestion reaction may be performed to digest non-template DNA that has been separated from the template DNA source molecule following the restriction endonuclease digestion of the template DNA source molecule. In some aspects, the non-template DNA portion of the template DNA source molecule in the cell-free reaction mixture is degraded after a single exonuclease digestion reaction.

[0105] In some aspects, the exonuclease may be selected from T7 exonuclease, Exonuclease I, Exonuclease III, Exonuclease VII, or Exonuclease VIII. In some aspects, the exonuclease may be Exonuclease III or T7 exonuclease. In some aspects, the first exonuclease is Exonuclease III. In some aspects, the second exonuclease is T7 exonuclease.

[0106] In some aspects, the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both comprises anti-sense digestion. In some aspects, the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both comprises sense digestion.

[0107] In some aspects, the first exonuclease, the second exonuclease, or both digests in the 3’ to 5’ direction. In some aspects, the first exonuclease, the second exonuclease, or both digests in the 5’ to 3 ' direction. In some aspects, the first exonuclease digests in the 3‘ to 5’ direction. In some aspects, the second exonuclease digests in the 5?to 3’ direction.

[0108] In some aspects, the first exonuclease digestion reaction is performed at a temperature from about 16°C to about 42°C, 35°C to about 40°C, about 36 °C to about 38 °C, or about 37°C.

[0109] In some aspects, the second exonuclease digestion reaction is performed at a temperature from about 16°C to about 42°C, about 20 °C to about 37 °C, about 23 °C to about 35 °C, or about 25 °C.

[0110] In some aspects, the duration of the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is shorter than the capping reaction. In some aspects, the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 0.5 to about 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 0.5 to about 1.5 hours. In some aspects, the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 5 minutes to about 1 hour, about 10 minutes to about 50 minutes, about 15 minutes to about 40 minutes, or about 20 minutes to about 30 minutes. In certain aspects.the first exonuclease digestion reaction is performed for 20 minutes to 60 minutes. In certain aspects, the second exonuclease digestion reaction is performed for 10 minutes to 30 minutes. In some aspects, the first exonuclease digestion reaction may be performed for 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some aspects, the second exonuclease digestion reaction may be performed for 5, 10, 15, 20, 25, or 30 minutes.[OHl] In some aspects, the method comprises inactivating endonuclease, inactivating exonuclease activity, or both with a heat inactivation step. In some aspects, the method comprises inactivating endonuclease, inactivating exonuclease activity, or both by adding a chelating agent to the cell-free reaction mixture. In some aspects, the method may comprise a heat inactivation step, adding a chelating agent, or both prior to the digestion step. In some aspects, the method may comprise a heat inactivation step, adding a chelating agent, or both following the digestion step. In some aspects, the method may not include any heat inactivation step. In some aspects, the heat inactivation step may be conducted at a temperature of at 70 °C or higher, 80 °C or higher, or 90 °C or higher.

[0112] The chelating agent is added in an amount sufficient to inactivate exonuclease activity by inactivation of the enzyme. In some aspects, the addition of the chelating agent added during the DNA precipitation step or as part of the DNA precipitation buffer may be in addition to the chelating agent added to inactivate exonuclease activity. An amount of chelating agent sufficient to inactivate exonuclease activity may be 5 mM to 100 mM, 7.5 mM to 80 mM. 10 mM to 60 mM. 12.5 mM to 40 mM, or 15 mM to 20 mM. In some aspects, the chelating agent for inactivating exonuclease activity is EDTA, (ethylene glycol- bis(P-aminoethyl ether)-N,N,N',N’ -tetraacetic acid) (EGTA), 1,4,7-triazacyclononane- N,N',N"-triacetic (NOTA), 1,4,7, 10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA). cyclohexyl 1,2-diamine tetra-acetic acid (CDTA). NiN'-bis(hydroxybenzyl)- ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA), 1, 4, 8, 1 l-tetraazacyclotetradecane-N,N',N'',N'" -tetraacetic acid (TETA), diethylenetriaminepentaacetic acid (DTP A), aminophenol-N,N,O-triacetate (APTRA), hydroxy ethyl ethylenediaminetriacetic acid (HEDTA), hydroxy ethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA). oxalic acid, ethylenediaminetetra(methylenephosphonic acid) (EDTMP), and derivatives, salts and mixtures thereof. In some aspects, the chelating agent for inactivating exonuclease activity is EDTA.

[0113] In some aspects, the cell-free reaction mixture comprises each of the first and second capping oligonucleotides independently in a ratio of about lx to about 400x, about 5x to about 200x (i.e., fold excess relative to the template DNA source molecule) including anynumber or range within the range of about lx to about 400x 5x to about 200x. In some aspects, the cell-free reaction mixture comprises each of the first and second capping oligonucleotides independently in a ratio of about 1-fold to about 400-fold, about 5-fold to about 200-fold relative to the template DNA source molecule, including any number or range within the range of about 1-fold to about 400-fold or about 5-fold to about 200-fold. In some aspects, the cell-free reaction mixture may comprise each of the first and second capping oligonucleotides independently in a ratio of at least 20-fold excess relative to the template DNA source molecule. In some aspects, the cell-free reaction mixture may comprise each of the first and second capping oligonucleotides independently in a ratio of 20-fold to 400-fold relative to the template DNA source molecule, including any number or range within the range of about 20-fold to about 400-fold. In some aspects, the cell-free reaction mixture comprises each of the first and second capping oligonucleotides independently in a ratio of about 1-fold to 400-fold, 5-fold to 200-fold, 10-fold to 100-fold, 15-fold to 80-fold, 20-fold to 70-fold, 25-fold to 60-fold, 30-fold to 50-fold, or 35-fold to 45-fold excess relative to the template DNA source molecule. More specifically, the cell-free reaction mixture may comprise each of the first and second capping oligonucleotides independently in a ratio of 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30- fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130- fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold. 190-fold, or 200-fold excess relative to the template DNA source molecule.

[0114] In some aspects, the yield of the exonuclease-resistant single stranded template DNA product is 50% or more, 60% or more, 70% or more, 80% or more 90% or more, 95% or more, or 99% or more relative to the total amount of template DNA source molecule after performing the capping reaction for 4 hours or less, 3 hours or less, or 2 hour or less.

[0115] Inactivating the first exonuclease, the second exonuclease, or both may be inactivated by a heat inactivation or a chelating agent inactivation. In some aspects, the heat inactivation is performed at about 40°C to about 99°C, about 45°C to about 95°C, about 50°C to about 90°C, about 60°C to about 80°C, about 65°C to about 75°C. In some aspects, the heat inactivation may be performed at 65°C, 70°C. 75°C, 80°C, 85°C. 90°C, 95°C, or 99°C. In some aspects, the heat inactivation may be performed at 70°C.

[0116] In some aspects, the method comprises cooling the heat inactivated reaction mixture to a temperature from about 20 °C to about 37 °C. about 23 °C to about 35 °C, or about 25 °C before adding the second exonuclease. In some aspects, the cooling step comprises cooling the heat inactivated reaction mixture from about 70°C to about 25°C.

[0117] In some aspects, the heat inactivation is performed for 1 minute or more, or 2 minutes or more, or 5 minutes or more. 10 minutes or more, 60 minutes or less, 30 minutes or less, about 5 minutes to about 20 minutes, about 7 minutes to about 15 minutes, or about 10 minutes. In some aspects, the heat inactivation is performed for 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes.

[0118] In some aspects, the chelating agent inactivation comprises adding a chelating agent at a concentration of 5 mM to 100 mM, 7.5 mM to 80 mM, 10 mM to 60 mM, 12.5 mM to 40 mM, or 15 mM to 20 mM. In some aspects, the chelating agent inactivation comprises adding a chelating agent at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35, mM, 40 mM, 45, mM or 50 mM. In some aspects, the chelating agent may be ethylenediaminetetraacetic acid (EDTA). In some aspects, inactivation of the first exonuclease may be performed by a heat inactivation and inactivation of the second exonuclease may be performed by adding a chelating agent.

[0119] In some aspects, the method comprises cutting the template DNA source molecule using at least one endonuclease that cuts the DNA outside of the recognition site of the at least one endonuclease recognition sequence. The at least one endonuclease may be a Type IIS enzyme.

[0120] The at least one endonuclease recognition sequence may be a recognition sequence for at least one endonuclease selected from Acul, Alw261, Alwl, Bael, BbsI, BbsI-HF, Bbvl, Bccl. BceAI. Bcgl, BciVI. BcoDI. BfuAI. BmrI, Bmsl, Bpil, Bpml, BpuEI, Bsal, Bsal-HF V2, BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-Mutl, Btsl-v2, CspCI, Earl, Ecil, Eco311, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lgul, Mval269I, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, SfaNI, or an isoschizomer thereof. More specifically, in some aspects, the at least one endonuclease recognition sequence may be a recognition sequence for at least one endonuclease selected from Bsal, BsaI-HFv2, BsmBI, BsmBI-v2, Esp3I, BbsI, BbsI-HF, SapI, or isoschizomers thereof.

[0121] In some aspects, the at least one endonuclease may be selected from Acul, Alw261. Alwl, Bael, BbsI, BbsI-HF, Bbvl, Bccl. BceAI. Bcgl. BciVI. BcoDI. BfuAI. BmrI, Bmsl, Bpil, Bpml, BpuEI, Bsal, Bsal-HF V2, BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI, BsmBI- v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-Mutl, Btsl-v2, CspCI, Earl, Ecil, Eco311, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lgul, Mval269I, MboII, Mlyl, Mmel. Mnll, NmeAIII, PaqCI, Piel, SapI, SfaNI. or an isoschizomer thereof. Morespecifically, in some aspects, the at least one endonuclease may be selected from Bsal, Bsal- HFv2, BsmBI, BsmBI-v2, Esp3I, BbsI, BbsI-HF, SapI, or isoschizomers thereof.

[0122] Suitable restriction endonucleases and reactions thereof for certain aspects described herein are disclosed in Pryor, John M., et al. "Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design." PLoS One 15.9 (2020): e0238592, which is incorporated herein by reference in its entirety.

[0123] In some aspects, the at least one endonuclease is added at a concentration of about 1- 10%, about 2-8%, about 2.5-5%, about 2, about 2.5, about 3, or about 3.5% v / v of the cell- free reaction mixture.

[0124] The ligase may be selected from T3 ligase, T4 ligase, T7 ligase. Taq ligase. DNA ligase I, DNA ligase. II, DNA ligase III. DNA ligase VI, Tth DNA ligase, or E. coli DNA ligase. In some aspects, the ligase is T4 ligase.

[0125] In some aspects, the ligase may be added at a concentration of about 0.05-5%, about 0.1-4%, about 0.125-3%, or about 0.1%, about 0.125%, about 0.15%. or about 0.2% v / v of the cell-free reaction mixture. In some aspects, the ligase is added at a concentration of 0.075% to 0. 125% v / v of the cell-free reaction mixture.

[0126] According to the methods of any one of the aspects described herein, the total yield of the exonuclease-resistant single stranded template DNA product may be 25 to 100 mg per liter of the cell-free reaction mixture within 6 hours, 100 to 400 mg per liter of the cell-free reaction mixture within 24 hours, or 200 to 800 mg per liter of the cell-free reaction mixture within 48 hours. In some aspects, the total yield may be based on the exonuclease-resistant single stranded template DNA product that comprises at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000. at least 9000, at least 10,000. at least 11,000. at least 12.000, at least 13,000, at least 14,000, or at least 15,000 nucleotides in length. In some aspects, the total yield may be based on the exonuclease-resistant single stranded template DNA product that comprises about 500 nucleotides to about 15,000 nucleotides, about 1000 nucleotides to about 14,000 nucleotides, about 2000 nucleotides to about 13,000 nucleotides, about 3000 nucleotides to about 12,000 nucleotides, about 4000 nucleotides to about 11,000 nucleotides, or about 5000 nucleotides to about 10,000 nucleotides.

[0127] In some aspects, the exonuclease-resistant single stranded template DNA product may be produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0. 1 L to 12.5 L, 0.25 L to 6.25 L. 0.5 L to 3.5 L, or 0.75 L to 1.5 L. In some aspects, the exonuclease-resistant singlestranded template DNA product may be produced in 1 hour to 48 hours, 2 hours to 24 hours, 3 hours to 18 hours, 4 hours to 12 hours, or 6 hours to 8 hours.

[0128] In some aspects, the exonuclease-resistant single stranded template DNA product may be produced in a single contiguous reaction solution. In some aspects, the exonucleaseresistant single stranded template DNA product may be produced in a reaction tube. In some aspects, the exonuclease-resistant single stranded template DNA product may be produced in a bioreactor.

[0129] In some aspects, the purification step comprises a silica-based purification procedure.

[0130] According to the methods of any one of the aspects described herein, purifying the exonuclease-resistant single stranded template DNA product may comprise or further comprise performing a high-performance liquid chromatography (HPLC) purification step using a HPLC system.

[0131] In the context of the HPLC purification step, a chromatographic column is used to house chromatographic support material (herein resin, support material, or solid phase). The chromatographic support material may be equilibrated in a buffer that is the same or substantially the same as the buffer used to load or inject the exonuclease-resistant single stranded template DNA product onto the HPLC system. A sample comprising the exonuclease-resistant single stranded template DNA product is loaded or injected onto the HPLC system comprising a particular chromatographic column. The column can then be subjected to one or more wash steps using a suitable buffer. Components of a sample that have not adsorbed onto the resin may flow through the column. Components that have adsorbed to the resin may be differentially eluted using one or more appropriate buffers.

[0132] A wash step is typically performed using conditions similar to the load conditions or alternatively by modifying one or more parameters, including, but not limited pH, ionic strength / conduct! vity, or ion pairing agents of the wash in a step.

[0133] In certain aspects, the HPLC purification step may be operated in bind-el ute mode, flowthrough mode, or a hy brid mode. In bind-elute mode, a HPLC column or membrane device is first conditioned with a buffer under conditions where the exonuclease-resistant single stranded template DNA product will adsorb to the resin-based matnx and then may be eluted using a buffer with different parameters, e g. as described above. In the flowthrough mode, a column or membrane device is operated under conditions such that the exonucleaseresistant single stranded template DNA product does not bind to the resin or the membrane. In the hybrid mode the exonuclease-resistant single stranded template DNA product mayexhibit binding to the chromatographic support material and yet can be washed from such material using a buffer that is the same or substantially similar to the loading buffer.

[0134] In some aspects, the HPLC purification step may use anion exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof.

[0135] Examples of anion exchange resins include, but are not limited to, PL-SAX, diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Additional non-limiting examples include: Poros 50PI and Poros 50HQ, which are a rigid polymeric bead with a backbone consisting of cross-linked poly |st rene-di vin l benzene |: Capto Q Impres and Capto DEAE, which are a high flow agarose bead: Toyopearl QAE-550, Toyopearl DEAE-650. and Toyopearl GigaCap Q-650, which are a polymeric base bead; Fractogel® EMD TMAE Hicap, which is a synthetic polymeric resin with a tentacle ion exchanger; Sartobind STIC® PA nano, which is a salt-tolerant chromatographic membrane with a primary amine ligand; Sartobind Q nano.

[0136] Examples of hydrophobic interaction chromatography resins include, but are not limited to, Phenyl Sepharose™ from GE Healthcare or a Phenyl Membrane from Sartorius). Many HIC resins are available commercially. Examples include, but are not limited to, Capto Phenyl, Phenyl Sepharose™ 6 Fast Flow with low or high substitution, Phenyl Sepharose™ High Performance. Octyl Sepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butyl columns (Bio-Rad, California); WP HLPropyl (C3)™ (J. T. Baker, New Jersey); and Toy opearl™ ether, phenyl or butyl (TosoHaas, PA); ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen and Butyl FF HiScreen Octyl FF.

[0137] Examples of hydrophilic interaction chromatography resins, include, but are not limited to, Superdex®, Sephadex®, Superose®, Sephacryl®, Sepharose®, cross-linked agarose, Toyopearl® Ether or Fractogel® EMD BioSEC.

[0138] Examples of size exclusion chromatography resin include, but are not limied to, Toyopearl® size exclusion protein chromatography resins, Superdex® or Sephadex® size exclusion protein chromatography resins, or TSKgel® size exclusion protein chromatography resins.

[0139] In some aspects, the purification step comprises: (i) injecting the exonuclease-resistant single stranded template DNA product onto the HPLC system comprising an anion exchange resin under conditions to bind the exonuclease-resistant single stranded template DNA productto the anion exchange resin; (ii) separating the exonuclease-resistant single stranded template DNA product from impurities using a ramping separation mode, a step- wise separation mode, or a combination thereof; and (iii) eluting the exonuclease-resistant single stranded template DNA product from the HPLC system.

[0140] In some aspects, the cell-free reaction mixture is fdtered prior to the purification step.

[0141] In some aspects, the HPLC purification step may comprise one or more separation modes. The separation modes may be a ramping separation mode, a step-wise separation mode, or a combination thereof. In certain aspects, the HPLC purification step may comprise a step-wise separation mode with two or more steps, three or more, or four or more steps.

[0142] In some aspects, the HPLC purification may comprise running the HPLC system with a mobile phase flow rate of about 1 mL / min to about 200 mL / min, about 5 mL / min to about 100 mL / min, or about 10 mL / min to about 50 mL / min. In some aspects the mobile phase flow rage may be about 1, 2, 3, 4, 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, or 50 mL / min.

[0143] In some aspects, the HPLC purification step may comprise one or more mobile phase buffers. In some aspects, the mobile phase buffers comprise an aqueous or organic solvent composition.

[0144] In some aspects, the one or more mobile phase buffers may comprise a chelating agent. The chelating agent may be EDTA, (ethylene glycol-bis([3-aminoethyl ether)-N,N,N',N’-tetraacetic acid) (EGTA), l,4,7-triazacyclononane-N,N',N"-triacetic (NOTA), 1,4, 7, 10-tetraazacyclododecane-N,N',N'',N' ''-tetraacetic acid (DOTA), cyclohexyl 1,2- diamine tetra-acetic acid (CDTA), NiN'-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexa-acetic acid (TTHA). 1,4,8,11- tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), diethylenetriaminepentaacetic acid (DTP A), aminophenol-N,N,O-triacetate (APTRA), hydroxy ethyl ethylenediaminetriacetic acid (HEDTA), hydroxy ethyliminodiacetic acid (HIDA), nitrilotriacetic acid (NTA), oxalic acid, ethylenediaminetetra(methylenephosphonic acid) (EDTMP). and derivatives, salts and mixtures thereof.

[0145] The chelating agent may be included at a concentration of 0.1 mM to 10 mM, or aboutO.5 mM to about 5 mM, or about 1 mM.

[0146] The mobile phase buffer may comprise one or more salts. In some embodiments, the mobile phase buffer may comprise NaCl, ammonium chloride (NH4CI), or magnesium chloride (MgCh).

[0147] In some aspects, the exonuclease-resistant single stranded template DNA product binds to the an anion exchange resin in a mobile phase buffer comprising less than about 600 mM NaCl or NH4CI, less than about 500 mM NaCl or NH4CI, less than about 250 mM NaCl or NH4CI, less than about 150 mM NaCl or NH4CI, less than about 100 mM NaCl or NH4CI, or less than about 50 mM NaCl or NH4CI. In some aspects, the exonuclease-resistant single stranded template DNA product binds to the an anion exchange resin in a mobile phase buffer comprising less than about 300 mM less than about MgCh, less than about 250 mM MgCh. less than about 125 mM MgCh, less than about 75 mM MgCh, less than about 50 mM MgCh, or less than about 25 mM MgCh. In some aspects, the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer without NaCl, NH4Q, or MgCh.

