DNA ligase composition, method, and use thereof

Abstract

While many CRISPR systems have emerged as useful tools for gene editing, such systems are limited by off-target effects, the length of the inserted sequence, and inefficient nucleic acid editing with low fidelity. DNA ligase compositions, guide polynucleotides, donor nucleic acids, systems, methods, and their uses are provided. Methods for modifying target nucleic acids and methods for genetically modifying cells are described. These methods may be used for biotechnology applications, therapeutic treatments, and the production of cell therapies for the treatment of various diseases and conditions. Scaffolds and kits, including the compositions and systems described herein, are also included.

Description

[Technical Field]

[0001] Related applications This application claims the benefit of U.S. Provisional Application No. 63 / 584,712, filed on 22 September 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The name of the ASCII copy created on September 19, 2024, is 219001_702601_SL.txt, and its size is 1,265,664 bytes. [Background technology]

[0003] background While many CRISPR systems have emerged as useful tools for gene editing, such systems are limited by off-target effects, the length of the inserted sequences, and inefficient nucleic acid editing with low fidelity. Therefore, there is a great unmet need for gene modification systems that can address these concerns. [Overview of the project]

[0004] overview A composition is provided herein, comprising (a) a donor nucleic acid; (b) a polynucleotide encoding a protein construct comprising a nickase or a variant thereof and a DNA ligase or a functional fragment thereof; (c) a guide polynucleotide, comprising (i) a target-directing region having complementarity with a target nucleic acid, (ii) a protein-binding region comprising a secondary structure that binds to an engineered protein construct comprising a nickase region; (iii) a ligation sprint 2 region, wherein the ligation sprint region has complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint region comprises at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region, wherein the ligation sprint 1 region comprises a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region has complementarity with the target nucleic acid, comprising a guide polynucleotide.

[0005] An engineered fusion protein is provided herein, comprising (a) a DNA ligase or a functional fragment thereof, and (b) an engineered nickase having three amino acid substitutions at positions corresponding to amino acid positions 221, 394, and 840 of a nuclease having the sequence of SEQ ID NO: 69.

[0006] A system for modifying a target nucleic acid is provided herein, comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; (d) a guide polynucleotide, comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; (iii) a ligation sprint 2 region, the ligation sprint 2 region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint 2 region having at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region, the ligation sprint 1 region having complementarity with the target nucleic acid, and the guide polynucleotide, wherein when introduced into a cell or cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

[0007] A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint region is A system is provided herein that comprises a guide polynucleotide comprising a ligation sprint 2 region having at least one change compared to a target nucleic acid, and (iv) a ligation sprint 1 region, the ligation sprint 1 region comprising one or more ribonucleotides or one or more deoxyribonucleotides, and the ligation sprint 1 region having complementarity with the target nucleic acid, wherein when introduced into a cell or cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

[0008] A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and ligation A system is provided herein that comprises a guide polynucleotide, including a ligation sprint region having at least one change compared to the target nucleic acid, a ligation sprint 2 region, and (iv) a ligation sprint 1 region, the ligation sprint 1 region comprising one or more deoxyribonucleotides, and the ligation sprint 1 region having complementarity with the target nucleic acid, wherein when introduced into cells or a cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

[0009] A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, A system is provided herein which, when introduced into cells or a cell-free system, incorporates a donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid. The system comprises a ligation sprint region having at least one change compared to the target nucleic acid, a ligation sprint 2 region, and (iv) a ligation sprint 1 region, wherein the ligation sprint 1 region comprises a guide polynucleotide comprising a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region having complementarity with the target nucleic acid.

[0010] A composition comprising a system provided herein, an engineered protein construct provided herein, a DNA ligase or a functional fragment thereof provided herein, or a guide polynucleotide provided herein is provided herein.

[0011] A composition comprising a system provided herein and a delivery vehicle is provided herein.

[0012] Polynucleotides that encode a system provided herein, an engineered protein provided herein, or a guide polynucleotide provided herein are provided herein.

[0013] A set of polynucleotides encoding a system provided herein, an engineered protein provided herein, or a guide polynucleotide provided herein is provided herein.

[0014] Nanoparticles comprising a polynucleotide provided herein, a set of polynucleotides provided herein, a system provided herein, a composition provided herein, a cell provided herein, a vector provided herein, or any part thereof, are provided herein.

[0015] A vector comprising a polynucleotide provided herein, or a set of polynucleotides provided herein, is provided herein.

[0016] Cells comprising a system provided herein, an engineered protein provided herein, a composition provided herein, a vector provided herein, or a guide polynucleotide provided herein are provided herein.

[0017] A composition is provided herein, comprising a donor nucleic acid, a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region having complementarity with a target nucleic acid, (b) a protein-binding region comprising a secondary structure that binds to a protein, (c) a ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand, and (d) a ligation sprint 1 region comprising one or more deoxyribonucleotides or one or more ribonucleotides, the guide polynucleotide, or a polynucleotide encoding a guide polynucleotide.