[0148] In some aspects, the HPLC purification step comprises eluting the exonucleaseresistant single stranded template DNA product from the anion exchange resin using a mobile phase buffer composition comprising a salt. In some aspects, the exonuclease-resistant single stranded template DNA product may be eluted from the anion exchange resin using NaCl, NH4CI, or MgCh. In some aspects, the exonuclease-resistant single stranded template DNA product may be eluted from the anion exchange resin using a mobile phase buffer composition comprising about 0.70 M to about 2.0 M NaCl or NH4CI, about 0.75 M to about 1.5 M NaCl or NH4CI, about 0.9 M to about 1. 1 M NaCl or NH4CI. or about 1 M NaCl or NH4CI. The exonuclease-resistant single stranded template DNA product may be eluted using a mobile phase buffer composition comprising about 0.70 M, 0.75 M, 0.80 M, 0.85 M, 0.90 M, 0.95 M, 1.0 M, 1.05 M, 1.1 M, 1.15 M, 1.20 M, 1.25 M, 1.30 M, 1.35 M, 1.40 M, 1.45 M, or about 1.50 M NaCl orNEUCl. In some aspects, the exonuclease-resistant single stranded template DNA product may be eluted from the anion exchange resin using a mobile phase buffer composition comprising about 0.35 M to about 1.0 M MgCh, about 0.375 M to about 0.75 M MgCh, about 0.45 M to about 0.55 M MgCh, or about 0.5 M MgCh. The exonuclease-resistant single stranded template DNA product may be eluted using a mobile phase buffer composition comprising about 0.35 M. 0.375 M. 0.40 M, 0.45 M, 0.50 M, 0.60 M, 0.65 M. 0.70 M, 0.75 M. 0.80 M, 0.85 M, 0.90 M. 0.95 M, or 1.0 M MgCh.

[0149] In some aspects, the salt concentration, e g., NaCl, NH4CI, MgCh concentration is based on the combination of one or more buffers comprising the mobile phase composition.

[0150] In some aspects, the mobile phase for the anion exchange resin may comprise one or more ion pairing agents. Examples of ion pairing reagents include, but are not limited to, triethylamine, tripropylamine, hexylamine, butylamine, dibutylamine, a tetraalkyd ammoniumsalt (e.g., triethylammonium acetate (TEAA)), and combinations thereof. In some aspects, the ion pairing agents may be present in one or more mobile phase buffers at a concentration of about 0.01 M to about 1 M, about 0.05 M to about 0.75 M, or about 0. 1 M to about 0.5 M.

[0151] In some aspects, the method may further comprise subjecting the purified exonuclease-resistant single stranded template DNA product to a desalting process. In some aspects, the desalting process comprises tangential flow filtration. The purified exonucleaseresistant single stranded template DNA product may be filtered for about 2 to about 20 diafiltration volumes, about 4 to about 10 diafiltration volumes, or about 6 to about 8 diafiltration volumes. In some aspects, the purified exonuclease-resistant single stranded template DNA product may be filtered for about 2, 3, 4. 5, 6, 7, 8, 9, or 10 diafiltration volumes.

[0152] In some aspects, after filtration, the purified exonuclease-resistant single stranded template DNA product has a conductivity less than 20,000 pS / cm or about less than 10,000 pS / cm. In some aspects, after filtration, the purified exonuclease-resistant single stranded template DNA product has a conductivity less than about 1.000 pS / cm. less than about 500 pS / cm. or less than about 100 pS / cm. In some aspects, after filtration, the purified exonuclease-resistant single stranded template DNA product has a conductivity of about deionized water. In some aspects, the purified exonuclease-resistant single stranded template DNA product has a conductivity of about 0.05 pS / cm to about 1,000 pS / cm, about 1 pS / cm to about 500 pS / cm, about 2 pS / cm to about 250 pS / cm, about 5 pS / cm to about 100 pS / cm, or about 10 pS / cm to about 50 pS / cm.

[0153] In some aspects, the purified exonuclease-resistant single stranded template DNA product undergoes a reduction in conductivity (pS / cm) of about 5,000-fold after about 4 to about 6 diafiltration volumes of filtration, about 10,000-fold after about 4 to about 6 diafiltration volumes of filtration, or about 20,000-fold after about 4 to about 6 diafiltration volumes of filtration

[0154] In some aspects, the exonuclease-resistant single stranded template DNA product may be concentrated using tangential flow filtration. In some aspects, the method may further comprise, after concentrating the exonuclease-resistant single stranded template DNA product using tangential flow filtration, freeze-drying the concentrated product.

[0155] In some aspects, the exonuclease-resistant single stranded template DNA product may be produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0. 1 L to 12.5 L, 0.25 L to 6.25 L. 0.5 L to 3.5 L, or 0.75 L to 1.5 L.

[0156] In some aspects, the volume of cell-free reaction mixture injected onto the HPLC system may be about 0. 1 mL to about 250 mL, about 1 mL to about 200 mL, about 5 mL to about 150 mL, about 10 mL to about 100 mL, about 15 mL to about 50 mL, or about 20 mL to about 40 mL.

[0157] In some aspects, the method may further comprise, after purification step (c), dialyzing the eluate comprising the exonuclease-resistant single stranded template DNA product into water, or into a buffer that does not contain salt or chelating agent, e.g., NaCl. NH4CI, MgCL, EDTA, or Tris-HCl, or has a reduced concentration of salt or chelating agent, e.g., NaCl, NH4CI, MgCL, EDTA, or Tris-HCl, to obtain a dialyzed product.

[0158] In some aspects, the method may further comprise, after dialyzing the eluate to obtain the dialyzed product, freeze-drying the dialyzed product.

[0159] In some aspects, the purification step further includes adding DNA-binding beads. Suitable DNA-binding beads may comprise a polystyrene core covered by a magnetite layer, and the magnetite layer is covered by a carboxylate-modified polymer coating. In some aspects, the beads are provided at a ratio of 0.5 to 0.9. 0.6 to 0.8, 0.65 to 0.75, or about 0.7 to the template DNA source molecule). In some aspects, the method may comprise purifying exonuclease-resistant single stranded template DNA product by performing multiple elutions of the beads.Template DNA Source, Double Stranded Template DNA, and Single Stranded Template DNA Product

[0160] The template DNA source molecule of the present disclosure comprises a double stranded template DNA that is processed to produce an exonuclease-resistant single stranded template DNA product. The exonuclease-resistant single stranded template DNA product may be produced by excising the double-stranded template DNA from the template DNA source molecule. According to the methods provided in the present disclosure, the doublestranded template DNA is further processed to produce the exonuclease-resistant single stranded template DNA product.

[0161] The ‘"template DNA source” may comprise at least one endonuclease recognition sequence and at least one template DNA sequence. In some aspects, the template DNA source molecule comprises a linear double stranded DNA, a plasmid DNA, a mini circle DNA, a cosmid DNA, a bacterial artificial chromosome (BAC), a molecular inversion probe, a doggy bone DNA, a cDNA, or a PCR product.

[0162] In some aspects, one or multiple double stranded "‘template DNA precursor” or double stranded ‘"template DNA precursors” may be produced from the same template DNAsource molecule. The double stranded template DNA precursor may be produced by excising the double stranded template DNA precursor from the template DNA source molecule followed by modification with capping oligonucleotides according to the aspects described herein. According to one or more aspects described here, the double stranded template DNA precursor is further processed to produce an exonuclease-resistant single stranded template DNA product.

[0163] In some aspects, the exonuclease-resistant single stranded template DNA product may be suitable to alter or insert at or near a target DNA site.

[0164] The amplification of the template DNA source molecule may be an in vitro or in vivo amplification. In some aspects, the amplification may be bacterial mediated amplification, for example using competent E. coli strains. In some aspects, the amplification is an in vitro amplification. For example, the amplification may be performed by traditional polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), MALBAC method, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HD A), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA).

[0165] In some aspects, the exonuclease-resistant single stranded template DNA product of the disclosure are capable of being inserted, at least in part, into the DNA of either a cycling or non-cycling cell that has been cleaved by gene editing system. For example, exonucleaseresistant single stranded template DNA product may be inserted following cleavage of target site DNA by non-homologous end joining repair (NHEJ) or homology-directed repair (HD).

[0166] In some aspects, the template sequence may comprise an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell. In some aspects, the endogenous sequence may be a genomic sequence of the cell. In some aspects, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some aspects, the endogenous sequence may be a plasmid sequence of the cell. In some aspects, the template sequence has no similarity7to the nucleic acid sequence near the cleavage site. In some aspects, the template or a portion of the template sequence is incorporated. In some aspects, the exonuclease-resistant single stranded template DNA product includes flanking inverted terminal repeat (ITR) sequences, a simian virus 40 (SV40) nuclear localization sequence, or Adeno- Associated Virus ITRs. In some aspects, the exonuclease-resistant single stranded template DNA product includes recognition sequences, or target sites, for DNA binding proteins. In certain aspects, the DNA binding protein may be a transcription factor.

[0167] In some aspects, the template DNA may be suitable to alter or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease.

[0168] In some aspects, the template DNA sequence may correspond to, comprise, or consist of an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell. In some aspects, the endogenous sequence may be a genomic sequence of the cell. In some aspects, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some aspects, the endogenous sequence may be a plasmid sequence of the cell.

[0169] In some aspects, the method comprises obtaining a yield of purified exonucleaseresistant single stranded template DNA product of 80% or more, 85% or more. 90% or more, 95% or more, or 99% or more relative to a total amount of the template DNA source molecule in the cell-free mixture. In some aspects, the method comprises obtaining a yield of purified exonuclease-resistant single stranded template DNA product of 80% or more, 85% or more, 90% or more. 95% or more, or 99% or more relative to a total amount of template DNA source molecule in the cell-free mixture without thermocy cling.

[0170] In some aspects, the exonuclease-resistant single stranded template DNA product may comprise at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000. at least 8000, at least 9000, at least 10,000, at least 11,000, at least 12.000. at least 13.000, at least 14,000, or at least 15,000 nucleotides in length. In some aspects, the exonuclease-resistant single stranded template DNA product may comprise at least 4,000 nucleotides in length. In some aspects, the exonuclease-resistant single stranded template DNA product may be 500 to 20,000 nucleotides in length, including any number or range within the range of 500 to 20,000 nucleotides in length. More specifically, the exonuclease-resistant single stranded template DNA product be about 500 to about 15,000 nucleotides in length, about 1000 to about 14,000 nucleotides in length, about 2000 to about 13,000 nucleotides in length, about 3000 to about 12,000 nucleotides in length, about 4000 to about 11.000 nucleotides in length, or about 5000 to about 10,000 nucleotides in length. In certain aspects, the exonuclease-resistant single stranded template DNA product may be about 500, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10,000, about 11,000, about 12,000, about 13,000, about 14,000. or about 15,000 nucleotides in length.Capping Oligonucleotides

[0171] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises at least 10 nucleotides. In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises 15 to 100, 20 to 80, 25 to 75, 30 to 60, or 40 to 50 nucleotides.

[0172] In some aspects, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified 5 ’-end. The 5’ phosphate may facilitate ligation to the linear doublestranded region (which may comprise a 3 ’-OH group at first end, a second end, or both). The first capping oligonucleotide, the second capping oligonucleotide, or both may comprise a 3’- OH. The 3 ’-OH may facilitate ligation to the linear double-stranded region (which may comprise a 5’ phosphate at first end, the second end, or both).

[0173] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a 5’ overhang, a 3’ overhang, or both. In some aspects, the 5’ overhang, the 3’ overhang, or both comprises 2 to 12, 4 to 10. or 6 to 8 nucleotides. In some aspects, the 5’ overhang. 3’ overhang, or both comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some aspects, the 5’ overhang, 3 ’ overhang, or both comprises 4 nucleotides. In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified overhang end.

[0174] In some aspects, the template sequence may comprise a 3’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides, a 5’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides, or both. The overhang(s) of the template sequence may act as a sticky end that is complimentary to the overhang of the first capping oligonucleotide, the second capping oligonucleotide, or both so the first capping oligonucleotide, the second capping oligonucleotide, or both may efficiently modify an end of the template sequence to form the double stranded template DNA precursor.

[0175] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide may comprise one or more nuclease resistant nucleotides. In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both comprise one or more hairpin structures. In some aspects, the nuclease resistant nucleotides may be selected from 2'-O-methyl nucleotides, 2'- O-methoxy ethyl (MOE) nucleotides, phosphorothioate nucleotides. 2’ -fluoro nucleotides, inverted nucleotides at a 3'-end, at a 5'-end, or both the 3' and the 5'-ends, thiophosphatenucleotides, phosphoroselenoate nucleotides, or selenophosphate nucleotides, locked nucleotides (e.g.. containing a 2'-O, 4'-C methylene bridge), or reversed nucleotide bases.

[0176] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide may comprise one or more functional groups. In some aspects, the functional groups may comprise a G- quadruplex structure, for example G-4 quadruplex, an aptamer, e.g., an RNA aptamer, e.g., an MS2 RNA aptamer, or a C spacer group. In some aspects, the C spacer is a C3 to C12 spacer group. In certain aspects, the C spacer group is a C3 spacer. In some aspects, the functional group is a fluorophore.

[0177] The first capping oligonucleotide, the second capping oligonucleotide, or both the first and second capping oligonucleotide may comprise a stable self-assembling nucleic acid nanostructure, comprising a plurality of oligonucleotides, a plurality of G-quadruplex forming nucleic acids linked to the plurality of oligonucleotides. The G-quadruplex forming nucleic acids may be TAGGGTT, may be a plurality of G-quadruplex stabilizing domains linked to the G-quadruplex forming nucleic acids. In some aspects, the G-quadruplex may be a sequence enriched with guanine, for example containing G-tetrads or G-quartets. In some aspects, the G-quadruplex forming nucleic acids may comprise GG, GGG, GGGG, or repeating units thereof. The self-assembling nucleic acid nanostructure may have an inorganic core, or the organic core may be absent. In some aspects, the G-quadruplex may have an N3'-P5' phosphoramidate linkage or a N3'-P5'thio-phosphoramidate linkage. In some aspects, the second capping oligonucleotide may comprise a closed-end. In some aspects, the second capping oligonucleotide comprises a hairpin structure, an RNA aptamer, e.g., an MS2 RNA aptamer, 2'-O-methyl nucleotides, 2'-O-methoxyethyl (MOE) nucleotides, phosphorothioate nucleotides, 2’-fluoro nucleotides, or inverted nucleotides at a 3'-end, at a 5'-end, or both the 3' and the 5'-ends, a C3 spacer, a G4 quadruplex structure, a phosphate modification, thiophosphate nucleotides, phosphoroselenoate nucleotides, selenophosphate nucleotides, a nuclear localization signal (NLS). In some aspects, the second capping oligonucleotide may comprise a hairpin structure. In some aspects, the hairpin structure is a hairpin loop. In some aspects, the hairpin loop comprises 4 to 40, 6 to 36, 8 to 32, 10 to 28, 12 to 24, 14 to 20, or 16 to 18 nucleotides. In some aspects, the hairpin loop comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0178] In some aspects, the hairpin loop may comprise one or more exonuclease resistant nucleotides according to any of the aspects disclosed herein. In some aspects, 2 to 20, 4 to 18, 6 to 16, 8 to 14, or 10 to 12 nucleotides of the hairpin loop comprise exonuclease resistantnucleotides according to any of the aspects disclosed herein. In some aspects, the hairpin loop may comprise phosphorothioate bonds. In some aspects. 2 to 20, 4 to 18, 6 to 16, 8 to 14, or 10 to 12 nucleotides of the hairpin loop comprise phosphorothioate bonds. In some aspects, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the hairpin loop may comprise phosphorothioate bonds.

[0179] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide are open-ended. In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a duplex oligonucleotide. In some aspects, the duplex oligonucleotide is prehybridized prior to modification of the first end, the second end, or both ends of the template sequence.

[0180] In some aspects, the prehybridized duplex oligonucleotide comprises two strands and wherein each strand of the prehybridized duplex oligonucleotide are different lengths. In some aspects, each strand of the prehybridized duplex oligonucleotide may independently comprises 2 to 200, 4 to 180, 6 to 160. 8 to 140, 10 to 120, 12 to 100. 10 to 80, 12 to 60, 14 to 40. 16 to 20 nucleotides. In some aspects, each strand of the prehybridized duplex oligonucleotide may independently comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0181] In some aspects, the prehybridized duplex oligonucleotide may comprise one or more exonuclease resistant nucleotides according to any of the aspects disclosed herein. In some aspects, 2 to 50, 4 to 40, 6 to 30, 8 to 20, or 10 to 12 nucleotides of one strand of the prehybridized duplex oligonucleotide comprise exonuclease resistant nucleotides according to any of the aspects disclosed herein. In some aspects, 2 to 50, 4 to 40, 6 to 30, 8 to 20. or 10 to 12 nucleotides of one strand of the prehybridized duplex oligonucleotide comprise phosphorothioate bonds. In some aspects, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of one strand of the prehybridized duplex oligonucleotide may comprise exonuclease resistant nucleotides, e.g., phosphorothioate bonds. In some aspects, the prehybridized duplex oligonucleotide comprises the phosphorothioate bonds positioned on the top or bottom strand forming a 3’ overhang of phosphorothioate bonded nucleotides.

[0182] In some aspects, the exonuclease-resistant single stranded template DNA product comprises no phosphorothioate modifications.Modified Nucleic acids

[0183] In some aspects, the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide may comprise one or more modified nucleic acids.

[0184] In some aspects, the nucleic acid has undergone a chemical or biological modification to render it more stable. Exemplary modifications to a nucleic acid include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase "chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA or nucleic acids, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such RNA or nucleic acid molecules).

[0185] In some aspects of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some aspects, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. In some aspects, a backbone modification may render the nucleic acid resistant to nucleases.

[0186] Examples of modified nucleic acid phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates, thiophosphates, selenophosphates, and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates). sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some aspects, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0187] The present disclosure also relates to an exonuclease-resistant single stranded template DNA product prepared according to any one of the aspects disclosed herein. In some aspects, the exonuclease-resistant single stranded template DNA product is resistant to Dnase. In some aspects, the exonuclease-resistant single stranded template DNA product has a frictional ratio of 4 to 20, 6 to 18, 8 to 16, or 10 to 12, as measured by analytical ultracentrifugation.

[0188] The present disclosure also provides a lipid nanoparticle comprising an exonucleaseresistant single stranded template DNA product prepared according to any of the aspects described herein. In certain aspects, a lipid nanoparticle may comprise (a) an ionizable lipid; (b) one or more helper lipids; (c) one or more neutral lipids; (d) optionally, a pegylated lipid; and (e) an exonuclease-resistant single stranded template DNA product, wherein the exonuclease-resistant single stranded template DNA product comprises a first end comprising a first modification and a second end comprising a second modification, wherein the first modification and the second modification are exonuclease resistant, and wherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15,000 nucleotides in length.

[0189] In certain aspects, a lipid nanoparticle may comprise (a) an ionizable lipid; (b) one or more helper lipids; (c) one or more neutral lipids; (d) optionally, a pegy lated lipid; and (e) an exonuclease-resistant single stranded template DNA product, wherein the exonucleaseresistant single stranded template DNA product, comprises a first end comprising a first modification and a second end comprising a second modification, wherein the first modification, the second modification, or both the first and second modification comprises a linear sequence comprising one or more phosphorothioate bonds or an exonuclease resistant functional group, wherein the exonuclease resistant functional group does not comprise biotinylation.