[0018] A composition is provided herein, comprising a donor nucleic acid, a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region having complementarity with a target nucleic acid, (b) a protein-binding region comprising a secondary structure that binds to a protein, (c) a ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and (d) a ligation sprint 1 region comprising a ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide.

[0019] A method for ligating a donor nucleic acid with a target nucleic acid, the method comprising: a cell or cell-free system; (a) a donor nucleic acid; (b) a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region having complementarity with the target nucleic acid; (ii) a protein-binding region comprising a secondary structure that binds to a nuclease or nickase; and (iii) a ligation sprint 2 region, the ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region comprising one or more deoxyribonucleotides or one or more ribonucleotides. A method is provided herein that comprises (c) a polynucleotide encoding a do polynucleotide or guide polynucleotide, and (c) an engineered protein or a polynucleotide encoding an engineered protein, wherein the engineered protein comprises (i) a nickase region and (ii) a DNA ligase region, wherein the guide polynucleotide complexes with the engineered protein via a protein-binding region, a target-directing sequence complexes with a complementary strand, the nickase region of the engineered protein causes cleavage at the target nucleic acid to generate a leading strand and a complementary strand, a ligation sprint 1 region complexes with the leading strand, and the DNA ligase region attaches the donor nucleic acid to the leading strand, thereby ligating the donor nucleic acid to the target nucleic acid.

[0020] A method is provided herein that comprises administering a system, a composition, or cells provided herein to a subject, organ, tissue, or cell, wherein the administration modifies genes in the subject, organ, tissue, or cell.

[0021] A method is provided herein, comprising contacting cells or a population of cells with a system or composition provided herein, thereby modifying genes in the cells.

[0022] A population of cells, which is produced by a method provided herein, is provided herein.

[0023] A kit is provided herein, comprising: a first container comprising a donor nucleic acid and a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises (i) a target-directing region complementary to the target nucleic acid, (ii) a protein-binding region comprising a secondary structure that binds to a nuclease or nickase, (iii) a ligation sprint 2 region, wherein the region that hybridizes to the donor is complementary to the donor nucleic acid and complementary to the target nucleic acid, and the ligation sprint 2 region comprises at least one mismatch nucleic acid base for the target nucleic acid, and (iv) a ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide, and a second container comprising an engineered polypeptide comprising a nickase operably linked to a DNA ligase.

[0024] A scaffold is provided herein, comprising a system or composition provided herein, and a solid surface on which the system or composition is fixed. [Brief explanation of the drawing]

[0025] Brief explanation of the drawing Novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings.

[0026] [Figure 1A] Figures 1A and 1B show schematic diagrams of one embodiment of the system provided herein. Figure 1A shows a schematic diagram of the DNA editing process involving an engineered protein containing Cas9 nickase-DNA ligase and a guide polynucleotide designed to edit the target nucleic acid by forming a complex with a double-stranded target nucleic acid and ligating the donor nucleic acid to the target nucleic acid sequence. Figure 1B shows a schematic diagram of a ligation editing guide polynucleotide (legRNA) for DNA editing by DNA ligase. The legRNA contains a ligation sprint 2 region (LS2) and a ligation sprint 1 region (LS1). Legend: D is deoxyribonucleotide (DNA) and R is ribonucleotide (RNA). [Figure 1B] Same as above.

[0027] [Figure 2]Figure 2 is an image of a denatured urea-polyacrylamide gel showing targeted DNA editing by a DNA ligase with trans-sprint oligonucleotides. Lanes 1 and 12 show oligoladders for unnicked (oligo1, 54 nucleotides (nt)) DNA and pre-nicked (oligo32, 34 nt) DNA. The band in lane 2 represents a Cas9 nickase (H840A) control using in vitro transcribed single guide RNA. The band in lane 3 represents a positive control, including a 15 nt DNA writing (49 nt) insertion (oligo32, oligo11, oligo22), as well as spike-in oligoladders for unnicked (oligo1, 54 nt) DNA and pre-nicked (oligo32, 34 nt) DNA. The band in lane 4 represents a positive control, including a 90nt DNA writing (49nt) insertion (oligo32, oligo11, oligo22), as well as spike-in oligo ladders for unnicked (oligo1, 54nt) DNA and pre-nicked (oligo32, 34nt) DNA. The band in lane 5 represents a negative control DNA ligase edit without Cas9 nickase (H840A). The bands in lanes 6-11 represent the products of DNA ligation editing using a trans-oligo22-containing DNA ligation sprint and a ligation donor oligonucleotide with oligo11 (lane 6), oligo12 (lane 7), oligo13 (lane 8), oligo14 (lane 9), oligo15 (lane 10), or oligo16 (lane 11).

[0028] [Figure 3]Figure 3 is an image of a denatured urea polyacrylamide gel showing targeted DNA editing by DNA ligases with synthetic single-guide polynucleotides. The bands in lanes 1 and 12 represent oligoladders showing unnicked (oligo 1, 54 nt) DNA and pre-nicked (oligo 2, 34 nt) DNA. The band in lane 2 represents a Cas9 nickase (H840A) control using guide 3. The bands in lanes 3-11 represent the products of DNA ligation editing using guide 1 (lane 3), guide 2 (lane 4), guide 3 (lane 5), guide 4 (lane 6), guide 5 (lane 7), guide 6 (lane 8), guide 7 (lane 9), guide 8 (lane 10), or guide 9 (lane 11).