[0190] The present disclosure also provides a method comprising encapsulating an exonuclease-resistant single stranded template DNA product in a lipid composition. In some aspects, the lipid composition further encapsulates one or more peptides, polypeptides, one more cationic salts, one or more anionic salts, one or more additional polynucleotides.Lipid Compositions

[0191] Lipid composition means a composition that includes, but is not limited to, a composition that comprises lipid vesicles, micelles, microspheres, liposomes, lipoplexes, or lipid nanoparticles. In some aspects, the lipid composition may be a lipid composition described in WO2022221695A1, WO2022221697A1, WO2022221697A1.W02020118041AL W02020072605A1, or WO2019067992AL which are incorporated herein by reference in their entireties.

[0192] In some aspects, lipid compositions may include at least one of a helper lipid, a neutral lipid, a conjugated lipid, or an ionizable lipid. In some aspects, lipid compositions may include at least one helper lipid, at least one neutral lipid, at least one ionizable lipid, and at least one conjugated lipid.

[0193] Compositions containing exonuclease-resistant single stranded template DNA product or lipid compositions thereof may be in various forms, including, but not limited to, particle forming delivery agents including microparticles, nanoparticles and transfection agents that are useful for delivering various molecules to cells. Compositions that are effective at transfecting or delivering biologically active agents to cells may be used. For example, Lipofectamine reagents and any derivatives thereof may be used.

[0194] Intracellular delivery of the exonuclease-resistant single stranded template DNA product may be facilitated by use of carrier systems including polymers, such as a polymer of glucosamine, e.g., polyethylenimine (PEI), polyamidoamine and polypropylamine dendrimers, polyallylamine, cationic dextran, chitosan, various proteins and peptides, amphipathic molecules, or by physical association or chemical modification of the construct, for example by the physical assembly or chemical ligation to cell penetrating peptides (CPPs) or cholesterol molecules. In some aspects, the delivery agent may comprise lipidoid-based formulations allowed for localized and systemic delivery.

[0195] In certain aspects, the formulations include one or more cell penetration agents, e.g., transfection agents. In one aspect, a modified messenger RNA (mmRNA) is mixed or admixed with a transfection agent (or mixture thereof) and the resulting mixture is employed to transfect cells. Exemplary and non-limiting transfection agents are cationic lipid compositions, particularly monovalent and polyvalent cationic lipid compositions, more particularly LIPOFECTIN®, LIPOFECTACE®, LIPOFECTAMINE™, CELLFECTIN®, DMRIE-C. (3- -dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium N-(2- hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l-propaniminium bromide (DMRIE), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N-(2-(2,5-Bis((3-aminopropyl)amino)pentanamido)ethyl)-N,N-dimethyl-2,3-bis((Z)-octadec-9-en-l- yloxy)propan-l-aminium chloride tetrahydrochloride (DOSPA), and 1.3-di-oleoyloxy-2-(6- carboxy-spermyl)-propylamide (DOSPER), and dendrimer compositions, particularly G5-G 10 dendrimers, including dense star dendrimers, P AMAM dendrimers, grafted dendrimers, and dendrimers known as dendrigrafts.Ionizable Lipids

[0196] "‘Ionizable lipids” suitable for use in a lipid composition of the disclosure include, for example, one or more "cationic" and / or amino (ionizable) lipids. As used herein, cationic and / or ionizable lipids include, for example, amine- containing lipids that can be readily protonated at a suitable pH, for example physiological pH. In some aspects, the ionizable / cationic lipid may be selected from, but not limited to, a cationic lipid described in WO2022221695A1, WO2022221697A1, WO2022221697A1, W02020118041 Al, W02020072605A1, or WO2019067992A1, which are incorporated herein by reference in their entireties.Conjugated Lipids

[0197] “Conjugated lipids” suitable for use in a lipid composition of the disclosure include, for example, lipids that inhibit aggregation of particles. In some aspects, the conjugated lipid comprises a lipid conjugated to one or more naturally occurring or non-naturally occurring amino acids. For example, in some aspects the conjugated lipid may comprise a polypeptide conjugate. In some aspects, the conjugated lipid comprises a polyalkyl, polyalkylene, or polyalkyleneoxide group. In some aspects, the conjugated lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. In some aspects, the alkyd chain length comprises about CIO to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain lengths may be symmetrical or asymmetric.

[0198] In some aspects, the conjugated lipid may be a PEG lipid. In some aspects, the conjugated lipid comprises a diacylglycerol and PEG group. In some aspects, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-di myristoylglycerol (PEG- DMG) (catalog # GM-020 from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG- distearoylglycerol (PEG-DSPE) (catalog # DSPE-020CN, NOF, Toky o, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide. PEG-dipalmitoylglycamide, and PEG- distearoylglycamide. PEG-cholesterol (l-[8’-(Cholest-5-en-3[beta]-oxy)carboxamido-3?,6’- dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- ditetradecoxylbenzyl-[omega]-methyl-poly(ethyleneglycol)ether), 1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (PEG2k-DMPE), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene gly col-2000 (PEG2k-DMG), 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (cat. #8801200 from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn- glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), polyethylene gly col)-2000-dimethacry late (PEG2k-DMA), and l,2-distearyloxypropyl-3- amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In certain such aspects, the PEG lipid may be PEG2k-DMG. In some aspects, the PEG lipid may be PEG2k-DSG. In other aspects, the PEG lipid may be PEG2k-DSPE. In some aspects, the PEG lipid may be PEG2k-DMA. In yet other aspects, the PEG lipid may be PEG2k-C- DMA. In certain aspects, the PEG lipid may be compound S027, disclosed in W02016 / 010840 (paragraphs

[0240] to [00244J ). In some aspects, the PEG lipid may be PEG2k-DSA. In other aspects, the PEG lipid may be PEG2k-Cn. In some aspects, the PEG lipid may be PEG2k-Ci4. In some aspects, the PEG lipid may be PEG2k-Ci6. In some aspects, the PEG lipid may be PEG2k-Cig.

[0199] In some aspects, the PEG lipid includes a glycerol group. In some aspects, the PEG lipid includes a dimyristoylglycerol (DMG) group. In some aspects, the PEG lipid comprises PEG-2L. In some aspects, the PEG lipid is a PEG-DMG. In some aspects, the PEG lipid is a PEG-2k-DMG. In some aspects, the PEG lipid is 1,2-dimyristoyl- rac-glycero-3 -methoxy poly ethylene glycol- 2000. In some aspects, the PEG-2k-DMG is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.Neutral Lipids

[0200] “Neutral lipids” suitable for use in a lipid composition of the disclosure include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC). phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC),dimyristoylphosphatidylcholine (DMPC), l-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl phosphatidylcholine (PMPC). l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1- stearoyl-2-palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In certain aspects, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE). or distearoylphosphatidylcholine (DSPC).Helper Lipids

[0201] “Helper lipids” include steroids, sterols, and alkyl resorcinols. In some aspects, helper lipids may include alkyl resoreinol, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, cholesterol -based derivatives, for example cholest-5-en-3[3-ol-based denvatives, cholesterin. or cholesteryl alcohol. In some aspects, helper lipids suitable for use in the present disclosure include, but are not limited to, 5- heptadecylresorcinol, and cholesterol hemisuccinate.Cargo

[0202] In some aspects, the lipid composition further comprises one or more components in addition to the exonuclease-resistant single stranded template DNA product. In some aspects one or more additional components may be delivered concurrently with or separately from the exonuclease-resistant single stranded template DNA product.

[0203] The cargo delivered via a lipid composition may be a biologically active agent. In certain aspects, the cargo is or comprises one or more biologically active agent, such as mRNA, gRNA, expression vector, template nucleic acid, RNA-guided DNA-binding agent, antibody (e.g. , monoclonal, chimeric, humanized, nanobody, and fragments thereof etc ), cholesterol, drug, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, low molecular weight drug, vitamin, co-factor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA:RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid and other ty pes of vectors,and small nucleic acid molecule. RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA). short hairpin RNA (shRNA) and "self-replicating RNA" (encoding a replicase enzyme activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), and iRNA (asymmetrical interfering RNA). The above list of biologically active agents is exemplary only, and is not intended to be limiting. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.

[0204] The cargo delivered via the lipid composition may be an RNA, such as an mRNA molecule encoding a protein of interest. For example, an mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA-binding agent, or a Cas nuclease is included. LNP compositions that include a Cas nuclease mRNA, for example a Class 2 Cas nuclease mRNA that allows for expression in a cell of a Class 2 Cas nuclease such as a Cas9 or Cpfl protein are provided. Further, the cargo may contain one or more gRNAs or nucleic acids encoding gRNAs. A template nucleic acid, e g., for repair or recombination, may also be included in the composition or a template nucleic acid may be used in the methods described herein. In a sub-embodiment, the cargo comprises an mRNA that encodes a Streptococcus pyogenes Cas9. optionally and an S. pyogenes gRNA. In a further subembodiment, the cargo comprises an mRNA that encodes a Neisseria meningitidis Cas9, optionally and an Nme (Neisseria meningitidis) gRNA.

[0205] In some aspects, the biologically active agent comprises a gene editing system. In certain aspects, the gene editing system comprises a DNA nuclease or an RNA-guided DNA nuclease. In certain aspects the gene editing system may comprise a CRISPR / Cas system; Tth Argonaute (TtAgo); zinc finger nuclease (ZFN) system; ARCUS nuclease system; megaTALs; and the transcription activator-like effector nuclease (TALEN) system.

[0206] As used herein, ‘"gene editing system” means an engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, or SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as engineered ZFN, TALENs, megaTALs, meganucleases (mns, also termed homing endonucleases), including an ARC nuclease of the ARCUS gene editing system, or using a guided system, for example, TtAgo or CRISPR / Cas system with an engineered guide DNA or RNA, respectively, to guide specific cleavage or nicking of a target DNA sequence.

[0207] In certain aspects, the disclosed compositions comprise an mRNA encoding an RNA- guided DNA-binding agent, such as a Cas nuclease. In particular aspects, the disclosed compositions comprise an mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9. In some aspects, RNA-guided DNA-binding agents include Cas cleavases / nickases and inactivated forms thereof (“dCas DNA-binding agents’"). “Cas nuclease”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA-binding agents. Cas cleavases / nickases and dCas DNA-binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas 10, Csml, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a “Class 2 Cas nuclease"’ is a single-chain polypeptide with RNA-guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA-binding agents, in which cleavase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e g., N497A, R661A, Q695A, Q926A variants). HypaCas9 (e.g., N692A. M694A, Q695A, H698A variants). eSPCas9(1.0) (e.g., K810A. K1003A. R1060A variants), and eSPCas9(l. 1) (e.g.. K848A, K1003A, R1060 A variants) proteins and modifications thereof. Cpfl protein, Zetsche et al, Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpfl sequences of Zetsche are incorporated by reference in their entirety. See, e.g.. Zetsche, Tables 2 and 4. See, e.g., Makarova et al, Nat Rev Microbiol, 13(11): 722- 36 (2015); Shmakov et al, Molecular Cell, 60:385-397 (2015).

[0208] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a gRNA together with an RNA-guided DNA-binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA-binding agent (e.g., Cas9). In some aspects, the gRNA guides the RNA-guided DNA-binding agent such as Cas9 to a target sequence, and the gRNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.

[0209] In some aspects of the present disclosure, the cargo for the LNP composition includes at least one gRNA comprising guide sequences that direct an RNA-guided DNA- binding agent, which can be a nuclease (e g., a Cas nuclease such as Cas9), to a target DNA. The gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some aspects, a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease. In some aspects, the gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex such as a CRISPR / Cas9complex. In some aspects, the CRISPR / Cas complex may be a Type- II CRISPR / Cas9 complex. In some aspects, the CRISPR / Cas complex may be a Type-V CRISPR / Cas complex, such as a Cpfl / gRNA complex. Cas nucleases and cognate gRNAs may be paired.

[0210] The gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR / Cas system.

[0211] “Guide RNA”, “gRNA”, and “guide” are used herein interchangeably to refer to a cognate guide nucleic acid for an RNA-guided DNA-binding agent. Guide RNAs can include modified RNAs as described herein. A gRNA may be either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also know n as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA).

[0212] “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally- occurring sequence, or a trRNA sequence with modifications or variations compared to naturally- occurring sequences.

[0213] As used herein, a “guide sequence” refers to a sequence within a gRNA that is complementary to a target sequence and functions to direct a gRNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding agent. A “guide sequence” may also be referred to as a “targeting sequence,” or a “spacer sequence.” A guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21 -, 22-, 23-, 24-, or 25 -nucleotides in length. In some aspects, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some aspects, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some aspects, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other aspects, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3. or 4 mismatches, w here the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some aspects, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some aspects, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.

[0214] Target sequences for RNA-guided DNA-binding proteins such as Cas proteins include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse compliment), as a nucleic acid substrate for a Cas protein is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be ’‘complementary to a target sequence”, it is to be understood that the guide sequence may direct a gRNA to bind to the reverse complement of a target sequence. Thus, in some aspects, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0215] The length of the targeting sequence may depend on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may comprise 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, 35, 40, 45, 50, or more than 50 nucleotides in length. In some aspects, the targeting sequence length is 0, 1, 2, 3, 4. or 5 nucleotides longer or shorter than the guide sequence of a naturally-occurring CRISPR / Cas system. In certain aspects, the Cas nuclease and gRNA scaffold will be derived from the same CRISPR / Cas system. In some aspects, the targeting sequence may comprise or consist of 18-24 nucleotides. In some aspects, the targeting sequence may comprise or consist of 19-21 nucleotides. In some aspects, the targeting sequence may comprise or consist of 20 nucleotides.

[0216] In some aspects, the sgRNA is a '‘Cas9 sgRNA” capable of mediating RNA- guided DNA cleavage by a Cas9 protein. In some aspects, the sgRNA is a “Cpfl sgRNA” capable of mediating RNA-guided DNA cleavage by a Cpfl protein. In certain aspects, the gRNA comprises a crRNA and tracr RNA sufficient for forming an active complex with a Cas9 protein and mediating RNA-guided DNA cleavage. In certain aspects, the gRNA comprises a crRNA sufficient for forming an active complex with a Cpfl protein and mediating RNA- guided DNA cleavage. See Zetsche 2015.

[0217] Certain aspects of the invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNA described herein. A “guide RNA nucleic acid” is used herein to refer to a gRNA (e g., an sgRNA or a dgRNA) and a gRNA expression cassette, which is a nucleic acid that encodes one or more gRNAs.

[0218] In certain aspects, a guide RNA nucleic acid and a Class 2 Cas nuclease mRNA, are provided in a ratio of the mRNA to the guide RNA nucleic acid from about 2: 1 to about 1 :4 by weight, or about 1 : 1 by weight.

[0219] In certain aspects, the gRNA is a modified gRNA, for example the modified gRNA comprises a modification at one or more of the first five nucleotides at a 5’ end, or the modified gRNA comprises a modification at one or more of the last five nucleotides at a 3’ end, or both.

[0220] In certain aspects, the gene editing system, for example a TtAgo system, may comprise one or more single stranded guide DNA molecules that are complimentary the DNA sequence to be cleaved or nicked. In some aspects, the guide DNA molecules are phosphorylated on the 5’-end. In some aspects, the guide DNA may be 10 to 20 nucleotides, 12 to 18 nucleotides, or 14 to 16 nucleotides in length.

[0221] In certain aspects, the lipid compositions may comprise modified nucleic acids, including modified RNAs. Modified nucleosides or nucleotides can be present in an RNA. for example a gRNA or mRNA. A gRNA or mRNA comprising one or more modified nucleosides or nucleotides, for example, is called a “modified” RNA to describe the presence of one or more non-naturally and / or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues.

[0222] In some aspects, a modified RNA is synthesized with a non-canomcal nucleoside or nucleotide, here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g. , replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g. , replacement, of a constituent of the ribose sugar, e.g. , of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary' backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3' end or 5' end of the polynucleotide, e.g. , removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications may comprise a sugar and / or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification). Certain aspects comprise a 5' end modification to an mRNA, gRNA, or nucleic acid. Certain aspects comprise a modification to an mRNA, gRNA, or nucleic acid. Certain aspects comprise a 3' end modification to an mRNA, gRNA, or nucleic acid. A modified RNA can contain 5' end and 3' end modifications. A modified RNA can contain one or more modified residues at non-terminal locations. In certain aspects, a gRNAincludes at least one modified residue. In certain aspects, an mRNA includes at least one modified residue. Unmodified nucleic acids can be prone to degradation by, e.g.. intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the RNAs (e.g. mRNAs, gRNAs) described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability’ toward intracellular or serum-based nucleases. In some aspects, the modified RNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term '‘innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0223] In some aspects, LNP compositions are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethylether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. A pharmaceutically acceptable buffer, e.g., for in vivo administration of a lipid composition, e.g., an LNP composition, may be used. In certain aspects, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 6.5. In certain aspects, a buffer is used to maintain the pH of the composition comprising LNPs at or above pH 7.0. In certain aspects, the composition has a pH ranging from about 7.2 to about 7.7. In additional aspects, the composition has a pH ranging from about 7.3 to about 7.7 or ranging from about 7.4 to about 7.6. In further aspects, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of a composition may be measured with a micro pH probe. In certain aspects, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as, for example, sucrose. In certain aspects, the composition may comprise tris saline sucrose (TSS). In certain aspects, the LNP composition may include about 1, 2. 3, 4, 5, 6, 7, 8, 9. or 10% cryoprotectant. In certain aspects, the LNP composition may include about I. 2, 3, 4. 5, 6, 7.8, 9, or 10% sucrose. In some aspects, the LNP composition may include a buffer. In some aspects, the buffer may comprise a phosphate buffer (PBS), a Tris buffer, a citrate buffer, and mixtures thereof. In certain exemplary aspects, the buffer comprises NaCl. In certain aspects, the buffer lacks NaCl. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may range from about 40 mM to about 50 mM. In someaspects, the amount of NaCl is about 45 mM. In some aspects, the buffer is a Tris buffer. Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may range from about 40 mM to about 60 mM. In some aspects, the amount of Tris is about 50 mM. In some aspects, the buffer comprises NaCl and Tris. Certain exemplary7aspects of the LNP compositions contain 5% sucrose and 45 mMNaCl in Tris buffer. In other exemplary aspects, compositions contain sucrose in an amount of about 5% w / v, about 45 mM NaCl. and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts may be varied such that the osmolality of the overall composition is maintained. For example, the final osmolality may be maintained at less than 450 mOsm / L. In further aspects, the osmolality is between 350 and 250 mOsm / L. Certain aspects have a final osmolality of 300 + / - 20 mOsm / L or 310 + / - 40 mOsm / L.

[0224] In certain aspects, the disclosure relates to a method of delivering a biologically active agent to a cell, comprising contacting a cell with an LNP composition described herein.

[0225] In certain aspects, the disclosure relates to a method of cleaving DNA, comprising contacting a cell with an lipid composition described herein. In certain aspects, the cleaving step comprises introducing a single stranded DNA nick. In other aspects, the cleaving step comprises introducing a double-stranded DNA break. In certain aspects, the LNP composition comprises a Class 2 Cas mRNA and a gRNA nucleic acid. In certain aspects, the methods further comprise introducing at least one exonuclease-resistant single stranded template DNA product into the cell.

[0226] In certain aspects, the disclosure relates to any method of gene editing described herein, comprising administering the LNP composition to an animal, for example a human. In certain aspects, the method comprises administering the LNP composition to a cell, such as a eukaryotic cell, and in particular a human cell. In some aspects, the cell is a type of cell useful in a therapy, for example, adoptive cell therapy (ACT). Examples of ACT include autologous and allogeneic cell therapies. In some aspects, the cell is a stem cell, such as a hematopoietic stem cell, an induced pluripotent stem cell, or another multipotent or pluripotent cell. In some aspects, the cell is a stem cell, for example, a mesenchymal stem cell that can develop into a bone, cartilage, muscle, or fat cell. In some aspects, the stem cells comprise ocular stem cells. In certain aspects, the cell is selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbal stem cells (LSCs), tissue-specific primary' cells or cells derived therefrom (TSCs), induced pluripotent stem cells (iPSCs). ocular stem cells,pluripotent stem cells (PSCs), embry onic stem cells (ESCs), and cells for organ or tissue transplantations.

[0227] In certain aspects, the cell is a liver cell. In other aspects, the cell is an immune cell, for example, a leukocyte or a lymphocyte, a lymphocyte, a T cell, a B cell, an NK cell, an activated T cell or a non- activated T cell.

[0228] The present disclosure also provides a method for inserting a custom DNA template into a cell comprising introducing an exonuclease-resistant single stranded template DNA product into a cell comprising a) producing the exonuclease-resistant single stranded template DNA product by the method of any one of aspects described herein; b) contacting the cell with the exonuclease-resistant single stranded template DNA product and a gene editing system; and c) inserting at least a part of the template sequence from the exonucleaseresistant single stranded template DNA product into DNA of the cell that has been cleaved by the gene editing system.

[0229] In some aspects, the cell is a non-cycling cell. In some aspects, the cell is a cycling cell. In some aspects, the method for inserting a custom DNA template into a cell further comprises performing non-homologous end joining (NHEJ) repair or homology directed (HD) repair in the cell. In some aspects, the template DNA source molecule is >1 kB, >2 kB, >3 kB, >4 kB, >5 kB, >6 kB, >7 kB, >8 kB, >9 kB, or >10 kB in size. In some aspects, the exonuclease-resistant single stranded template DNA product is encapsulated in a lipid nanoparticle. In some aspects, the inserting is performed in vitro, is performed ex vivo, or is performed in vivo.

[0230] In some aspects, the exonuclease-resistant single stranded template DNA product may be used to alter or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA-binding protein such as a Cas nuclease, e.g., a Class 2 Cas nuclease. In some aspects, the methods comprise introducing the exonuclease-resistant single stranded template DNA product into a cell. In some aspects, a single exonuclease-resistant single stranded template DNA product may be provided. In other aspects, two or more exonuclease-resistant single stranded template DNA product may be provided. For example, different exonucleaseresistant single stranded template DNA product may be provided to edit a single gene in a cell, or two different genes in a cell.

[0231] In some aspects, the exonuclease-resistant single stranded template DNA product may be used in homologous recombination. In some aspects, the homologous recombination may result in the integration of the exonuclease-resistant single stranded template DNA product or a portion of the template sequence into the target nucleic acid molecule. In other aspects, thetemplate may be used in homology-directed repair, which involves DNA strand invasion at the site of the cleavage in the nucleic acid. In some aspects, the homology-directed repair may result in including the exonuclease-resistant single stranded template DNA product in the edited target nucleic acid molecule. In yet other aspects, the template may be used in gene editing mediated by non-homologous end joining. In some aspects, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some aspects, the exonuclease-resistant single stranded template DNA product or a portion of the exonucleaseresistant single stranded template DNA product is incorporated. In some aspects, the exonuclease-resistant single stranded template DNA product sequence may correspond to, comprise, or consist of an endogenous sequence of a target cell. It may also or alternatively correspond to, comprise, or consist of an exogenous sequence of a target cell. In some aspects, the endogenous sequence may be a genomic sequence of the cell. In some aspects, the endogenous sequence may be a chromosomal or extrachromosomal sequence. In some aspects, the endogenous sequence may be a plasmid sequence of the cell.

[0232] Intracellular delivery of the exonuclease-resistant single stranded template DNA product may be used or administered in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By "in combination with," it is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and / or on a time schedule determined for that agent. In some aspects, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability. reduce and / or modify their metabolism, inhibit their excretion, and / or modify their distribution within the body.

[0233] Delivery of the exonuclease-resistant single stranded template DNA product may be in the form of an injectable preparation. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils may be employedas a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0234] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0235] Delivery of the exonuclease-resistant single stranded template DNA product may be in the form of a parenteral preparation. Parenteral formulations are typically aqueous or oily solutions or suspensions. Where the formulation is aqueous, excipients such as sugars (including but not restricted to glucose, mannitol, sorbitol, etc.) salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated with a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water (WFI).

[0236] As shown in FIG. 5, double stranded template DNA precursors A. to E. are postligation products obtained from an excised template sequence modified with a capping oligonucleotide on each end to form the double stranded template DNA precursor. Double stranded template DNA precursors A. and B. have a capping oligonucleotide comprising a phosphorothioate modified hairpin on one end and a capping oligonucleotide comprising a double stranded phosphorothioate modified prehybridized duplex oligo on the other end. In A. and B. the phosphorothioate bonds are positioned on the top or bottom strand of the prehybrized duplex forming a 3’ overhang of phosphorothioate bonded nucleotides. Double stranded template DNA precursors A. and B. are then processed by T7 exonuclease digestion in the 5' to 3’ direction of only the bottom strand (A.) or only the top strand (B.) to form an exonuclease-resistant single stranded template DNA product that corresponds to the top strand of the double stranded template DNA precursor (A.) or the bottom strand of the double stranded template DNA precursor (B.).

[0237] Double stranded template DNA precursor C. is obtained by ligating an excised template sequence with capping oligonucleotides comprising a double stranded phosphorothioate modified prehybridized duplex oligo on each end such that the 3’ overhang of phosphorothioate bonded nucleotides is formed on both the top strand and bottom strand of the double stranded template DNA precursor. T7 exonuclease digests a strand in the 5’ 3’ direction if the other strand has a 3’ overhang. As a result, further processing of the doublestranded template DNA precursor C. by T7 exonuclease digestion in the 5’ to 3’ direction on both the top strand and bottom strand results in degradation of both strands.

[0238] Double stranded template DNA precursors D. and E. have a capping oligonucleotide comprising a double stranded phosphorothioate modified prehybridized duplex oligo on each end such that an overhang of phosphorothioate bonded nucleotides is formed on either the top strand or bottom strand, but not both, of the double stranded template DNA precursor. For example, the top strand has both a 5‘ and 3’ overhang of phosphorothioate bonded nucleotides (D.) or the bottom strand has both a 3’ and 5’ overhang of phosphorothioate bonded nucleotides (E.). Double stranded template DNA precursors D. and E. are then processed by T7 exonuclease digestion in the 5' to 3’ direction of only the bottom strand (D.) and only top strand (E.) to form an exonuclease-resistant single stranded template DNA product that corresponds to the top strand of the double stranded template DNA precursor (D.) or the bottom strand of the double stranded template DNA precursor (E.).

[0239] As shown in FIG. 6, a template DNA source molecule, e.g., a plasmid, comprising a template sequence and non-template DNA vector sequence is digested with an endonuclease to excise the template sequence from the template DNA source molecule resulting in a double stranded excised template sequence and a leftover vector sequence. A capping oligonucleotide comprising a hairpin structure and a capping oligonucleotide comprising a double stranded phosphorothioate modified prehybridized duplex oligo are added in a molar excess over the starting amount of plasmid to modify each end of the template sequence and prevent or reduce religation of the template sequence and left over vector with the addition of T4 ligase. The phosphorothioate bonds are positioned on the top strand of the double stranded phosphorothioate modified prehybridized duplex forming a 3’ overhang of phosphorothioate bonded nucleotides. The resulting double stranded template DNA precursor may then be processed by T7 exonuclease digestion in the 5’ to 3’ direction of only the bottom strand to form an exonuclease-resistant single stranded template DNA product. The leftover vector is further digested using Exonuclease III prior to purification of the exonuclease-resistant single stranded template DNA product.EXAMPLES

[0240] The following examples are intended to exemplify the present disclosure and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples form non-limiting parts of the present disclosure.

[0241] Example 1: Scalable exonuclease-resistant single stranded template DNA product preparation

[0242] Exonuclease-resistant single stranded template DNA product was prepared by excision of a template sequence (3.9kbp in length) from a plasmid and ligation of the excised template sequence with either a modified or an unmodified Left Hairpin (LHP). The unmodified LHP contained no phosphorothioate modifications while the modified version contained four phosphorothioate modifications in the loop portion of the hairpin loop as shown in Table I.

[0243] Table I. Exemplary sequences of oligonucleotides.

[0244] Specifically, the template sequence was comprised in a plasmid sequence (Genscript) and flanked by two Esp3I restriction enzyme cut sites. Plasmids (18.5 micrograms) were combined with Esp3I (NEB, Cat. R0734B, 5 microliters), T4 ligase (Promega, Cat. M1794, 1.25 microliters), and either 2.5 microliters of 100 micromolar unmodified LHP (mixture 1) or 2.5 microliters of 100 micromolar modified LHP (mixture 2), in 0.5X T4 ligase buffer (Promega, Cat. C126A). Then, 12 microliters of Buffer 4 (NEB, Cat. B7004S), and 10 microliters of T7 exonuclease (NEB, Cat. M0263L) were added to each reaction mixture to digest the antisense strand of the double stranded template DNA precursor in order to produce the exonuclease-resistant single stranded template DNA product. During the T7 exonuclease digestion, 1 microliter samples were collected and inactivated with 0.5 microliters of 0.5M EDTA (Invitrogen, Cat. AM9260G) at various timepoints to determine digestion progress. An agarose gel (Lonza, Cat. 57023) of samples from each mixture at 5. 10, 15, 20 and 30 minutes of digestion reveals two main product bands as shown in FIG. 1A; one band corresponding to a long exonuclease-resistant single stranded template DNA product and the other band corresponding to a large byproduct. There was no significant difference between the bands observed with the use of the modified LHP (mixture 2) and the bands observed with the use of the unmodified LHP (mixture 1).

[0245] In a second experiment, long exonuclease-resistant single stranded template DNA product was prepared with the template DNA source molecule and the modified LHP as described above, and an open-ended phosphorothioate modified prehybridized duplex oligonucleotide consisting of strands “duplex strand A” and “duplex strand B” as listed in Table I. The phosphorothioate modified prehybridized duplex oligonucleotide was prepared by incubating 100 micromolar of duplex strand A in molecular biology grade water (Coming, Cat. 46-000-CI) and 100 micromolar of duplex strand B in molecular biology grade water with IM magnesium chloride solution (Sigma, Cat. M1028-100ML) in a volume ratio of 1 : 1 :0.02 overnight at room temperature.

[0246] The reaction mixture was prepared as follows by adding reagents in the following order. First, 18.5 micrograms of plasmid (1 mg / mL) were pipetted into a 2.0 mL DNA LoBind tube (Eppendorf, Cat. 022431048), then 65 microliters of water, 5 microliters of Promega T4 ligase buffer (Promega, Cat. Cl 26 A), 5 microliters of the prehybridized phosphorothioate modified prehybridized duplex oligonucleotide, 2.5 microliters of the modified LHP. 5 microliters of Esp3I (NEB, Cat. R0734B). and 1.25 microliters of T4 ligase (Promega, Cat. M1794) were added. The reaction mixture was incubated at 37°C for 90 minutes. The mixture was then incubated for 30 minutes at 16°C to allow- for ligation of the modified LHP and the phosphorothioate modified prehybridized duplex oligonucleotide to the template sequence, thereby producing a double stranded template DNA precursor of the final exonuclease-resistant single stranded template DNA product. Following this ligation. 11 microliters of Buffer 4 (NEB, Cat. B7004S) and 10 microliters of T7 exonuclease (NEB, Cat. M0263L) w ere added to digest the anti-sense template strand and to produce a exonucleaseresistant single stranded template DNA product with both ends bearing protective phosphorothioate modifications, i.e., the exonuclease-resistant single stranded template DNA product. An agarose gel (Lonza, Cat. 57023) of samples collected either after the enzyme cutting step, after the T4 ligation step, or after the T7 digestion step is shown in FIG. IB.

[0247] Example 2: Digestion of leftover vector using two exonucleases

[0248] A double stranded template DNA precursor of exonuclease-resistant single stranded template DNA product was prepared by excision of a template sequence from a template DNA source molecule (4kbp in length) from a plasmid, and ligation of the excised template sequence w ith a modified Left Hairpin (LHP) and an open-ended phosphorothioate modified prehybridized duplex oligonucleotide. The modified Left Hairpin contained four phosphorothioate modifications in the loop portion of the hairpin loop as shown in Table I. The phosphorothioate modified prehybridized duplex oligonucleotide consisted of strands“duplex strand A” and “duplex strand B” as listed in Table I. The duplex was prepared by incubating 100 micromolar duplex strand A in molecular grade water (Coming, Cat. 46-000- CI) and 100 micromolar duplex strand B in molecular grade water with IM magnesium chloride solution (Sigma, Cat. M1028-100ML) in a volume ratio of 1: 1:0.02 for 30 minutes at 37°C, followed by 30 minutes at room temperature.

[0249] The reaction mixture was prepared as follows by adding reagents in the following order. First, 18.5 micrograms of plasmid (1 mg / mL) was pipetted into a 2.0 mL DNA LoBind tube (Eppendorf, Cat. 022431048), then 60 microliters of water, 5 microliters of T4 ligase buffer (Promega, Cat. C126A), 5 microliters of the prehybridized phosphorothioate modified prehybridized duplex oligonucleotide, 5 microliters of the modified LHP, and 5 microliters of Esp3I (NEB. Cat. R0734B) were added. Next. 1.25 microliters of T4 ligase (Promega, Cat. Ml 794) was added to the mixture. The reaction mixture was incubated at 37°C for 90 minutes. The mixture was then incubated for 30 minutes at 16°C to allow for ligation of the modified LHP and the phosphorothioate modified prehybridized duplex oligonucleotide to the template sequence, thereby producing a double stranded template DNA precursor of the exonuclease-resistant single stranded template DNA product. The double stranded template DNA precursor does not have hairpins at both ends. One microliter of this reaction mixture was set aside for agarose gel electrophoresis.

[0250] Eleven microliters of rCutsmart Buffer (NEB, Cat. B6004S), 2 microliters of Exonuclease III (NEB. Cat. M0206L). and 8 microliters of RecJF (NEB, Cat. M0264L), were added to the sample and thoroughly mixed. The mixture was incubated for 30 minutes at 37°C for digestion. One microliter was subsequently removed for agarose gel electrophoresis.

[0251] Agarose gel (Lonza, Cat. 57023) electrophoresis shows that after the T4 ligation step, a re-ligated template forms, corresponding to the excised template sequence ligating to the leftover vector backbone (FIG. 2). After Exonuclease III and RecJF treatment, a nearcomplete digestion of the leftover vector and re-ligated template is observed, with preservation of the double stranded template DNA precursor of the exonuclease-resistant single stranded template DNA product. A fraction of the excised template sequence religated back onto the leftover vector on one end, resulting in a linearized product of the starting plasmid.

[0252] Example 3: Digestion of leftover vector using ExoIII followed by T7 exonuclease digestion

[0253] Long exonuclease-resistant single stranded template DNA product was prepared by excision of a template sequence (3,350 bp in length,) from a plasmid, and ligation of theexcised template sequence with a modified Left Hairpin (LHP) and an open-ended phosphorothioate modified prehybridized duplex oligonucleotide. The modified Left Hairpin contained four phosphorothioate modifications in the loop portion of the hairpin loop as shown in Table I. The phosphorothioate modified prehybridized duplex oligonucleotide consisted of strands “duplex strand A” and “duplex strand B’" as listed in Table I. The duplex was prepared by incubating 100 micromolar duplex strand A in molecular grade water (Coming, Cat. 46-000-CI) and 100 micromolar duplex strand B in molecular grade water with IM magnesium chloride solution (Sigma, Cat. M1028-100ML) in a volume ratio of 1 : 1 :0.02 for 30 minutes at 37°C, followed by 30 minutes at room temperature.

[0254] A reaction mixture for three samples was prepared as follows by adding reagents in the following order. First, 58.8 micrograms of plasmid (1 mg / mL) was pipetted into a 2.0 mL DNA LoBind tube (Eppendorf, Cat. 022431048), then 196 microliters of w ater, 15 microliters of T4 ligase buffer (Promega, Cat. C126A), 15 microliters of the prehybridized phosphorothioate modified prehybridized duplex oligonucleotide, 7.5 microliters of the modified LHP. and 7.5 microliters of BsaI-HFv2 (NEB. Cat. R3733B) were added. Next, 0.375 microliters of T4 ligase (Promega, Cat. Ml 794) were added to the mixture. The reaction mixture w as split equally into 3 separate 2 mL LoBind tubes. Each of the three samples was incubated at 37°C for 45 minutes. The samples w ere then incubated for 60 minutes at 16°C to allow for ligation of the modified LHP and the phosphorothioate modified prehybridized duplex oligonucleotide to template sequence, thereby producing a double stranded template DNA precursor of the final exonuclease-resistant single stranded template DNA product. One microliter from one of the samples was set aside for agarose gel electrophoresis.

[0255] Then. 11 microliters of rCutsmart Buffer (NEB, Cat. B6004S), and 300 units (3 microliters) of Exonuclease III (NEB, Cat. M0206L) were added to each sample and thoroughly mixed. The samples were incubated for 20 (sample C), 40 (sample B), or 60 (sample A) minutes at 37°C for digestion. One microliter from each sample w as subsequently removed for agarose gel electrophoresis. Agarose gel (Lonza. Cat. 57023) electrophoresis in FIG. 3A reveals preservation of the double stranded template DNA precursor of exonucleaseresistant single stranded template DNA product in all three samples with full digestion of leftover vector in samples A and B, and with partial leftover vector digestion in sample C. A fraction of the excised template sequence re-ligated back onto the leftover vector on one end, resulting in a linearized product of the starting plasmid.

[0256] Fifty units (5 microliters) of T7 exonuclease (NEB, Cat. M0263L) were then added to each of samples A. B, and C, and all three samples were incubated at 25°C. One microliter was removed from sample A at 10, 20, and 30 minutes and mixed with 1 microliter of 11 mM EDTA for enzyme inactivation before agarose gel electrophoresis. Samples B and C were both digested with T7 exonuclease for 30 minutes before addition of EDTA and removal of 1 microliter from each sample for agarose gel electrophoresis. Agarose gel (Lonza. Cat. 57023) electrophoresis in FIG. 3B reveals sufficient conversion of double stranded template DNA precursor of exonuclease-resistant single stranded template DNA product within 10 minutes of T7 exonuclease digestion.

[0257] Example 4: Heat inactivation step reduces unwanted dsDNA contaminants

[0258] Long exonuclease-resistant single stranded template DNA product L was prepared byexcision of a template sequence (3,946 bp in length) from a plasmid, and ligation of the excised template sequence with a modified Left Hairpin (LHP) and an open-ended phosphorothioate modified prehybridized duplex oligonucleotide. The modified LHP comprised four phosphorothioate modifications in the loop portion of the hairpin loop as shown in Table I. The phosphorothioate modified prehybridized duplex oligonucleotide consisted of strands ‘'duplex strand A" and ‘'duplex strand B” as listed in Table I. This duplex was prepared by incubating 100 micromolar duplex strand A in molecular biology- grade water (Coming, Cat. 46-000-CI) and 100 micromolar duplex strand B in molecular biologygrade water with IM magnesium chloride solution (Sigma, Cat. M1028-100ML) in a volume ratio of 1 : 1 .0.02 for 5 minutes at 95°C, followed by a gradual 20-minute ramp cool to room temperature.