[0029] [Figure 4] Figure 4 is a bar graph showing the efficiency of DNA ligase genome editing in human cells using three manipulated protein constructs. The Y-axis represents the efficiency (%) of the desired editing. The X-axis represents the manipulated protein constructs (constructs 10-12, sequence numbers 98-100).

[0030] [Figure 5] Figure 5 is a bar graph showing the DNA ligase genome editing efficiency in human cells at FANCF site 1 (gRVB_3) using the Chlorella DNA ligase editor construct (construction 10) and various combinations of sprint modification and DNA donor modification. The Y-axis represents the efficiency (%) of the desired editing. The X-axis represents the combinations of sprint modification and DNA donor modification, including oRVB_61 / oRVB_95 (sequence ID 184 / sequence ID 218), oRVB_61 / oRVB_96 (sequence ID 184 / sequence ID 219), oRVB_61 / oRVB_97 (sequence ID 184 / sequence ID 220), oRVB_62 / oRVB_95 (sequence ID 185 / sequence ID 218), oRVB_62 / oRVB_96 (sequence ID 185 / sequence ID 219), and oRVB_62 / oRVB_97 (sequence ID 185 / sequence ID 220).

[0031] [Figure 6] Figure 6 is a bar graph showing the DNA ligase genome editing efficiency at HEK site 3 in human cells using the Chlorella DNA ligase editor construct (construction 10) and various sprint constructs containing RNA and various sprint constructs without RNA. The Y-axis represents the efficiency (%) of the desired editing. The X-axis represents various sprint constructs containing RNA and various sprint constructs without RNA, including oRVB_81 / oRVB_82 (sequence number 204 / sequence number 205), oRVB_69 / oRVB_70 (sequence number 192 / sequence number 193), oRVB_77 / oRVB_78 (sequence number 200 / sequence number 201), oRVB_65 / oRVB_66 (sequence number 188 / sequence number 189), and oRVB_73 / oRVB_74 (sequence number 196 / sequence number 197).

[0032] [Figure 7] Figure 7 is a bar graph showing the DNA ligase genome editing efficiency at AAV site 1 in human cells using the Chlorella DNA ligase editor construct (construction 10) and various sprint constructs containing RNA and various sprint constructs without RNA. The Y-axis represents the efficiency (%) of the desired editing. The X-axis represents sprint constructs containing RNA and sprint constructs without RNA, including oRVB_79 / oRVB_80 (sequence number 202 / sequence number 203), oRVB_75 / oRVB_76 (sequence number 198 / sequence number 199), oRVB_63 / oRVB_64 (sequence number 186 / sequence number 187), oRVB_67 / oRVB_68 (sequence number 190 / sequence number 191), and oRVB_71 / oRVB_72 (sequence number 194 / sequence number 195).

[0033] [Figure 8]Figure 8 is a bar graph showing the DNA ligase genome editing efficiency at AAV site 1 in human cells using a Chlorella DNA ligase editor construct (construction 10) and guide RNAs (legRNAs) with sprint regions incorporating varying amounts of RNA. The Y-axis represents the efficiency (%) of the desired editing. The X-axis represents the guide RNA constructs, including gRVB_9 / gRVB_13 (sequence ID 110 / sequence ID 114), gRVB_16 / gRVB_20 (sequence ID 117 / sequence ID 121), and gRVB_17 / gRVB_21 (sequence ID 118 / sequence ID 122).

[0034] Various embodiments will be described in more detail below. However, such embodiments may be embodied in many different forms and should not be construed as being limited to the embodiments described herein. [Modes for carrying out the invention]

[0035] Detailed description of the invention Compositions, kits, methods and uses for editing donor nucleic acid sequences using DNA ligases and ligating them to target genes are provided herein. Briefly, this specification further describes: (1) gene editing systems, (2) delivery vehicles and vectors, (3) cell and cell-free systems, (4) pharmaceutical compositions, dosing and administration, (5) scaffolds and systems, (6) kits, (7) gene editing activities, and (8) applications.

[0036] This specification provides compositions and systems for gene editing that can be used in a variety of applications, including gene editing, diagnostics, and biopharmaceutical manufacturing. The compositions and systems provided herein comprise engineered proteins having nickase activity and DNA ligase activity, the activity of which enables the engineered proteins to bind to dsDNA and form nicks on non-target DNA strands to modify target nucleic acids.