[0259] The reaction mixture for 3 samples was prepared as follows by adding reagents in the following order. First, 61.9 micrograms of plasmid (1 mg / mL) was pipetted into a 1.5 mL DNA LoBind tube (Eppendorf, Cat. 022431048), then 201.9 microliters of water, 15 microliters of T4 ligase buffer (Promega, Cat. C126A), 9 microliters of the prehybridized phosphorothioate modified prehybridized duplex oligonucleotide, 4.5 microliters of the modified LHP, and 7.5 microliters of BsaI-HFv2 (NEB. Cat. R3733B) were added. Next, 0.375 microliters of T4 ligase (Promega, Cat. M1794) were added to the mixture. The reaction mixture was mixed thoroughly then split equally into three separate 1.5 mL DNA LoBind tubes. Sample 1 was incubated at 37°C for 60 minutes, and then incubated for 60 minutes at 16°C to allow for ligation of the modified LHP and the phosphorothioate modified prehybridized duplex oligonucleotide to the template sequence, thereby producing a double stranded template DNA precursor of the final exonuclease-resistant single stranded templateDNA product. Samples 2 and 3 were incubated at 37°C for 120 minutes to allow for both excision of template from vector as well as ligation of modified LHP and phosphorothioate modified prehybridized duplex oligonucleotide. One microliter from each sample was set aside for agarose gel electrophoresis.

[0260] Eleven microliters of rCutsmart buffer (NEB, Cat. B6004S) and 4 microliters of Exonuclease III (NEB. Cat. M0206L) were added to each sample, mixed thoroughly, and incubated at 37°C for 60 minutes. Sample 1 was temporarily stored at -20°C while samples 2 and 3 were heat inactivated at 70°C for 10 minutes. One microliter was collected from all three samples for agarose gel electrophoresis and they were all equilibrated to 25°C. Ten microliters of T7 exonuclease (NEB, Cat. M0263L) were added to each sample and incubated for 30 minutes at 25°C. One microliter was collected from each sample for agarose gel electrophoresis.

[0261] Agarose gel (Lonza, Cat. 57023) electrophoresis in FIG. 4 reveals no visible leftover dsDNA band in samples 2 and 3 as compared to sample 1.Example 5: HPLC-based purification of exonuclease-resistant single stranded template DNA product using strong anion exchange mobile phase

[0262] A exonuclease-resistant single stranded template DNA product was enzymatically produced as follows. A capping reaction mixture was prepared by adding reagents in the following order. First, 3262.8 micrograms of plasmid (1 mg / mL) comprising a template sequence (4.3kb in length) was pipetted into a 50 mL DNA LoBind tube (Eppendorf, Cat. 0030122232), then l OmL of water, 750 microliters of T4 ligase buffer (Promega, Cat. C126A), 450 microliters of a first capping oligonucleotide comprising a phosphorothioate modified prehybridized duplex oligonucleotide (consisting of strands “duplex strand A” and “duplex strand B” as listed in Table I), 225 microliters of a second capping oligonucleotide comprising a modified LHP (as listed in Table I), and 375 microliters of BsaI-HFv2 (NEB, Cat. R3733B) were added. Next, 18.75 microliters of T4 ligase (Promega, Cat. M1794) were added, and the mixture was incubated at 37°C for 120 minutes. Then, 1.65 mL of Buffer 4 (NEB, Cat. B7004S) and 600 microliters of Exonuclease III (NEB, Cat. M0206L) were added to each sample, mixed thoroughly, and incubated at 37°C for 60 minutes. The mixture was heat inactivated at 70°C for 10 minutes. Finally, 1.5 mL of T7 exonuclease (NEB, Cat. M0263L) was added and the reaction mixture was incubated for 30 minutes at 25°C.

[0263] A high performance liquid chromatography (HPLC) system was then used to purify the exonuclease-resistant single stranded template DNA product and remove unwanted proteins and oligonucleotides from the crude reaction mixture.

[0264] First, 4 mL of the crude reaction mixture was filtered via syringe filtration (ThermoFisher, Cat. 720-1320) to remove solid precipitates. Then, HPLC-based purification of the filtered reaction mixture was performed on an Agilent 1290 Infinity II Preparative HPLC system equipped with a PL-SAX column (Agilent Technologies, Cat. PL-1551-3803). Two buffers were used for the mobile phase, namely Buffer A and Buffer B. Buffer A was composed of 1 mM EDTA (Invitrogen, Cat. AM9260G) and 10 mM TRIS-HC1 (Teknova, Cat. T1080) in nanopure water (MilliporeSigma, MilliQ IQ® 7005) at pH 8. Buffer B was composed of 1 mM EDTA, 10 mM TRIS-HC1, and 2 M NaCl (Sigma Aldrich, Cat. S9888) in nanopure water at pH 8. HPLC purification was performed by injecting 2000 pL of the filtered reaction mixture onto the HPLC system and then following the method outlined in Table II, where the mobile phase was isocratic in the first 3 minutes being composed of 100% Buffer A, then the mobile phase composition was gradually changed using a ramping phase between the 3-minute and the 23.99-minute timepoints to reach 50% Buffer A and 50% Buffer B, then, between 23.99 and 28.00 minutes, the mobile phase composition was held at 50% Buffer A and 50% Buffer B and finally, the mobile phase composition was changed again gradually to reach 100% Buffer A at the 30-minute timepoint. The flow rate of the mobile phase was 8 mL / minute.

[0265] The resulting HPLC chromatogram is shown in FIG. 7. Fractions were manually collected at the following times during the HPLC purification process: 0.699, 1.532, 8.759, 17.905. 19.797, and 21.227 minutes. Identification of the exonuclease-resistant single stranded template DNA product in the fractions was performed using atomic force microscopy. A4 grade mica (Fisher Scientific, NC2080433) was adhered to steel support discs (Fisher Scientific, NC2080444) with two-component quick-set epoxy (DevCon) and allowed to dry overnight. Scotch tape was used to cleave mica several times until surface cleavage looked clean, without visible flakes. Each mica surface was dosed with 10 microliters of 250 mM magnesium chloride solution diluted from a IM solution (Sigma, Cat. M1028-100ML), and the discs were incubated at room temperature in a humidity chamber for 5 minutes. Next, 5 microliters of HPLC purified product were added directly to the buffer droplet on the mica and allowed to incubate for 2 minutes. The mica was then rinsed with 500 microliters of molecular biology grade water (Coming, 46-000-CI) before immediately being blown dry with gas duster can. Samples were imaged with an Asylum Cypher VRS atomic force microscope (Oxford Instruments) using FS-1500 probe tips (Oxford Instruments, ID 805.FS1500) in tapping mode. FIG. 8 shows the results of atomic force microscopy,indicating that the exonuclease-resistant single stranded template DNA product was contained in the HPLC peak corresponding to the fraction collected at 21.227 minutes.

[0266] Table II: HPLC ramping method using PL-SAX column (Agilent Technologies, Cat. PL-1551-3803).

[0267] In a second HPLC purification experiment, a shorter protocol was used to purify the exonuclease-resistant single stranded template DNA product from the crude reaction mixture. First, 4 mL of the crude reaction mixture was filtered via syringe filtration (ThermoFisher, Cat. 720-1320). Then, 3000 pL of the filtered reaction mixture was injected onto the HPLC system, and purification was performed using the ramping method outlined in Table III, where the mobile phase composition was held at 80% Buffer A in the first 2 minutes as indicated, then the mobile phase composition was changed in a ramping phase between the 2-minute and the 16-minute timepoints to reach 55% Buffer A and 45% Buffer B, and held at 55% Buffer A and 45% Buffer B to the 19.99 minute timepoint, until it was returned to 80% Buffer A again at the 20-minute timepoint. The flow rate of the mobile phase was 8 mL / minute. The resulting HPLC chromatogram is shown in FIG. 9. Fractions were manually collected at the following times during the HPLC purification process: 9.374, 11.872, and 14.079 minutes.

[0268] Table III: HPLC ramping method using PL-SAX column (Agilent Technologies, Cat. PL-1551-3803).

[0269] In a third HPLC purification a shorter protocol was used to purify the exonucleaseresistant single stranded template DNA product from the crude reaction mixture. First, 4 mL of the crude reaction mixture was filtered via syringe filtration (ThermoFisher, Cat. 720-1320). Then, 3000 pL of the filtered reaction mixture was injected onto the HPLC system, andpurification was performed using the ramping method outlined in Table IV, where the mobile phase composition was held at 75% Buffer A in the first 2 minutes as indicated, then the mobile phase composition was changed in a ramping phase between the 2-minute and the 12-minute timepoints to reach 55% Buffer A and 45% Buffer B, was held at 55% Buffer A and 45% Buffer B until the 14-minute timepoint, and was finally returned to 75% Buffer A again at the 15 -minute timepoint. The flow rate of the mobile phase was 8 mL / minute. The resulting HPLC chromatogram is shown in FIG. 10. Fractions were manually collected at the following times during the HPLC purification process: 6.267, 8.871, and 10.662 minutes.Table IV: HPLC ramping method using PL-SAX column (Agilent Technologies, Cat. PL- 1551-3803).

[0270] In a fourth HPLC experiment, a shorter protocol was used to purify the exonucleaseresistant single stranded template DNA product from the crude reaction mixture. First, 4 mL of the crude reaction mixture was filtered via syringe filtration (ThermoFisher, Cat. 720-1320). Then, 3000 pL of the filtered reaction mixture was injected onto the HPLC system, and purification was performed using the ramping method outlined in Table V, where the mobile phase composition was held at 100% Buffer A in the first 2 minutes as indicated, then the mobile phase composition was changed between the 2-minute and the 12-minute timepoints to reach 55% Buffer A and 45% Buffer B until it was returned to 75% Buffer A and 25% Buffer B again at the 13-minute timepoint. The flow rate of the mobile phase was 7 mL / minute. This protocol was repeated twice more using the same method with an injection volume of 2500 pL. The resulting HPLC chromatogram in FIG. 11 shows the 3 separate runs of the HPLC method described in Table V. For the first run, fractions were manually collected at the following times during the HPLC purification process: 9.454 and 11.075 minutes. For the second run, one fraction was manually collected at 11.110 minutes. For the third run. one fraction was manually collected at 11.131 minutes.

[0271] Table V: HPLC ramping method using PL-SAX column (Agilent Technologies, Cat. PL-1551-3803).

[0272] Following HPLC purification, fractions collected from the above-described HPLC processes and containing the exonuclease-resistant single stranded template DNA product (namely, the 21.227-minute collected fraction from the first experiment, the 14.079-minute collected fraction from the second experiment, the 10.662-minute collected fraction from the third experiment, the 11.075-minute collected fraction from the first run of the fourth experiment, and the 11.110-minute and the 11. 131 -minute fractions collected from the second and third runs of the fourth experiment) were pooled to produce a “HPLC purified product” sample. The HPLC purified product was injected into a 3.5-kDa molecular weight cutoff dialysis cassette (Thermofisher, A52969). This cassette was placed into 4.25 liters of nanopure water (MilliporeSigma, MilliQ IQ® 7005). The water volume of 4.25 liters was replaced every hour for a total of 3 hours. Identification of the exonuclease-resistant single stranded template DNA product after dialysis, was performed using gel electrophoresis (Lonza, Cat. 57023). FIG. 12 shows the results of the gel. indicating that the exonuclease-resistant single stranded template DNA product was contained in the HPLC peaks and was conserved post dialysis.

[0273] The dialyzed HPLC purified product was removed from the dialysis cassette and frozen at -80°C. Once frozen, the sample was lyophilized on a Labconco Freeze Dry er (Avantar, 75999-778) for 48 hours, resulting in the production of a white powder. The white powder was resuspended in 1 mL of nanopure water (MilliporeSigma, MilliQ IQ® 7005).

[0274] Identification of the exonuclease-resistant single stranded template DNA product post lyophilization was performed using gel electrophoresis (Lonza, Cat. 57023). FIG. 13 shows the results of the gel, indicating that the exonuclease-resistant single stranded template DNA product was conserved post dialysis and lyophilization. Qubit was used to determine the concentration of the exonuclease-resistant single stranded template DNA product, measured at 422 ng / pL.Example 6; HPLC-based purification of exonuclease-resistant single stranded template DNA product using an organic ion pairing mobile phase

[0275] A exonuclease-resistant single stranded template DNA product was enzymatically produced as described in Example 5.

[0276] An HPLC system was then used to purify the exonuclease-resistant single stranded template DNA product and remove unwanted proteins and oligonucleotides from the crude reaction mixture.

[0277] First, 4 mL of the crude reaction mixture was filtered via syringe filtration (ThermoFisher, Cat. 720-1320) to remove solid precipitates. Then, HPLC-based purification of the filtered reaction mixture was performed on an Agilent 1290 Infinity II Preparative HPLC system equipped with a PL-SAX column (Agilent Technologies, Cat. PL-1551-3803). Two buffers were used for the mobile phase, namely Buffer A and Buffer B. Buffer A was composed of 93% 0.1 M tetraethylammonium acetate (MilliporeSigma, 69372- IL) and 7% acetonitrile (Fisher Chemical, A996-4) (v / v) at pH 8.5. Buffer B was composed of 93% 0.1 M tetraethylammonium acetate (MilliporeSigma, 69372-1L), 7% acetonitrile (Fisher Chemical, A996-4) (v / v), and 1 M ammonium chloride (SigmaAldrich, 21330-500G) at pH 8.5. HPLC purification was performed by injecting 2000 pL of the filtered reaction mixture onto the HPLC system and then following the method outlined in Table VL where the mobile phase in the first 3 minutes was composed of 100% Buffer A, then the mobile phase composition was gradually changed in a ramping phase between the 3-minute and the 28-minute timepoints to reach 0% Buffer A and 100% Buffer B. and finally the mobile phase composition was changed again gradually to reach 100% Buffer A at the 30-minute timepoint. The flow rate of the mobile phase was 6 mL / minute.

[0278] The resulting HPLC chromatogram is shown in FIG. 14. Fractions were manually collected at the following times during the HPLC purification process: 0.927, 9.306, 10.532, 10.961. 15.407, 15.854, 16.871, and 17.645 minutes. Identification of the exonuclease-resistant single stranded template DNA product in the fractions was performed using atomic force microscopy as described in Example 5. FIGS. 15A-15B show the results of atomic force microscopy, indicating that the exonuclease-resistant single stranded template DNA product was contained in the HPLC peak corresponding to the fraction collected at 17.645 minutes.

[0279] Table VI: HPLC ramping method using PL-SAX column (Agilent Technologies, Cat. PL-1551-3803).Example 7; HPLC-based purification, tangential flow filtration, and freeze drying of exonuclease-resistant single stranded template DNA product

[0280] A exonuclease-resistant single stranded template DNA product was enzymatically produced as follows. A capping reaction mixture was prepared by adding reagents in the following order. First, 8343.3 micrograms of plasmid (1 mg / mL) comprising a template sequence (4.3kb in length) was pipetted into a 50 mL DNA LoBind tube (Eppendorf, Cat. 0030122232), then 33.4 mL of water, 1951 microliters of T4 ligase buffer (Promega, Cat. C126A), 1170.6 microliters of a first capping oligonucleotide comprising a phosphorothioate modified prehybridized duplex oligonucleotide (consisting of strands “duplex strand A” and “duplex strand B” as listed in Table I), 585.3 microliters of a second capping oligonucleotide comprising a modified LHP (as listed in Table I), and 975.5 microliters of BsaI-HFv2 (NEB, Cat. R3733B) were added. Next, 48.78 microliters of T4 ligase (Promega, Cat. Ml 794) were added, and the mixture was incubated at 37°C for 120 minutes. Then, 4.5 mL of Buffer 4 (NEB, Cat. B7004S) and 1561 microliters of Exonuclease III (NEB, Cat. M0206L) were added to each sample, mixed thoroughly, and incubated at 37°C for 60 minutes. The mixture was heat inactivated at 70°C for 20 minutes after which it was cooled to 25°C for 15 minutes. Finally, 2.73 mL of T7 exonuclease (NEB, Cat. M0263L) was added and the reaction mixture was incubated for 60 minutes at 25°C.

[0281] A HPLC system was then used to purify the exonuclease-resistant single stranded template DNA product and remove unwanted proteins and oligonucleotides from the crude reaction mixture. Following HPLC, tangential flow filtration was used to desalt the HPLC peak containing the exonuclease-resistant single stranded template DNA product, then freeze-dry ing was used to concentrate the exonuclease-resistant single stranded template DNA product.

[0282] First, the crude reaction mixture was filtered via syringe filtration (Millipore Sigma, Cat.# Z358193-1CS) to remove solid precipitates. Then, HPLC-based purification of the filtered reaction mixture was performed on an Agilent 1290 Infinity' II Preparative HPLC system equipped with a PL-SAX column (Agilent Technologies, Cat. PL-1551-3703). Two buffers were used for the mobile phase, namely Buffer A and Buffer B. Buffer A was composedof 1 mM EDTA (Invitrogen, Cat. AM9260G) and 10 mM TRIS-HC1 (Teknova, Cat. T1080) in nanopure water (MilliporeSigma, MilliQ IQ® 7005) at pH 8. Buffer B was composed of 1 mM EDTA, 10 mM TRIS-HC1, and 2 M NaCl (Sigma Aldrich, Cat. S9888) in nanopure water at pH 8. HPLC purification was performed by injecting 5000 pL of the filtered reaction mixture onto the HPLC system and then following the step-wise method outlined in Table VII, where the mobile phase was isocratic in the first 3 minutes and was composed of 75% Buffer A and 25% Buffer B. then the mobile phase composition was changed to 65% Buffer A and 35% Buffer B at 3.01 minutes and then held at 65% Buffer A and 35% Buffer B between 3.01 and 8.00 minutes in the first step, then the mobile phase composition was changed again to 75% Buffer A and 25% Buffer B at the 8.01 minute timepoint and held at 75% Buffer A and 25% Buffer B until the 10.00 minute timepoint in the second step. The flow rate of the mobile phase was 10 mL / minute. This HPLC step-wise protocol was repeated back-to-back 8 more times on the same column with the same injection volume of filtered reaction mix.

[0283] The resulting nine HPLC chromatograms were overlaid in FIG. 16. One fraction was collected from each chromatogram within the peak located between the 5.7-minute and the 6.2- minute timepoints during the HPLC purification process. Identification of the exonucleaseresistant single stranded template DNA product in the nine collected fractions was performed using gel electrophoresis (Lonza, Cat. 57023). FIG. 17 shows the results of the gel, indicating that the exonuclease-resistant single stranded template DNA product was contained in each HPLC peak corresponding to the fraction collected from each of the nine chromatograms between 5.7 minutes and 6.2 minutes.

[0284] Table VII: HPLC stepwise method using PL-SAX column (Agilent Technologies, Cat. PL-1551-3703).

[0285] The collected fractions from the nine HPLC processes were pooled together into a “HPLC purified product”. Then, tangential flow filtration (KR2I TFF system, Repligen Cat # SYR2-U50) was used to remove the NaCl and EDTA from the HPLC purified product. Following TFF, the exonuclease-resistant single stranded template DNA product was filteredusing a 0.2 micron PES Nalgene filter (Millipore Sigma, Cat.# Z358193-1CS). Identification of the exonuclease-resistant single stranded template DNA product post TFF is shown in FIG. 18 (Lonza, Cat. 57023).

[0286] Following TFF, the exonuclease-resistant single stranded template DNA product was frozen at -80°C. Once frozen, the sample was lyophilized on a Labconco Freeze Dryer (Avantar, 75999-778) for 24 hours, resulting in the production of a white powder. The white powder was resuspended in 1 mL of nanopure water (MilliporeSigma. MilliQ IQ® 7005). Quantification of the exonuclease-resistant single stranded template DNA product following HPLC purification was performed using a NanoDrop 8000 Spectrophotometer (Thermo Scientific, Cat. ND-8000-GL) per manufacturer protocol. The measured concentration of the exonuclease-resistant single stranded template DNA product was 570 ng / pL.NUMBERED ASPECTS

[0287] The following numbered aspects also form part of the instant disclosure.1. A method for producing an exonuclease-resistant single stranded template deoxyribonucleic acid (DNA) product for non-viral delivery to a cell, comprising: a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide configured to modify a second end of the template sequence, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonucleaseresistant single stranded template DNA product, e) inactivating the second exonuclease, andI performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture. wherein the second capping oligonucleotide is resistant to exonuclease digestion,wherein one strand of the first capping oligonucleotide is resistant to exonuclease digestion, and wherein the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.2. The method of any one or combination of numbered aspects disclosed herein, wherein the second capping oligonucleotide comprises a duplex structure modified with one or both of an exonuclease resistant chemical modification or an exonuclease resistant secondary structure.3. The method of any one or combination of numbered aspects disclosed herein, wherein the second capping oligonucleotide comprises a hairpin structure, a prehybridized duplex oligonucleotide, an RNA aptamer, 2'-O-methyl nucleotides. 2'-O-methoxyethyl (MOE) nucleotides, phosphorothioate nucleotides, or a combination thereof.4. The method of any one or combination of numbered aspects disclosed herein, wherein the second capping oligonucleotide comprises a hairpin structure with one or more phosphorothioate nucleotides.5. The method of any one or combination of numbered aspects disclosed herein, wherein the second capping oligonucleotide comprises an MS2 RNA aptamer.6. A method for producing an exonuclease-resistant single stranded template DNA product for non-viral delivery to a cell, comprising: a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide comprising a hairpin structure and configured to modify a second end of the template sequence to produce a double stranded template DNA closed on one end, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell -free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonucleaseresistant single stranded template DNA product,e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture to obtain the exonuclease-resistant single stranded template DNA product, wherein one strand of the first capping oligonucleotide is resistant to exonuclease digestion, and wherein the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.7. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction comprises sequentially adding a) to c) to the cell-free reaction mixture in the specified order: a) the template DNA source molecule; b) an aqueous solvent; c) a buffer; mixing after c) is added; then adding one or both of d) and e): d) the first capping oligonucleotide; e) the second capping oligonucleotide; and then adding one or both of f) and g): f) the at least one endonuclease; g) the DNA ligase.8. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction comprises: i) incubating the template DNA source molecule comprising the least one endonuclease recognition sequence and the template sequence in a cell- free reaction mixture comprising a first endonuclease, a first DNA ligase, and the first capping oligonucleotide to modify the first end of the template sequence; optionally inactivating the first endonuclease prior to step ii); and ii) incubating the template sequence modified on the first end with the second capping oligonucleotide to modify the second end of the templatesequence, and optionally adding a second endonuclease, a second DNA ligase, or both after step i).9. The method of any one or combination of numbered aspects disclosed herein, wherein the first and second endonuclease are the same or different.10. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are performed sequentially in the same reaction.11. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are performed as separate reactions.12. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are performed in the same reaction vessel.13. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are performed in separate reaction vessels, optionally the template sequence modified on the first end is purified prior to step ii).14. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are performed at the same or different temperature.15. The method of any one or combination of numbered aspects disclosed herein, wherein step i) and ii) are each independently performed at one or more temperatures.16. The method of any one or combination of numbered aspects disclosed herein m, wherein the yield of the exonuclease-resistant single stranded template DNA product is 50% or more relative to the total amount of template DNA source molecule after performing the capping reaction for 4 hours or less, 3 hours or less, or 2 hour or less.17. The method of any one or combination of numbered aspects disclosed herein, wherein the hairpin structure comprises a hairpin loop with 4 to 40, 6 to 36, 8 to 32, 10 to 28, 12 to 24, 14 to 20, or 16 to 18 nucleotides.18. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprise one or more hairpin structures.19. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises a 3’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides, or both the first capping oligonucleotide and the second capping oligonucleotide comprises a 3’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides.20. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises a 5’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides.21. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises 15 to 100. 20 to 80, 25 to 75, 30 to 60, or 40 to 50 nucleotides, or both the first capping oligonucleotide and the second capping oligonucleotide comprises 15 to 100, 20 to 80, 25 to 75, 30 to 60, or 40 to 50 nucleotides.22. The method of any one or combination of numbered aspects disclosed herein, wherein the digestion reaction step comprises anti-sense digestion.23. The method of any one or combination of numbered aspects disclosed herein, wherein the digestion reaction step comprises sense digestion.24. The method of any one or combination of numbered aspects disclosed herein, wherein the cell-free reaction mixture comprises each of the first and second capping oligonucleotides independently in a ratio of 5-fold to 200-fold, 10-fold to 100-fold, 15-fold to 80-fold, 20-fold to 70-fold, 25-fold to 60-fold. 30-fold to 50-fold, or 35-fold to 45-fold excess relative to the template DNA source molecule excess relative to the template DNA source molecule.25. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is at least partially performed at a first temperature and optionally at a second temperature that is different than the first temperature.26. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed at a first temperature after adding the at least one endonuclease and a second temperature after adding the ligase.27. The method of any one or combination of numbered aspects disclosed herein, wherein the first temperature and the second temperature are each independently selected from a temperature in a range of about 10°C to about 42°C, about 14°C to about 40°C, about 15°C to about 39°C, about 16°C to about 37°C, or about 37°C.28. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is conducted at the first temperature for about 1 hour to about 72 hours.29. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is conducted at the second temperature for about 1 hour to about 72 hours.30. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed at a single temperature.31. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed at about 37 °C.32. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed for about 0.5 to about 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 1 hour to about 2 hours.33. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed for about 2 hours.34. The method of any one or combination of numbered aspects disclosed herein, wherein the first exonuclease digestion reaction is performed at a temperature from about 35°C to about 40°C, about 36 °C to about 38 °C, or about 37°C.35. The method of any one or combination of numbered aspects disclosed herein, wherein the second exonuclease digestion reaction is performed at a temperature from about 20 °C to about 37 °C, about 23 °C to about 35 °C. or about 25 °C.36. The method of any one or combination of numbered aspects disclosed herein, wherein the duration of the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is shorter than the capping reaction.37. The method of any one or combination of numbered aspects disclosed herein, wherein the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 0.5 to about 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 0.5 to about 1.5 hours.38. The method of any one or combination of numbered aspects disclosed herein, wherein the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 5 minutes to about 1 hour, about 10 minutes to about 50 minutes, about 15 minutes to about 40 minutes, or about 20 minutes to about 30 minutes.39. The method of any one or combination of numbered aspects disclosed herein, wherein the first exonuclease digestion reaction is performed for 20 minutes to 60 minutes.40. The method of any one or combination of numbered aspects disclosed herein, wherein the second exonuclease digestion reaction is performed for 10 minutes to 30 minutes.41. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product comprises about 500 to about 15,000 nucleotides in length, about 1000 to about 14,000 nucleotides in length, about 2000 to about 13,000 nucleotides in length, about 3000 to about 12,000 nucleotides inlength, about 4000 to about 11,000 nucleotides in length, or about 5000 to about 10,000 nucleotides in length.42. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product comprises about 5000 to about 10,000 nucleotides in length.43. The method of any one or combination of numbered aspects disclosed herein, wherein the capping reaction is performed for a longer duration than the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both.44. The method of any one or combination of numbered aspects disclosed herein, wherein the step of inactivating the first exonuclease, the second exonuclease, or both comprises a heat inactivation or a chelating agent inactivation.45. The method of any one or combination of numbered aspects disclosed herein, wherein the step of inactivating the first exonuclease comprises a heat inactivation.46. The method of any one or combination of numbered aspects disclosed herein, wherein the heat inactivation is performed at about 50°C to about 90°C, about 60°C to about 80°C, about 65°C to about 75°C.47. The method of any one or combination of numbered aspects disclosed herein, wherein the heat inactivation step is performed at about 70°C.48. The method of any one or combination of numbered aspects disclosed herein, wherein the heat inactivation is performed for 1 minute or more, or 2 minutes or more, or 5 minutes or more, 10 minutes or more, 60 minutes or less, 30 minutes or less, about 5 minutes to about 20 minutes, about 7 minutes to about 15 minutes, or about 10 minutes.49. The method of any one or combination of numbered aspects disclosed herein, wherein the chelating agent inactivation comprises adding a chelating agent at a concentration of 5 mM to 100 mM, 7.5 mM to 80 mM, 10 mM to 60 mM, 12.5 mM to 40 mM, or 15 mM to 20 mM.50. The method of any one or combination of numbered aspects disclosed herein, wherein the chelating agent is EDTA.51. The method of any one or combination of numbered aspects disclosed herein, wherein the step of inactivating the first exonuclease comprises a heat inactivation and the step of inactivating the second exonuclease comprises a chelating agent inactivation.52. The method of any one or combination of numbered aspects disclosed herein, wherein non-template plasmid DNA in the cell-free reaction mixture is degraded after a single digestion reaction.53. The method of any one or combination of numbered aspects disclosed herein, wherein a single digestion reaction produces the exonuclease-resistant single stranded template DNA product.54. The method of any one or combination of numbered aspects disclosed herein, wherein the non-template plasmid DNA is digested without damaging at least one strand of the template DNA molecule.55. The method of any one or combination of numbered aspects disclosed herein, comprising cooling the heat inactivated reaction mixture to a temperature from about 20 °C to about 37 °C, about 23 °C to about 35 °C, or about 25 °C before adding the second exonuclease.56. The method of any one or combination of numbered aspects disclosed herein, wherein the cooling step comprises cooling the heat inactivated reaction mixture from about 70°C to about 25°C.57. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease is selected from T7 exonuclease, Exonuclease I. Exonuclease III, Exonuclease VII, or Exonuclease VIII.58. The method of any one or combination of numbered aspects disclosed herein, wherein the first exonuclease is Exonuclease III.59. The method of any one or combination of numbered aspects disclosed herein, wherein the second exonuclease is T7 Exonuclease or further comprises T7 Exonuclease as a second exonuclease.60. The method of any one or combination of numbered aspects disclosed herein, wherein a) Lambda Exonuclease is not used, b) biotin modification is not used, c) rolling circle amplification is not used, d) streptavidin-coated beads are not used, or any combination of a)-d) are not used.61. The method of any one or combination of numbered aspects disclosed herein, wherein the second capping oligonucleotide comprises a hairpin loop with 4-20 base pairs.62. The method of any one or combination of numbered aspects disclosed herein, wherein the hairpin structure comprises phosphorothioate bonds.63. The method of any one or combination of numbered aspects disclosed herein, wherein one strand of the prehybridized duplex oligonucleotide comprises phosphorothioate bonds.64. The method of any one or combination of numbered aspects disclosed herein, wherein 2 to 20, 4 to 18, 6 to 16. 8 to 14, or 10 to 12 nucleotides of the hairpin loop comprise phosphorothioate bonds.65. The method of any one or combination of numbered aspects disclosed herein, wherein the prehybridized duplex oligonucleotide comprises two strands and wherein each strand of the prehybridized duplex oligonucleotide independently comprises 2 to 200, 4 to 180, 6 to 160, 8 to 140, 10 to 120, 12 to 100, 10 to 80, 12 to 60. 14 to 40. 16 to 20 nucleotides.66. The method of any one or combination of numbered aspects disclosed herein, wherein the strands of the prehybridized duplex oligonucleotide are different lengths.67. The method of any one or combination of numbered aspects disclosed herein, wherein the prehybridized duplex oligonucleotide comprises a nucleotide overhang, optionally on each end.68. The method of any one or combination of numbered aspects disclosed herein, wherein the prehybridized duplex oligonucleotide comprises an overhang of 2 to 12, 4 to 10. or 6 to 8 nucleotides.69. The method of any one or combination of numbered aspects disclosed herein, wherein the prehybridized duplex oligonucleotide comprises a functional group attached at one end.70. The method of any one or combination of numbered aspects disclosed herein, wherein the functional group is exonuclease resistant.71. The method of any one or combination of numbered aspects disclosed herein, wherein the functional group comprises a G-quadruplex structure, a hairpin structure, an RNA aptamer, a C3 to C12 spacer group, or PEG.72. The method of any one or combination of numbered aspects disclosed herein, wherein the functional group comprises an MS2 RNA aptamer.73. The method of any one or combination of numbered aspects disclosed herein, wherein the functional group comprises one or more phosphorothioate bonds.74. The method of any one or combination of numbered aspects disclosed herein, wherein 2 to 50, 4 to 40, 6 to 30, 8 to 20, or 10 to 12 nucleotides of one strand of the prehybridized duplex oligonucleotide comprises phosphorothioate bonds.75. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or the first and the second capping oligonucleotide comprises phosphorothioate bonds.76. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified 5 ’-end.77. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified 3 ’-end.78. The method of any one or combination of numbered aspects disclosed herein, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified overhang end.79. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease is a Type IIS enzyme.80. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease recognition sequence is a recognition sequence for the at least one endonuclease selected from Acul, Alw261, Alwl, Bael, BbsI, BbsI-HF, Bbvl, Bccl. BceAI. Bcgl, BciVI, BcoDI. BfuAI. BmrI, Bmsl, Bpil, Bpml, BpuEI, Bsal, Bsal-HF V2. BsaXI. BseGI. BseRL Bsgl. BsmAI, BsmBI, BsmBI-v2, BsmFI. BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-Mutl, Btsl-v2, CspCI, Earl, Ecil, Eco311, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lgul, Mval269I, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, or SfaNI.81. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease recognition sequence is a recognition sequence for at least one endonuclease selected from Bsal, BsaI-HFv2, BsmBI, BsmBI-v2, Esp3I, BbsI, BbsI-HF, SapI, or isoschizomers thereof.82. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease is selected from Acul, Alw26L Alwl, Bael, BbsI, BbsI- HF, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, BmrI, Bmsl, Bpil, Bpml, BpuEI, Bsal, Bsal-HF V2, BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI. BspQI. BsrDI, BsrI, BtgZI, BtsCI. Btsl-Mutl, Btsl-v2, CspCI, Earl, Ecil. Eco311, Esp3I. Faul. FokI. Hgal. HphI, HpyAV. Lgul, Mval269I, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, or SfaNI.83. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease is selected from Bsal, BsaI-HFv2, BsmBI, BsmBI-v2, Esp3I, BbsI, BbsI-HF, SapI, or isoschizomers thereof.84. The method of any one or combination of numbered aspects disclosed herein, wherein the at least one endonuclease is added at a concentration of about 1-10%, about 2- 8%, about 2.5-5%, about 2%, about 2.5%, about 3%, or about 3.5% v / v of the cell-free reaction mixture.85. The method of any one or combination of numbered aspects disclosed herein, wherein the ligase is selected from T3 ligase, T4 ligase, T7 ligase, Taq ligase, DNA ligase I, DNA ligase, II, DNA ligase III, DNA ligase VI. Tth DNA ligase, or E. coli DNA ligase.86. The method of any one or combination of numbered aspects disclosed herein, wherein the ligase is T4 ligase.87. The method of any one or combination of numbered aspects disclosed herein, wherein the ligase is added at a concentration of about 0.05-5%, about 0.1-4%. about 0. 125- 3%, or about 0.1%, about 0.125%, about 0.15%, or about 0.2% v / v of the cell-free reaction mixture.88. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is produced isothermally in steps a) to e).89. The method of any one or combination of numbered aspects disclosed herein, wherein steps a) performing a capping reaction, b) performing a first exonuclease digestion reaction, and c) inactivating the first exonuclease are performed at a temperature from about I0°C to about 42°C, about 14 °C to about 40 °C, about 15 °C to about 39 °C. or about 16 °C to about 37° C.90. The method of any one or combination of numbered aspects disclosed herein, wherein steps a) to c) are performed at about 37°C.91. The method of any one or combination of numbered aspects disclosed herein, wherein the total yield of the exonuclease-resistant single stranded template DNA product is 25 to 100 mg per liter of the cell-free reaction mixture within 6 hours, 100 to 400 mg per liter of the cell-free reaction mixture within 24 hours, or 200 to 800 mg per liter of the cell-free reaction mixture within 48 hours.92. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product comprises 3,000 to 4,000 nucleotides in length.93. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is produced in 1hour to 48 hours, 2 hours to 24 hours, 3 hours to 18 hours, 4 hours to 12 hours, or 6 hours to 8 hours.94. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is produced in a single contiguous reaction solution.95. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0.1 L to 12.5 L, 0.25 L to 6.25 L, 0.5 L to 3.5 L, or 0.75 L to 1.5 L.96. The method of any one or combination of numbered aspects disclosed herein, comprising producing the exonuclease-resistant single stranded template DNA product in a bioreactor.97. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises a silica-based purification procedure.98. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises performing a high-performance liquid chromatography (HPLC) purification step using a HPLC system, optionally wherein: a) the HPLC purification step comprises anion exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof, b) the HPLC purification step comprises one or more separation modes, c) the HPLC purification step comprises a ramping separation mode, a step- wise separation mode, or a combination thereof, or d) the HPLC purification step comprises a combination of any one or more of a), b), and c).99. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is resistant to Dnase.100. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product has a frictional ratio of 4 to 20, 6 to 18, 8 to 16, or 10 to 12, as measured by analytical ultracentrifugation.101. The method of any one or combination of numbered aspects disclosed herein, further comprising the exonuclease-resistant single stranded template DNA product in a lipid composition.102. The method of any one or combination of numbered aspects disclosed herein, wherein the template DNA source molecule comprises a linear double stranded DNA, a plasmid DNA, a mini circle DNA, a cosmid DNA. a bacterial artificial chromosome, a molecular inversion probe, a doggybone DNA, a vector, a cDNA, an AAV vector, or a PCR product.103. The method of any one or combination of numbered aspects disclosed herein, comprising adding an RNA aptamer at the first or second end, adding a G4 quadruplex at the first or second end. or adding an RNA aptamer at the first end and adding a G4 quadruplex at the second end.104. The method of any one or combination of numbered aspects disclosed herein, wherein the RNA aptamer comprises 2’-O-methyl groups.105. The method of any one or combination of numbered aspects disclosed herein, wherein the aptamer is an MS2 RNA aptamer.106. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step further includes adding DNA-binding beads.107. The method of any one or combination of numbered aspects disclosed herein, wherein the DNA-binding beads comprise a polystyrene core covered by a magnetite layer, and the magnetite layer is covered by a carboxyl at e-modified polymer coating.108. The method of any one or combination of numbered aspects disclosed herein, wherein the beads are provided at a ratio of 0.5 to 0.9. 0.6 to 0.8, 0.65 to 0.75, or about 0.7 to the exonuclease-resistant single stranded template DNA product.109. The method of any one or combination of numbered aspects disclosed herein, further comprising purifying the exonuclease-resistant single stranded template DNA product by performing multiple elutions of the beads.110. The method of any one or combination of numbered aspects disclosed herein, wherein exonuclease-resistant single stranded template DNA product comprises no phosphorothioate modifications.111. The method of any one or combination of numbered aspects disclosed herein, wherein the template DNA source molecule comprises a first endonuclease recognition sequence at the first end of the template sequence and a second endonuclease recognition sequence at the second end of the template sequence.112. The method of any one or combination of numbered aspects disclosed herein, wherein the first end and the second end of the template sequence comprises a different overhang end following digestion of the template DNA source molecule by two different endonucleases.113. An exonuclease-resistant single stranded template DNA product prepared by the method of any one or combination of numbered aspects disclosed herein.114. A lipid composition comprising the exonuclease-resistant single stranded template DNA product prepared by the method of any one or combination of numbered aspects disclosed herein.115. The lipid composition of any one or combination of numbered aspects disclosed herein, wherein the lipid composition further encapsulates one or more peptides, polypeptides, one more cationic salts, one or more anionic salts, one or more additional polynucleotides.116. The lipid composition of any one or combination of numbered aspects disclosed herein, wherein the lipid composition comprises microspheres, liposomes, lipoplexes. or lipid nanoparticles.117. A exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification and the second modification are exonuclease resistant, and wherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15,000 nucleotides in length.118. An exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification, the second modification, or both the first and second modification comprises a linear sequence comprising one or more phosphorothioate bonds or an exonuclease resistant functional group, wherein the exonuclease resistant functional group does not comprise biotinylation.119. The exonuclease-resistant single stranded template DNA product of any one or combination of numbered aspects disclosed herein, wherein the first modification and the second modification comprises a linear sequence comprising one or more phosphorothioate bonds.120. The exonuclease-resistant single stranded template DNA product of any one or combination of numbered aspects disclosed herein, wherein the first modification or the second modification comprises an exonuclease resistant functional group.121. The exonuclease-resistant single stranded template DNA product of any one or combination of numbered aspects disclosed herein, wherein the exonuclease resistant functional group comprises a hairpin structure.122. The exonuclease-resistant single stranded template DNA product of any one or combination of numbered aspects disclosed herein, wherein the exonuclease resistant functional group comprises a polynucleotide secondary structure comprising phosphorothioate bonds.123. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) one or more helper lipids;(c) one or more neutral lipids;(d) optionally, a pegylated lipid; and(e) an exonuclease-resistant single stranded template DNA product, wherein the exonuclease-resistant single stranded template DNA product comprises a first end comprising a first modification and a second end comprising a second modification, wherein the first modification and the second modification are exonuclease resistant. and wherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15,000 nucleotides in length.124. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) one or more helper lipids;(c) one or more neutral lipids;(d) optionally, a pegylated lipid; and(e) the exonuclease-resistant single stranded template DNA product of any one of any one or combination of numbered aspects disclosed herein.125. A method for inserting a custom DNA template into a cell comprising introducing an exonuclease-resistant single stranded template DNA product into a cell comprising: a) producing the exonuclease-resistant single stranded template DNA product by the method of any one of any one or combination of numbered aspects disclosed herein; b) contacting the cell with the exonuclease-resistant single stranded template DNA product and a gene editing system; and inserting at least a part of atemplate sequence from the exonuclease-resistant single stranded template DNA product into DNA of the cell that has been cleaved by the gene editing system.126. The method of any one or combination of numbered aspects disclosed herein, wherein the gene editing system comprises a CRISPR / Cas system; a Tth Argonaute (TtAgo) system; a zinc finger nuclease (ZFN) system; ARCUS nuclease system; megaTALs; or a transcription activator-like effector nuclease (TALEN) system.127. The method of any one or combination of numbered aspects disclosed herein, wherein the cell is a non-cycling cell.128. The method of any one or combination of numbered aspects disclosed herein, wherein the cell is a cycling cell.129. The method of any one or combination of numbered aspects disclosed herein, further comprising performing non-homologous end joining (NHEJ) repair or homology directed (HD) repair in the cell.130. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the custom DNA template is >5 kB, >6 kB. >7 kB. >8 kB. >9 kB, or >10 kB in size.131. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is encapsulated in a lipid nanoparticle.132. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the inserting is performed in vitro.133. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the inserting is performed ex vivo.134. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the inserting is performed in vivo.135. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises anion exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof.136. The method of any one of any one or combination of numbered aspects disclosed herein, wherein the HPLC purification step comprises using one or more separation modes.137. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises a ramping separation mode, a step-wise separation mode, or a combination thereof138. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises:(i) injecting the exonuclease-resistant single stranded template DNA product onto the HPLC system comprising an anion exchange resin under conditions to bind the exonuclease-resistant single stranded template DNA product to the anion exchange resin;(ii) separating the exonuclease-resistant single stranded template DNA product from impurities using a ramping separation mode, a step-wise separation mode, or a combination thereof; and(iii) eluting the exonuclease-resistant single stranded template DNA product from the HPLC system.139. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises using one or more mobile phase buffers.140. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises using two mobile phase buffers.141. The method of any one or combination of numbered aspects disclosed herein, wherein the mobile phase buffers comprise an aqueous or organic solvent composition.142. The method of any one or combination of numbered aspects disclosed herein, wherein the mobile phase buffers comprise an ion pairing agent.143. The method of any one or combination of numbered aspects disclosed herein, wherein the ion pairing agent comprises triethylamine, tripropylamine, hexylamine, butylamine, dibutylamine, or a tetraalkyl ammonium salt, optionally wherein the ion pairing agent is triethylammonium acetate (TEAA).144. The method of any one or combination of numbered aspects disclosed herein, wherein the ion pairing agent is present at a concentration of about 0.01 M to about 1 M, about 0.05 M to about 0.75 M, or about 0. 1 M to about 0.5 M145. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer comprising less than about 600 mM NaCl or NH4CI or less than about 500 mM NaCl or NH4CI.146. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer comprising less than about 300 mM MgCh or less than about 250 mM MgCh.147. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer without NaCl, NH4CI, or MgCh.148. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises running the HPLC system with a mobile phase flow rate of about 1 mL / min to about 200 mL / min, about 5 mL / min to about 100 mL / min, or about 10 mL / min to about 50 mL / min.149. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises a step-wise separation mode with two or more steps.150. The method of any one or combination of numbered aspects disclosed herein, wherein each mobile phase buffer comprises a chelating agent.151. The method of any one or combination of numbered aspects disclosed herein, wherein the chelating agent is EDTA.152. The method of any one or combination of numbered aspects disclosed herein, wherein the chelating agent is included at a concentration of 0.1 mM to 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.153. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resin using a mobile phase buffer composition comprising NaCl. NH4CI, or MgCh.154. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resin in a mobile phase buffer comprising about 0.70 M to about 2.0 M NaCl or NH4CI or about 0.75 M to about 1.5 M NaCl or NH4CI.155. The method of any one or combination of numbered aspects disclosed herein, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resin in a mobile phase buffer comprising about 0.35 M to about 1.0 M MgCh or about 0.375 M to about 0.75 M MgCh.156. The method of any one or combination of numbered aspects disclosed herein, further comprising subjecting the exonuclease-resistant single stranded template DNA product to a desalting process.157. The method of any one or combination of numbered aspects disclosed herein, wherein the desalting process comprises tangential flow filtration.158. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is filtered for about 2 to about 20 diafiltration volumes, about 4 to about 10 diafiltration volumes, or about 6 to about 8 diafiltration volumes.159. The method of any one or combination of numbered aspects disclosed herein, wherein after the desalting process the exonuclease-resistant single stranded template DNA product has a conductivity less than 20,000 pS / cm or about less than 10,000 pS / cm.160. The method of any one or combination of numbered aspects disclosed herein, wherein after the desalting process the exonuclease-resistant double stranded template DNA product has a conductivity less than about 1,000 pS / cm, less than about 500 pS / cm, or less than about 100 pS / cm.161. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is concentrated using tangential flow filtration.162. The method of any one or combination of numbered aspects disclosed herein, wherein the exonuclease-resistant single stranded template DNA product is produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0.1 L to 12.5 L, 0.25 L to 6.25 L, 0.5 L to 3.5 L, or 0.75 L to 1.5 L.163. The method of any one or combination of numbered aspects disclosed herein, wherein the cell-free reaction mixture has a volume of about 0. 1 mL to about 250 mL, about1 mL to about 200 mL, about 5 mL to about 150 mL, about 10 mL to about 100 mL, about 15 mL to about 50 mL, or about 20 mL to about 40 mL.164. The method of any one or combination of numbered aspects disclosed herein, wherein the cell-free reaction mixture is filtered prior to the HPLC purification step.165. The method of any one or combination of numbered aspects disclosed herein, further comprising, after the purification step, dialyzing the exonuclease-resistant single stranded template DNA product into water to obtain a dialyzed product.166. The method of any one or combination of numbered aspects disclosed herein, further comprising, after purifying, dialyzing the exonuclease-resistant single strandedtemplate DNA product into a buffer that does not contain one or more of NaCl. NH4CI, MgCh. EDTA. and Tris-HCl to obtain a dialyzed product.167. The method of any one or combination of numbered aspects disclosed herein, further comprising freeze-drying the dialyzed product.168. The method of any one or combination of numbered aspects disclosed herein, wherein prior to the injection step, the HPLC system is equilibrated with one or more mobile phase buffers.OTHER ASPECTS