[0037] The compositions and systems provided herein also include a guide polynucleotide and a donor nucleic acid for integration into the target nucleic acid. The guide polynucleotide binds to the manipulated protein and the target nucleic acid to facilitate loop formation between the cleaved target nucleic acid and the guide polynucleotide, thereby promoting high-fidelity gene editing. The guide polynucleotide mediates hybridization of the donor nucleic acid, bringing the 3' end of the cleavage site of the target nucleic acid and the 5' end of the donor nucleic acid into close proximity. The target nucleic acid and the guide polynucleotide form a complex, providing a substrate for the DNA ligase to directly attach the donor nucleic acid sequence of the guide nucleic acid to the 3' end of the cleavage site. The compositions and systems provided herein enable controlled editing results and have only limited off-target effects. Furthermore, the compositions and systems provided herein enable the insertion of large nucleic acid sequences that can be precisely integrated into the target nucleic acid.

[0038] definition All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meanings of the defined terms.

[0039] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter from which they are cited, and in some cases may encompass the entire document. All references disclosed herein, including patent and non-patent references, are incorporated by reference as if each were incorporated individually. However, where patents, patent applications, or publications containing explicit definitions are incorporated by reference, those explicit definitions should be understood to apply to the incorporated patent, patent application, or publication in which the definition is found, but not necessarily to the text of this application, in particular to the claims, in which case the definitions provided herein shall supersede them.

[0040] In this application, the indefinite articles “a” and “an” used in the specification and claims should be understood to mean “at least one” unless explicitly stated otherwise.

[0041] In this application, the phrases “and / or” as used in the specification and claims should be understood to mean “either or both” of the elements thus combined (i.e., elements that exist conjunctively in some cases and disjunctly in others). Multiple elements enumerated by “and / or” should be interpreted similarly (i.e., “one or more” of the elements thus combined). In addition to the elements specifically identified by the “and / or” clause, other elements may be present as needed, whether or not they are related to the specifically identified element. Thus, as a non-restrictive example, the reference “A and / or B” when used in combination with open-ended language such as “includes” may refer to A only in one embodiment (including elements other than B as needed), B only in another embodiment (including elements other than A as needed), and both A and B in yet another embodiment (including other elements as needed), and so on.

[0042] Where used in the specification and claims in this application, “or” should be understood to have the same meaning as “and / or” as defined above. For example, where items in a list are separated, “or” or “and / or” shall be interpreted as inclusive (i.e., including not only at least one of multiple elements or lists of elements, but more than one, and, where applicable, additional unlisted items). Only terms that are explicitly indicated as not being inclusive (such as “only one of” or “exactly one of” or, as used in the claims, “consisting of”) refer to the inclusion of exactly one element of multiple elements or lists of elements. In general, where used herein, the term “or” shall be interpreted only as indicating an exclusive choice (i.e., “one or the other, not both”) when preceded by an exclusive term such as “either,” “one of,” “only one of,” or “exactly one of.” When used in patent claims, "essentially derived from" shall have its ordinary meaning as it is used in the field of patent law.

[0043] As used herein, “as needed” or “as required” means that the circumstances described thereafter may or may not occur, and the description includes both cases in which such circumstances occur and cases in which they do not.

[0044] As used herein, the terms “about” or “approximately” mean a range of up to ±20% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within one order of magnitude, preferably two times, of a given value. Where a specific value is stated in this application and claims, unless otherwise specified, the term “about” is implied and, in this context, means within an acceptable margin of error for that specific value.

[0045] The term "effective dose" or "therapeutic dose" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0046] As used herein, the term “leading strand” and its grammatical equivalent refer to a single-stranded nucleic acid sequence bound to a ligation sprint 1 region (also referred to herein as a sprint) or ligation sprint 2 region of the guide polynucleotide provided herein.

[0047] As used herein, the term “complementary strand” and its grammatical equivalent refer to a single-stranded nucleic acid sequence that includes a protospacer adjacent motif (PAM), is complementary to the leading strand, and / or binds to a target-directed region of a guide polynucleotide provided herein.

[0048] The term "effective dose" or "therapeutic dose" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0049] (1) Gene editing systems A system comprising a donor nucleic acid, a guide polynucleotide, and an engineered protein is provided herein. In some embodiments, the system is for use in nucleic acid editing. In some embodiments, the system cleaves a target nucleic acid. In some embodiments, the system generates a single-strand break in the target nucleic acid. In some embodiments, the system incorporates the donor nucleic acid into the target nucleic acid, thereby replacing an abnormal nucleic acid sequence compared to a reference sequence.

[0050] The systems provided herein include the following elements: (1) a donor nucleic acid, (2) a guide polynucleotide, (3) a nuclease, nickase, or a variant thereof, and (4) a DNA ligase or a functional fragment thereof. An exemplary system is shown in Figure 1A. The components of the system include an engineered protein containing Cas9 nickase, a DNA ligase, a donor nucleic acid, and a guide polynucleotide containing a DNA ligation sprint complex for forming a ribonucleoprotein (RNP). The DNA ligation sprint may include a ligation sprint 2 region and / or a ligation sprint 1 region provided herein. The RNP can target double-stranded DNA (dsDNA) in the presence of a protospacer adjacent motif (PAM) and high sequence homology between the guide polynucleotide and the dsDNA. After binding of the Cas9-ligase:guide polynucleotide complex to the dsDNA, Cas9 nickase generates a single-strand break in the non-target strand (NTS), allowing the release of the single-strand DNA (Step 1). The DNA ligation sprint of the guide polynucleotide then hybridizes to this single-strand DNA along with the ligation donor nucleic acid, providing a substrate for the DNA ligase to directly attach the 5' end of the donor nucleic acid to the 3' end of the NTS (Step 2). After the DNA ligation editing step, the Cas9-ligase:guide polynucleotide complex dissociates from the target dsDNA, resulting in different three-dimensional structures in which the DNA ligation edited strand is either not hybridized to the dsDNA or is hybridized (Step 3). Subsequently, endogenous cellular enzymes for DNA repair incorporate either the original DNA strand or the newly attached DNA ligation edited strand into the dsDNA, resulting in either unedited dsDNA (Step 4) or edited dsDNA (Step 5), in that order, respectively.

[0051] In some configurations, the guide polynucleotide for DNA editing by DNA ligase comprises two elements: (1) a ligation sprint 1 region (LS1) and (2) a ligation sprint 2 region (LS2). LS1 mediates hybridization with the non-target strand (NTS), while LS2 mediates hybridization with the ligation donor oligonucleotide (Figure 1B). Following hybridization of LS1 with the NTS and LS2 with the donor oligonucleotide, this provides a substrate for the DNA ligase to directly attach the donor nucleic acid to the non-target strand (also referred to herein as the leading strand) by the DNA ligation event. LS1 contains RNA (typically at the 3' end) and DNA (typically at the 5' end) in varying proportions to enhance DNA ligation efficiency. LS2 typically contains a majority of DNA bases to facilitate efficient DNA ligation by the DNA ligase. In some embodiments, the LS2 region contains only RNA. In some embodiments, the LS1 region contains only DNA.

[0052] The systems provided herein are designed to insert nucleic acids with high fidelity and high processability. The systems provided herein are useful for enabling precise targeted insertion of polynucleotides into target nucleic acids. In some embodiments, the systems provided herein enable targeted insertion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, at least 100,000, or more polynucleotides. In some embodiments, the systems provided herein enable targeted insertion of single-stranded polynucleotides of 10 kb or more.

[0053] Donor nucleic acid Compositions and systems comprising donor nucleic acids are provided herein. Donor nucleic acids provide high-fidelity DNA insertions for introducing modifications to target nucleic acids, and the donor nucleic acids can further correct abnormalities in the sequence compared to the wild-type sequence. In some embodiments, the wild-type sequence is a reference sequence, a nucleic acid sequence derived from a healthy subject, or a nucleic acid sequence derived from healthy cells. In some embodiments, the donor nucleic acid comprises the reverse complementary sequence of the target nucleic acid. In some embodiments, the donor nucleic acid comprises one or more variations in nucleic acid bases, nucleosides, or nucleotides. In some embodiments, the donor nucleic acid comprises the reverse complementary sequence of a sequence encoding an intron or a variant thereof. In some embodiments, the donor nucleic acid comprises the reverse complementary sequence of a sequence encoding an exon or a variant thereof. In some embodiments, the donor nucleic acid comprises the reverse complementary sequence of sequences encoding exons and introns. In some embodiments, the donor nucleic acid comprises the complementary sequence of a sequence encoding an intron or a variant thereof. In some embodiments, the donor nucleic acid comprises the complementary sequence of a sequence encoding an exon or a variant thereof. In some embodiments, the donor nucleic acid includes a complementary sequence of sequences encoding exons and introns. In some embodiments, the donor nucleic acid includes a reverse complementary sequence of sequences encoding non-coding elements (e.g., promoters, enhancers) or variants thereof. In some embodiments, the donor nucleic acid includes a complementary sequence of sequences encoding non-coding elements (e.g., promoters, enhancers) or variants thereof. In some embodiments, the donor nucleic acid includes a variant. In some embodiments, the donor nucleic acid includes a sequence comprising at least one nucleic acid base that is complementary or mismatched with the sequence encoding the splice acceptor site.In some embodiments, the donor nucleic acid sequence includes A / C mismatches, A / T mismatches, A / G mismatches, T / C mismatches, T / G mismatches, T / A mismatches, C / G mismatches, C / A mismatches, C / T mismatches, G / C mismatches, G / T mismatches, G / A mismatches, or any combination thereof with respect to the target nucleic acid sequence or complementary strand (also called the lagging strand) provided herein. In some embodiments, the donor nucleic acid includes epigenetic changes (e.g., methylation).

[0054] Guide polynucleotides Compositions and systems comprising a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, are provided herein. The guide polynucleotide provided herein binds to a target nucleic acid and a nuclease or nickase provided herein to form a complex. In some embodiments, the complex facilitates the cleavage of the target nucleic acid.