[0288] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative aspects, including various combinations and sub-combinations of features, those skilled in the art will readily appreciate other aspects and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such aspects, combinations, and subcombinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims. Section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0289] Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for producing an exonuclease-resistant single stranded template deoxyribonucleic acid (DNA) product for non-viral delivery to a cell, comprising: a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide configured to modify a second end of the template sequence, b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonucleaseresistant single stranded template DNA product. e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture, wherein the second capping oligonucleotide is resistant to exonuclease digestion, wherein one strand of the first capping oligonucleotide is resistant to exonuclease digestion, and wherein the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.

2. The method of claim 1, wherein the second capping oligonucleotide comprises a duplex structure modified with one or both of an exonuclease resistant chemical modification or an exonuclease resistant secondary structure.

3. The method of claim 1 , wherein the second capping oligonucleotide comprises a hairpin structure, a prehybridized duplex oligonucleotide, an RNA aptamer, 2'-O-methylnucleotides, 2'-O-methoxyethyl (MOE) nucleotides, phosphorothioate nucleotides, or a combination thereof.

4. The method of any preceding claim, wherein the second capping oligonucleotide comprises a hairpin structure with one or more phosphorothioate nucleotides.

5. The method of any preceding claim, wherein the second capping oligonucleotide comprises an MS2 RNA aptamer.

6. A method for producing an exonuclease-resistant single stranded template DNA product for non-viral delivery to a cell, comprising: a) performing a capping reaction by incubating a template DNA source molecule comprising at least one endonuclease recognition sequence and a template sequence in a cell-free reaction mixture comprising at least one endonuclease, a DNA ligase, a first capping oligonucleotide comprising a prehybridized duplex oligonucleotide and configured to modify a first end of the template sequence, and a second capping oligonucleotide comprising a hairpin structure and configured to modify a second end of the template sequence to produce a double stranded template DNA closed on one end. b) performing a first exonuclease digestion reaction by adding a first exonuclease to the cell-free reaction mixture, c) inactivating the first exonuclease, d) performing a second exonuclease digestion reaction by adding a second exonuclease to the cell-free reaction mixture to produce an exonucleaseresistant single stranded template DNA product, e) inactivating the second exonuclease, and f) performing a purification step by purifying the exonuclease-resistant single stranded template DNA product from the cell-free reaction mixture to obtain the exonuclease-resistant single stranded template DNA product, wherein one strand of the first capping oligonucleotide is resistant to exonuclease digestion, andwherein the first and second capping oligonucleotides are added in a molar excess over the template DNA source molecule.

7. The method of any preceding claim, wherein the capping reaction comprises sequentially adding a) to c) to the cell-free reaction mixture in the specified order: a) the template DNA source molecule; b) an aqueous solvent; c) a buffer; mixing after c) is added; then adding one or both of d) and e): d) the first capping oligonucleotide; e) the second capping oligonucleotide; and then adding one or both of f) and g): f) the at least one endonuclease; g) the DNA ligase.

8. The method of any preceding claim, wherein the capping reaction comprises: i) incubating the template DNA source molecule comprising the least one endonuclease recognition sequence and the template sequence in a cell-free reaction mixture comprising a first endonuclease, a first DNA ligase, and the first capping oligonucleotide to modify the first end of the template sequence; optionally inactivating the first endonuclease prior to step ii); and ii) incubating the template sequence modified on the first end with the second capping oligonucleotide to modify the second end of the template sequence, and optionally adding a second endonuclease, a second DNA ligase, or both after step i).

9. The method of claim 8. wherein the first and second endonuclease are the same or different.

10. The method of claim 8, wherein step i) and ii) are performed sequentially in the same reaction.

11. The method of claim 8. wherein step i) and ii) are performed as separate reactions.

12. The method of claim 8, wherein step i) and ii) are performed in the same reaction vessel.

13. The method of claim 8, wherein step i) and ii) are performed in separate reaction vessels, optionally the template sequence modified on the first end is purified prior to step ii).

14. The method of any one of claims 8 to 13, wherein step i) and ii) are performed at the same or different temperature.

15. The method of any one of claims 8 to 13, wherein step i) and ii) are each independently performed at one or more temperatures.

16. The method of any preceding claim, wherein the yield of the exonucleaseresistant single stranded template DNA product is 50% or more relative to the total amount of template DNA source molecule after performing the capping reaction for 4 hours or less. 3 hours or less, or 2 hour or less.

17. The method of any preceding claim, wherein the hairpin structure comprises a hairpin loop with 4 to 40, 6 to 36, 8 to 32, 10 to 28, 12 to 24, 14 to 20, or 16 to 18 nucleotides.

18. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both compnse one or more hairpin structures.

19. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises a 3’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides, or both the first capping oligonucleotide and the second capping oligonucleotide comprises a 3’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides.

20. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises a 5’ overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides.

21. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both comprises 15 to 100, 20 to 80, 25 to 75. 30 to 60, or 40 to 50 nucleotides, or both the first capping oligonucleotide and the second capping oligonucleotide comprises 15 to 100, 20 to 80, 25 to 75, 30 to 60, or 40 to 50 nucleotides.

22. The method of any preceding claim, wherein the digestion reaction step comprises anti-sense digestion.

23. The method any one of claims 1-21, wherein the digestion reaction step comprises sense digestion.

24. The method of any preceding claim, wherein the cell-free reaction mixture comprises each of the first and second capping oligonucleotides independently in a ratio of 5- fold to 200-fold, 10-fold to 100-fold, 15-fold to 80-fold, 20-fold to 70-fold, 25-fold to 60- fold, 30-fold to 50-fold, or 35-fold to 45-fold excess relative to the template DNA source molecule excess relative to the template DNA source molecule.

25. The method of any preceding claim, wherein the capping reaction is at least partially performed at a first temperature and optionally at a second temperature that is different than the first temperature.

26. The method of any preceding claim, wherein the capping reaction is performed at a first temperature after adding the at least one endonuclease and a second temperature after adding the ligase.

27. The method of claim 26, wherein the first temperature and the second temperature are each independently selected from a temperature in a range of about 10°C to about 42°C, about 14°C to about 40°C, about 15°C to about 39°C, about 16°C to about 37°C, or about 37°C.

28. The method of any one of claims 26 to 27, wherein the capping reaction is conducted at the first temperature for about 1 hour to about 72 hours.

29. The method of any one of claims 26 to 28, wherein the capping reaction is conducted at the second temperature for about 1 hour to about 72 hours.

30. The method of any one of claims 1 to 25, wherein the capping reaction is performed at a single temperature.

31. The method of any one of claims 1 to 25, wherein the capping reaction is performed at about 37 °C.

32. The method of any preceding claim, wherein the capping reaction is performed for about 0.5 to about 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 1 hour to about 2 hours.

33. The method of any preceding claim, wherein the capping reaction is performed for about 2 hours.

34. The method of any preceding claim, wherein the first exonuclease digestion reaction is performed at a temperature from about 35°C to about 40°C, about 36 °C to about 38 °C, or about 37°C.

35. The method of any preceding claim, wherein the second exonuclease digestion reaction is performed at a temperature from about 20 °C to about 37 °C, about 23 °C to about 35 °C, or about 25 °C.

36. The method of any preceding claim, wherein the duration of the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is shorter than the capping reaction.

37. The method of any preceding claim, wherein the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 0.5 toabout 10 hours, about 1 to about 8 hours, about 2 to about 6 hours, about 4 to about 5 hours, or about 0.5 to about 1.5 hours.

38. The method of any one of claims 1-37, wherein the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both is performed for about 5 minutes to about 1 hour, about 10 minutes to about 50 minutes, about 15 minutes to about 40 minutes, or about 20 minutes to about 30 minutes.

39. The method of any preceding claim, wherein the first exonuclease digestion reaction is performed for 20 minutes to 60 minutes.

40. The method of any preceding claim, wherein the second exonuclease digestion reaction is performed for 10 minutes to 30 minutes.

41. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product comprises about 500 to about 15,000 nucleotides in length, about 1000 to about 14,000 nucleotides in length, about 2000 to about 13,000 nucleotides in length, about 3000 to about 12,000 nucleotides in length, about 4000 to about 11,000 nucleotides in length, or about 5000 to about 10,000 nucleotides in length.

42. The method of claim 41, wherein the exonuclease-resistant single stranded template DNA product comprises about 5000 to about 10,000 nucleotides in length.

43. The method of any preceding claim, wherein the capping reaction is performed for a longer duration than the first exonuclease digestion reaction, the second exonuclease digestion reaction, or both.

44. The method of any preceding claim, wherein the step of inactivating the first exonuclease, the second exonuclease, or both comprises a heat inactivation or a chelating agent inactivation.

45. The method of claim 44, wherein the step of inactivating the first exonuclease comprises a heat inactivation.

46. The method of claim 45, wherein the heat inactivation is performed at about50°C to about 90°C, about 60°C to about 80°C, about 65°C to about 75°C.

47. The method of claim 44, wherein the heat inactivation step is performed at about 70°C.

48. The method of any one of claims 44-47, wherein the heat inactivation is performed for 1 minute or more, or 2 minutes or more, or 5 minutes or more, 10 minutes or more, 60 minutes or less, 30 minutes or less, about 5 minutes to about 20 minutes, about 7 minutes to about 15 minutes, or about 10 minutes.

49. The method of claim 44, wherein the chelating agent inactivation comprises adding a chelating agent at a concentration of 5 mM to 100 mM, 7.5 mM to 80 mM, 10 mM to 60 mM, 12.5 mM to 40 mM, or 15 mM to 20 mM.

50. The method of any one of claims 44-49, wherein the chelating agent is EDTA.

51. The method of any preceding claim, wherein the step of inactivating the first exonuclease comprises a heat inactivation and the step of inactivating the second exonuclease comprises a chelating agent inactivation.

52. The method of any preceding claim, wherein non-template plasmid DNA in the cell-free reaction mixture is degraded after a single digestion reaction.

53. The method of any preceding claim, wherein a single digestion reaction produces the exonuclease-resistant single stranded template DNA product.

54. The method of any preceding claim, wherein the non-template plasmid DNA is digested without damaging at least one strand of the template DNA molecule.

55. The method of any preceding claim, comprising cooling the heat inactivated reaction mixture to a temperature from about 20 °C to about 37 °C, about 23 °C to about 35 °C. or about 25 °C before adding the second exonuclease.