[0055] In some embodiments, the guide polynucleotide comprises an RNA nucleoside and a DNA nucleoside. In some embodiments, the guide polynucleotide comprises an RNA nucleotide and a DNA nucleotide. In some embodiments, the guide polynucleotide comprises an RNA nucleic acid base and a DNA nucleic acid base. Examples of nucleic acid bases include, but are not limited to, adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). The nucleic acid bases of the nucleotide can be independently selected from purines, pyrimidines, and purine or pyrimidine analogs. In one embodiment, the nucleic acid bases may include, for example, naturally occurring and synthetic derivatives of the bases. The guide polynucleotides provided herein may include sequences that do not exist in nature or manipulated sequences. In some embodiments, the polynucleotide encoding the guide polynucleotide provided herein includes a promoter region or a localization sequence. For example, a U6 promoter can be used to drive the expression of the guide polynucleotide in cells.

[0056] In some embodiments, the guide polynucleotide or donor nucleic acid provided herein comprises a modified nucleic acid base, a modified nucleotide, or a modified nucleoside. The modified nucleosides and modified nucleotides described herein, which can be incorporated into a target nucleic acid, may include a modified nucleic acid base. In some embodiments, the nucleic acid base or nucleotide provided herein is chemically modified. The nucleic acid base may be modified or substituted to provide a modified nucleoside and modified nucleotide that can be incorporated into a target nucleic acid.

[0057] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include, but are not limited to, 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (mC), N4-acetylcytidine (act), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethyl- Cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (kC), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl- This includes 2'-O-methylcytidine (ac4Cm), N4,2'-O-dimethylcytidine (m4Cm), 5-formyl-2'-O-methylcytidine (f5Cm), N4,N4,2'-O-trimethylcytidine (m42Cm), 1-thiocytidine, 2'-F-alacytidine, 2'-F-cytidine, and 2'-OH-alacytidine, 2'-OMe-exNA-cytidine, and 2'-F-exNA-cytidine.

[0058] In some embodiments, the modified nucleic acid base is modified adenine. Exemplary nucleic acid bases and nucleosides having modified adenine include, but are not limited to, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-Deaza-2,6-diaminopurine, 7-Deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (iA), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methyl Thio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine ( hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6-methyl-2'-deoxyadenosine, N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,It contains 2'-O-dimethyladenosine (iAm), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-ala-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, N6-(19-amino-pentaoxanonadecyl)-adenosine, 2'-OMe-exNA-adenosine, and 2'-F-exNA-adenosine.

[0059] In some embodiments, the modified nucleic acid base is modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanine include, but are not limited to, inosine (I), 1-methyl-inosine (iVl), waiosine (imG), methylwaiosine (mimG), 4-demethylwaiosine (imG-14), isowyosine (imG2), waibutosine (yW), peroxywaibutosine (o2yW), hydroxywaibutosine (OHyW), and undermodified hydroxywaibutosine (OHyW) *), 7-deaza-guanosine, quosin (Q), epoxyquosin (oQ), galactosylquosin (galQ), mannosylquosin (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQO), alkaeosin (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m'G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methylthio-guanosine (1-meth thio guanosine), N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2''Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m'', It contains 7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m'Im), 06-phenyl-2'-deoxyinosine, 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 06-methyl-2'-deoxyguanosine, ZF-ala-guanosine, 2'-F-guanosine, 2'-OMe-exNA-guanosine, and 2'-F-exNA-guanosine.

[0060] In some embodiments, the modified nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include, but are not limited to, pseudouridine (ψ), pyridine-4-onyribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thiouridine (s4U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo -Uridine), 3-methyluridine (m3U), 5-methoxyuridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethyl-pseudruridine, 5-carboxyhydroxymethyluridine (chm5U), 5-carboxyhydroxymethyluridine methyl ester (mchm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyluridine Bonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5-methylaminomethyluridine (mnm5U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s 2U), 5-propynyl-uridine, 1-propynyl-pseuduridine, 5-taurinomethyl-uridine (xcm5U), 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thio-uridine (Tm5s2U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseuduridine (η'ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseuduridine (m xi / ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (mψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine (D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseuduridine, 4-methoxy-2-thio-pseuduridine, N1-methyl-pseuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U) , 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2'-0-methyluridine (Um), 5,2'-0-dimethyluridine (m5Um), 2'-0-methylpseudridine (ψηι), 2-thio-2'-0-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mem5Um), 5-carbamoylmethyl-2'-0-methyluridine (ncm 5Um), 5-carboxymethylaminomethyl-2'-0-methyluridine (cmnm 5Um), 3,2'-0-dimethyluridine (m 3Um), 5-(isopentenylaminomethyl)-2'-0-methyluridine (inm 5Um), 1-thiouridine, deoxythymidine, 2'-F-alar-uridine, 2'-F-uridine, 2'-OH-alar-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)]uridine, pyrazolo[3,4-d]pyrimidine, xanthine, hypoxanthine, 2'-OMe-exNA-uridine, and 2'-F-exNA-uridine.