56. The method of claim 55, wherein the cooling step comprises cooling the heat inactivated reaction mixture from about 70°C to about 25°C.

57. The method of any preceding claim, wherein the exonuclease is selected from T7 exonuclease, Exonuclease I, Exonuclease III, Exonuclease VII, or Exonuclease VIII.

58. The method of any preceding claim, wherein the first exonuclease is Exonuclease III.

59. The method of any preceding claim, wherein the second exonuclease is T7 Exonuclease or further comprises T7 Exonuclease as a second exonuclease.

60. The method of any preceding claim, wherein a) Lambda Exonuclease is not used, b) biotin modification is not used, c) rolling circle amplification is not used, d) streptavidin-coated beads are not used, or any combination of a)-d) are not used.

61. The method of any preceding claim, wherein the second capping oligonucleotide comprises a hairpin loop with 4-20 base pairs.

62. The method of any preceding claim, wherein the hairpin structure comprises phosphorothioate bonds.

63. The method of any preceding claim, wherein one strand of the prehybridized duplex oligonucleotide comprises phosphorothioate bonds.

64. The method of any preceding claim, wherein 2 to 20, 4 to 18, 6 to 16, 8 to 14, or 10 to 12 nucleotides of the hairpin loop comprise phosphorothioate bonds.

65. The method of any preceding claim, wherein the prehybridized duplex oligonucleotide comprises two strands and wherein each strand of the prehybridized duplex oligonucleotide independently comprises 2 to 200, 4 to 180, 6 to 160, 8 to 140, 10 to 120, 12 to 100, 10 to 80, 12 to 60, 14 to 40, 16 to 20 nucleotides.

66. The method of any preceding claim, wherein the strands of the prehybridized duplex oligonucleotide are different lengths.

67. The method of any preceding claim, wherein the prehybridized duplex oligonucleotide comprises a nucleotide overhang, optionally on each end.

68. The method of any preceding claim, wherein the prehybridized duplex oligonucleotide comprises an overhang of 2 to 12, 4 to 10, or 6 to 8 nucleotides.

69. The method of any preceding claim, wherein the prehybridized duplex oligonucleotide comprises a functional group attached at one end.

70. The method of claim 69, wherein the functional group is exonuclease resistant.

71. The method of claim 70, wherein the functional group comprises a G- quadruplex structure, a hairpin structure, an RNA aptamer, a C3 to C12 spacer group, or PEG.

72. The method of claim 71, wherein the functional group comprises an MS2 RNA aptamer.

73. The method of any one of claims 69-72, wherein the functional group comprises one or more phosphorothioate bonds.

74. The method of any preceding claim, wherein 2 to 50, 4 to 40, 6 to 30, 8 to 20, or 10 to 12 nucleotides of one strand of the prehybridized duplex oligonucleotide comprises phosphorothioate bonds.

75. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or the first and the second capping oligonucleotide comprises phosphorothioate bonds.

76. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified 5’-end.

77. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified 3?-end.

78. The method of any preceding claim, wherein the first capping oligonucleotide, the second capping oligonucleotide, or both the first and the second capping oligonucleotide comprises a phosphate-modified overhang end.

79. The method of any preceding claim, wherein the at least one endonuclease is a Type IIS enzy me.

80. The method of any preceding claim, wherein the at least one endonuclease recognition sequence is a recognition sequence for the at least one endonuclease selected from Acul, Alw261, Alwl, Bael, BbsI, BbsI-HF, Bbvl, Bed, BceAI, Bcgl, BciVI, BcoDI, BfuAI, BmrI, Bmsl, Bpil, BpmI. BpuEI. Bsal. Bsal-HF V2. BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI, BsmBI-v2, BsmFI, BsmI, BspCNI. BspMI, BspQI, BsrDI, BsrI. BtgZI, BtsCI, Btsl-Mutl, Btsl-v2, CspCI, Earl, Ecil, Eco311, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lgul, Mval269I, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, or SfaNI.

81. The method of claim 80, wherein the at least one endonuclease recognition sequence is a recognition sequence for at least one endonuclease selected from Bsal, Bsal- HFv2, BsmBI, BsmBI-v2, Esp3I, BbsI, BbsI-HF, SapI, or isoschizomers thereof.

82. The method of any preceding claim, wherein the at least one endonuclease is selected from Acul, Alw261. Alwl, Bael, BbsI, BbsI-HF, Bbvl. Bccl. BceAI. Bcgl. BciVI. BcoDI, BfuAI, BmrI, Bmsl, Bpil, BpmI, BpuEI, Bsal, Bsal -HF V2, BsaXI, BseGI, BseRI, Bsgl, BsmAI, BsmBI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-Mutl. Btsl-v2, CspCI, Earl, Ecil, Eco311, Esp3I, Faul, FokI, Hgal, HphI, HpyAV, Lgul, Mval269I, MboII, Mlyl. Mmel, Mnll. NmeAIII, PaqCI, Piel. SapI. or SfaNI.

83. The method of claim 82, wherein the at least one endonuclease is selected from Bsal, BsaI-HFv2, BsmBI, BsmBI-v2, Esp3I. BbsI, BbsI-HF, SapI, or isoschizomers thereof.

84. The method of any preceding claim, wherein the at least one endonuclease is added at a concentration of about 1-10%, about 2-8%. about 2.5-5%, about 2%, about 2.5%, about 3%, or about 3.5% v / v of the cell-free reaction mixture.

85. The method of any preceding claim, wherein the ligase is selected from T3 ligase, T4 ligase. T7 ligase, Taq ligase, DNA ligase I. DNA ligase, II, DNA ligase III, DNA ligase VI, Tth DNA ligase, or E. coli DNA ligase.

86. The method of any preceding claim, wherein the ligase is T4 ligase.

87. The method of any preceding claim, wherein the ligase is added at a concentration of about 0.05-5%, about 0.1-4%, about 0.125-3%, or about 0.1%, about 0.125%, about 0.15%, or about 0.2% v / v of the cell-free reaction mixture.

88. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is produced isothermally in steps a) to e).

89. The method of any preceding claim, wherein steps a) performing a capping reaction, b) performing a first exonuclease digestion reaction, and c) inactivating the first exonuclease are performed at a temperature from about 10°C to about 42°C, about 14 °C to about 40 °C, about 15 °C to about 39 °C, or about 16 °C to about 37°C.

90. The method of any preceding claim, wherein steps a) to c) are performed at about 37°C.

91. The method of any preceding claim, wherein the total yield of the exonuclease-resistant single stranded template DNA product is 25 to 100 mg per liter of the cell-free reaction mixture within 6 hours, 100 to 400 mg per liter of the cell-free reaction mixture within 24 hours, or 200 to 800 mg per liter of the cell-free reaction mixture within 48 hours.

92. The method of claim 91, wherein the exonuclease-resistant single stranded template DNA product comprises 3,000 to 4,000 nucleotides in length.

93. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is produced in 1 hour to 48 hours, 2 hours to 24 hours. 3 hours to 18 hours, 4 hours to 12 hours, or 6 hours to 8 hours.

94. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is produced in a single contiguous reaction solution.

95. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0.1 L to 12.5 L, 0.25 L to 6.25 L, 0.5 L to 3.5 L, or 0.75 L to 1.5 L.

96. The method of any preceding claim, comprising producing the exonucleaseresistant single stranded template DNA product in a bioreactor.

97. The method of any preceding claim, wherein the purification step comprises a silica-based purification procedure.

98. The method of any preceding claim, wherein the purification step comprises performing a high-performance liquid chromatography (HPLC) purification step using a HPLC system, optionally wherein: a) the HPLC purification step comprises anion exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof, b) the HPLC purification step comprises one or more separation modes, c) the HPLC purification step comprises a ramping separation mode, a step- wise separation mode, or a combination thereof, or d) the HPLC purification step comprises a combination of any one or more of a), b), and c).

99. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is resistant to Dnase.

100. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product has a frictional ratio of 4 to 20, 6 to 18, 8 to 16, or 10 to 12, as measured by analytical ultracentrifugation.

101. The method of any preceding claim, further comprising the exonucleaseresistant single stranded template DNA product in a lipid composition.

102. The method of any preceding claim, wherein the template DNA source molecule comprises a linear double stranded DNA, a plasmid DNA, a minicircle DNA, a cosmid DNA, a bacterial artificial chromosome, a molecular inversion probe, a doggybone DNA, a vector, a cDNA, an AAV vector, or a PCR product.

103. The method of any preceding claim, comprising adding an RNA aptamer at the first or second end, adding a G4 quadruplex at the first or second end, or adding an RNA aptamer at the first end and adding a G4 quadruplex at the second end.

104. The method of any preceding claim, wherein the RNA aptamer comprises 2’- O-methyl groups.

105. The method of claim 104, wherein the aptamer is an MS2 RNA aptamer.

106. The method of any preceding claim, wherein the purification step further includes adding DNA-binding beads.

107. The method of claim 106, wherein the DNA-binding beads comprise a polystyrene core covered by a magnetite layer, and the magnetite layer is covered by a carboxylate-modified polymer coating.

108. The method of claim 106 or claim 107, wherein the beads are provided at a ratio of 0.5 to 0.9, 0.6 to 0.

8. 0.65 to 0.75, or about 0.7 to the exonuclease-resistant single stranded template DNA product.

109. The method of any one of claims 106-108. further comprising purifying the exonuclease-resistant single stranded template DNA product by performing multiple elutions of the beads.

110. The method of any preceding claim, wherein exonuclease-resistant single stranded template DNA product comprises no phosphorothioate modifications.

111. The method of any preceding claim, wherein the template DNA source molecule comprises a first endonuclease recognition sequence at the first end of the template sequence and a second endonuclease recognition sequence at the second end of the template sequence.

112. The method of any preceding claim, wherein the first end and the second end of the template sequence comprises a different overhang end following digestion of the template DNA source molecule by two different endonucleases.

113. An exonuclease-resistant single stranded template DNA product prepared by the method of any preceding claim.1 14. A lipid composition comprising the exonuclease-resistant single stranded template DNA product prepared by the method of any preceding claim.

115. The lipid composition of claim 1 14, wherein the lipid composition further encapsulates one or more peptides, polypeptides, one more cationic salts, one or more anionic salts, one or more additional polynucleotides.

116. The lipid composition of claim 1 14, wherein the lipid composition comprises microspheres, liposomes, lipoplexes, or lipid nanoparticles.

117. A exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification and the second modification are exonuclease resistant, andwherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15.000 nucleotides in length.

118. An exonuclease-resistant single stranded template DNA product, comprising a first end comprising a first modification and a second end comprising a second modification, wherein the first modification, the second modification, or both the first and second modification comprises a linear sequence comprising one or more phosphorothioate bonds or an exonuclease resistant functional group, wherein the exonuclease resistant functional group does not comprise biotinylation.

119. The exonuclease-resistant single stranded template DNA product of claim 117 or claim 118, wherein the first modification and the second modification comprises a linear sequence comprising one or more phosphorothioate bonds.

120. The exonuclease-resistant single stranded template DNA product of claim 117 or claim 118, wherein the first modification or the second modification comprises an exonuclease resistant functional group.

121. The exonuclease-resistant single stranded template DNA product of claim 120, wherein the exonuclease resistant functional group comprises a hairpin structure.

122. The exonuclease-resistant single stranded template DNA product of claim 120, wherein the exonuclease resistant functional group comprises a polynucleotide secondary’ structure comprising phosphorothioate bonds.

123. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) one or more helper lipids;(c) one or more neutral lipids;(d) optionally, a pegylated lipid; and(e) an exonuclease-resistant single stranded template DNA product, wherein the exonuclease-resistant single stranded template DNA product comprises a first end comprising a first modification and a second end comprising a second modification,wherein the first modification and the second modification are exonuclease resistant, and wherein the exonuclease-resistant single stranded template DNA product is about 5000 to about 15,000 nucleotides in length.

124. A lipid nanoparticle comprising:(a) an ionizable lipid;(b) one or more helper lipids;(c) one or more neutral lipids;(d) optionally, a pegylated lipid; and(e) the exonuclease-resistant single stranded template DNA product of any one of claims 117-122.

125. A method for inserting a custom DNA template into a cell comprising introducing an exonuclease-resistant single stranded template DNA product into a cell comprising: a) producing the exonuclease-resistant single stranded template DNA product by the method of any one of claims 1-112; b) contacting the cell with the exonuclease-resistant single stranded template DNA product and a gene editing system; and inserting at least a part of a template sequence from the exonuclease-resistant single stranded template DNA product into DNA of the cell that has been cleaved by the gene editing system.

126. The method of claim 125, wherein the gene editing system comprises a CRISPR / Cas system; a Tth Argonaute (TtAgo) system; a zinc finger nuclease (ZFN) system; ARCUS nuclease system; megaTALs; or a transcription activator-like effector nuclease (TALEN) system.

127. The method of claim 125 or claim 126, wherein the cell is a non-cycling cell.

128. The method of claim 125 or claim 126, wherein the cell is a cycling cell.

129. The method of claim 125, further comprising performing non-homologous end joining (NHEJ) repair or homology directed (HD) repair in the cell.

130. The method of any one of claims 125-129, wherein the custom DNA template is >5 kB, >6 kB, >7 kB, >8 kB, >9 kB, or >10 kB in size.

131. The method of any one of claims 125-130. wherein the exonuclease-resistant single stranded template DNA product is encapsulated in a lipid nanoparticle.

132. The method of any one of claims 125-131, wherein the inserting is performed in vitro.

133. The method of any one of claims 125-131, wherein the inserting is performed ex vivo.

134. The method of any one of claims 125-131, wherein the inserting is performed in vivo.

135. The method of claim 98, wherein the purification step comprises anion exchange chromatography, hydrophilic interaction chromatography, size exclusion chromatography, hydrophobic interaction chromatography, or any combination thereof.

136. The method of any one of claims 98 and 135, wherein the HPLC purification step comprises using one or more separation modes.

137. The method of any one of claims 98 and 135-136, wherein the purification step comprises a ramping separation mode, a step-wise separation mode, or a combination thereof.

138. The method of any one of claims 98 and 135-137, wherein the purification step comprises:(i) injecting the exonuclease-resistant single stranded template DNA product onto the HPLC system comprising an anion exchange resin under conditions to bind the exonuclease-resistant single stranded template DNA product to the anion exchange resin;(ii) separating the exonuclease-resistant single stranded template DNA product from impurities using a ramping separation mode, a step-wise separation mode, or a combination thereof; and(iii) eluting the exonuclease-resistant single stranded template DNA product from the HPLC system.

139. The method of any one of claims 98 and 135-138. wherein the purification step comprises using one or more mobile phase buffers.

140. The method of any one of claims 98 and 135-139, wherein the purification step comprises using two mobile phase buffers.

141. The method of any one of claims 139 and 140, wherein the mobile phase buffers comprise an aqueous or organic solvent composition.

142. The method of any one of claims 139-141. wherein the mobile phase buffers comprise an ion pairing agent.

143. The method of claim 142, wherein the ion pairing agent comprises triethylamine, tripropylamine, hexylamine, butylamine, dibutylamine, or a tetraalkyl ammonium salt, optionally wherein the ion pairing agent is triethylammonium acetate (TEAA).

144. The method of any one of claims 142-143, wherein the ion pairing agent is present at a concentration of about 0.01 M to about 1 M, about 0.05 M to about 0.75 M, or about 0. 1 M to about 0.5 M145. The method of any one of claims 138-144. wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer comprising less than about 600 mM NaCl or NH4CI or less than about 500 mM NaCl or NTUCl .

146. The method of any one of claims 138-144, wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer comprising less than about 300 mM MgCb or less than about 250 mM MgCk.

147. The method of any one of claims 138-144, wherein the exonuclease-resistant single stranded template DNA product binds to the anion exchange resin in a mobile phase buffer without NaCl. NTUCl, or MgCh.

148. The method of any one of claims 98 and 138-147, wherein the purification step comprises running the HPLC system with a mobile phase flow rate of about 1 mL / min to about 200 mL / min, about 5 mL / min to about 100 mL / min, or about 10 mL / min to about 50 mL / min.

149. The method of any one of claims 98 and 138-148, wherein the purification step comprises a step-wise separation mode with two or more steps.

150. The method of any one of claims 139 or 140, wherein each mobile phase buffer comprises a chelating agent.

151. The method of claim 150, wherein the chelating agent is EDTA.

152. The method of any one of claims 150-151, wherein the chelating agent is included at a concentrarion of 0. 1 mM to 10 mM, or about 0.5 mM to about 5 mM, or about 1 mM.

153. The method of any one of claims 98 and 138-152, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resin using a mobile phase buffer composition comprising NaCl, NH4CI, or MgCh.

154. The method of claim 153, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resinin a mobile phase buffer comprising about 0.70 M to about 2.0 M NaCl or NH4CI or about 0.75 M to about 1.5 M NaCl or NH4Cl.

155. The method of claim 153, wherein the purification step comprises eluting the exonuclease-resistant single stranded template DNA product from the anion exchange resin in a mobile phase buffer comprising about 0.35 M to about 1.0 M MgCh or about 0.375 M to about 0.75 M MgCb.

156. The method of any one of claims 98 and 138-155, further comprising subjecting the exonuclease-resistant single stranded template DNA product to a desalting process.

157. The method of claim 156, wherein the desalting process comprises tangential flow filtration.

158. The method of any one of claims 156-157. wherein the exonuclease-resistant single stranded template DNA product is filtered for about 2 to about 20 diafiltration volumes, about 4 to about 10 diafiltration volumes, or about 6 to about 8 diafiltration volumes.

159. The method of any one of claims 156-157, wherein after the desalting process the exonuclease-resistant single stranded template DNA product has a conductivity less than 20,000 pS / cm or about less than 10,000 pS / cm.

160. The method of any one of claims 156-159, wherein after the desalting process the exonuclease-resistant double stranded template DNA product has a conductivity less than about 1,000 pS / cm, less than about 500 pS / cm, or less than about 100 pS / cm.

161. The method of any preceding claim, wherein the exonuclease-resistant single stranded template DNA product is concentrated using tangential flow filtration.

162. The method of any one of claims 138-161, wherein the exonuclease-resistant single stranded template DNA product is produced in a reaction volume 0.01 L to 50 L, 0.5 L to 25 L, 0.1 L to 12.5 L, 0.25 L to 6.25 L, 0.5 L to 3.5 L, or 0.75 L to 1.5 L.

163. The method of any one of claims 138-162. wherein the cell-free reaction mixture has a volume of about 0. 1 mL to about 250 mL, about 1 mL to about 200 mL, about 5 mL to about 1 0 mL, about 10 mL to about 100 mL, about 15 mL to about 50 mL, or about 20 mL to about 40 mL.

164. The method of any one of claims 98 and 138-163. wherein the cell-free reaction mixture is filtered prior to the HPLC purification step.

165. The method of any one of claims 98 and 138-164, further comprising, after the purification step, dialyzing the exonuclease-resistant single stranded template DNA product into water to obtain a dialyzed product.

166. The method of any one of claims 98 and 138-164, further comprising, after purifying, dialyzing the exonuclease-resistant single stranded template DNA product into a buffer that does not contain one or more of NaCl, NHiCl, MgCh, EDTA, and Tris-HCl to obtain a dialyzed product.

167. The method of any one of claims 161 and 165-166, further comprising freeze- drying the dialyzed product.

168. The method of any one or claims 138-167 wherein prior to the injection step, the HPLC system is equilibrated with one or more mobile phase buffers.

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