[0061] In some embodiments, the modified nucleic acid base is a modified thymidine. In some embodiments, the modified nucleoside is a modified thymidine. Non-limiting examples of modified thymines and thymidines include 6-(azo)thymine, 3'-azido-3'-deoxythymidine, 2',3'-didehydro-2',3'-dideoxythymidine, 1-(2,3-dideoxy-β-D-glyceropenta-2-enofuranosyl)thymine, 3-(2-chloroethyl)thymidine, 3'-fluoro-3'-deoxythymidine, β-L-2'-deoxythymidine, thieno[3,4-d]-pyrimidine T-mimicking deoxynucleoside, 1-(2-de It contains oxy-β-D-threopentofuranosyl)thymine, 5-ethynyl-2'-deoxyuridine, bromodeoxyuridine, tritiated thymidine, 5-chlorodeoxyuridine (CldU), 5-iododeoxyuridine (IdU), 2-thiothymidine triphosphate, 5-(α-tert-butylortho-bromobenzyloxy)methyl-2'-deoxyuridine, 5-(α-methylbenzyloxy)methyluracil, and 5-ethyldeoxyuridine.

[0062] Nucleic acids can be modified using a variety of chemical and modification methods. In some embodiments, the regular nucleoside bonds between nucleotides can be altered by mono-thiolation or di-thiolation of phosphodiester bonds to produce phosphorothioate esters or phosphorodithioate esters, in that order, respectively. Other modifications of nucleoside bonds include amidation or peptide linkers. Ribose sugars can be modified by substituting the 2'-O portion with a lower alkyl (C1-4, e.g., 2'-O-Me), alkenyl (C2-4), alkynyl (C2-4), methoxyethyl (2'-MOE), or other substituents. In some cases, the substituent on the 2'OH group can include a methyl group, a methoxyethyl group, or a 3,3'-dimethylallyl group. In some cases, locked nucleic acid sequences (LNAs) containing 2'-4' intramolecular crosslinks (such as a methylene crosslink between the 2' oxygen and 4' carbon) inside the ribose ring can be applied. Purine nucleotides and / or pyrimidine nucleotides can be modified to alter their properties, for example, by heterocyclic amination or deamination. Many of these modified nucleotides and their corresponding ribonucleosides are available from commercial suppliers. If desired, guide polynucleotides and / or donor nucleic acids may contain phosphoramidate bonds, phosphorothioate bonds, and / or methylphosphonate bonds. Several suitable methods can be used to produce nucleic acid molecules and nucleic acids containing modified nucleotides. For example, guide polynucleotides containing modified nucleotides and / or donor nucleic acids containing modified nucleotides can be prepared by transcribing the DNA encoding the guide polynucleotide and / or donor nucleic acid using a suitable DNA-dependent RNA polymerase, such as T7 phage RNA polymerase, SP6 phage RNA polymerase, T3 phage RNA polymerase, or variants of these polymerases that allow for efficient incorporation of modified nucleotides into RNA molecules. In some embodiments, guide polynucleotides containing modified nucleotides and / or donor nucleic acids containing modified nucleotides can be prepared by in vitro transcription.The transcription reaction may involve nucleotides and modified nucleotides, as well as other components that support the activity of the selected polymerase, such as appropriate buffers and appropriate salts. The incorporation of nucleotide analogs into guide polynucleotides may be manipulated, for example, to alter the stability of such RNA / DNA molecules or to increase their resistance to RNases.

[0063] In some embodiments, the guide polynucleotide includes a secondary or tertiary structure. In some embodiments, the secondary structure includes a bulge, stem, stem-loop, loop, tetra-loop, hairpin, fluctuating base pair, pseudoknot, nexus, or a combination thereof. In some embodiments, the bulge, stem-loop, or hairpin includes an unpaired region of nucleotides within the nucleic acid double helix.

[0064] In some embodiments, the guide polynucleotides provided herein include a target-directed region having complementarity with the target nucleic acid. In some embodiments, the target-directed region includes RNA. In some embodiments, the target-directed region has at least 80% complementarity with the target nucleic acid. In some embodiments, the target-directed region has at least 85% complementarity with the target nucleic acid. In some embodiments, the target-directed region has at least 90% complementarity with the target nucleic acid. In some embodiments, the target-directed region has at least 95% complementarity with the target nucleic acid. In some embodiments, the target-directed region has at least 99% complementarity with the target nucleic acid. In some embodiments, the target-directed region has 100% complementarity with the target nucleic acid.

[0065] In some embodiments, the target-directed region contains at least 10 ribonucleotides, at least 15 ribonucleotides, at least 20 ribonucleotides, at least 25 ribonucleotides, at least 30 ribonucleotides, at least 35 ribonucleotides, at least 40 ribonucleotides, at least 45 ribonucleotides, at least 50 ribonucleotides, or more. In some embodiments, the target-directed region contains at least about 10 to a maximum of 15 ribonucleotides for enhanced specificity. In some embodiments, the target-directed region contains at least about 20 to 30 ribonucleotides for enhanced structural stability and specificity.

[0066] In some embodiments, the target-directed region hybridizes to the target nucleic acid. In some embodiments, the nuclease or nickase cleaves the target nucleic acid to generate a leading strand and a complementary strand (also referred to herein as the lagging strand). In some embodiments, when the target nucleic acid is cleaved by the engineered protein construct or nickase provided herein, the target-directed region binds to at least a portion of the cleaved target nucleic acid. In some embodiments, the target-directed region binds to the complementary strand. In some embodiments, the complementary strand includes a protospacer adjacent motif (PAM). The PAM is a short nucleic acid sequence (typically 2–6 base pairs long) preceding the region targeted for cleavage by the nuclease or nickase provided herein. In some embodiments, the target-directed region binds to a target gene sequence of at least 20 nucleotides, at least 15 nucleotides, at least 10 nucleotides, or at least 5 nucleotides from the protospacer adjacent motif (PAM). Guide polynucleotides can bind to the upstream 5' side or the downstream 3' side of the protospacer-adjacent motif (PAM) sequence via the target region.

[0067] In some embodiments, the guide polynucleotide includes a protein-binding region. In some embodiments, the protein-binding region includes a secondary structure that binds to a protein. In some embodiments, the protein-binding region binds to a nuclease, nickase, endonuclease, or exonuclease. In some embodiments, the protein-binding region binds to a Cas protein. The secondary structure can minimize the possibility that the guide polynucleotide may interfere with nuclease or nickase activity when it forms a complex with the manipulated protein provided herein. In some embodiments, the secondary structure includes a bulge, stem, loop, hairpin, fluctuating base pair, pseudoknot, or a combination thereof.

[0068] In some embodiments, the guide polynucleotide includes a ligation sprint 2 (LS2) region. In some embodiments, the ligation sprint 2 region is complementary to the donor nucleic acid. In some embodiments, the ligation sprint 2 region is complementary to the target nucleic acid. In some embodiments, the LS2 region is located on the 5' side of the LS1 region. In some embodiments, the LS2 region is located within the protein-binding region. In some embodiments, the LS2 region contains a deoxyribonucleotide. In some embodiments, the LS2 region is located within the protein-binding region of the guide polynucleotide. In some embodiments, the LS2 region contains a secondary or tertiary structure that enhances the stability of the guide polynucleotide. In some embodiments, the ligation sprint 2 region contains at least one mismatched nucleic acid base relative to the target nucleic acid. In some embodiments, the LS2 region contains a length of at least about 3 nucleic acid bases to a maximum of 100,000 nucleic acid bases. In some embodiments, the ligation sprint 2 region contains at least about 5 nucleic acid bases to a maximum of 10,000 nucleic acid bases. In some embodiments, the ligation sprint 2 region contains at least about 7 nucleic acid bases to a maximum of 10,000 nucleic acid bases.

[0069] In some embodiments, the ligation sprint 2 region includes a reverse complementary sequence of the target nucleic acid or its chain. The LS2 region and / or donor nucleic acid may be designed to correct anomalies in the target sequence compared to the wild-type sequence. For example, the wild-type sequence may include a reference sequence, a nucleic acid sequence from a healthy subject, or a nucleic acid sequence from a healthy cell. Methods for obtaining a reference sequence for polynucleotides may include sequencing or sequence alignment tools and databases, such as NCBI BLAST or UniProt.

[0070] In some embodiments, the ligation sprint 2 region includes one or more variations in a nucleic acid base, nucleoside, or nucleotide. In some embodiments, the ligation sprint 2 region includes a reverse complementary sequence of a sequence encoding an intron or a variant thereof. In some embodiments, the ligation sprint 2 region includes a reverse complementary sequence of a sequence encoding an exon or a variant thereof. In some embodiments, the ligation sprint 2 region includes a reverse complementary sequence of sequences encoding an exon and an intron. In some embodiments, the ligation sprint 2 region includes a complementary sequence of a sequence encoding an intron or a variant thereof. In some embodiments, the ligation sprint 2 region includes a complementary sequence of a sequence encoding an exon or a variant thereof. In some embodiments, the ligation sprint 2 region includes a complementary sequence of sequences encoding an exon and an intron. In some embodiments, the ligation sprint 2 region includes a reverse complementary sequence of a sequence encoding a non-coding element (e.g., promoter, enhancer) or a variant thereof. In some embodiments, the ligation sprint 2 region includes a complementary sequence of sequences that encode non-coding elements (e.g., promoters, enhancers) or variants thereof.

[0071] In some embodiments, the guide polynucleotides provided herein include one or more variations. In some embodiments, the one or more variations include a variation in at least one nucleic acid base, nucleoside, or nucleotide in the LS2 or LS1 region. In some embodiments, the ligation sprint 2 region includes a sequence containing at least one nucleic acid base that is complementary or mismatched with the sequence encoding the splice acceptor site. In some embodiments, the ligation sprint 2 region includes A / C mismatch, A / T mismatch, A / G mismatch, T / C mismatch, T / G mismatch, T / A mismatch, C / G mismatch, C / A mismatch, C / T mismatch, G / C mismatch, G / T mismatch, G / A mismatch, or any combination thereof with respect to the target nucleic acid sequence or complementary strand (also called the lagging strand) provided herein.

[0072] In some embodiments, the guide polynucleotide provided herein includes a ligation sprint 1 region. In some embodiments, the ligation sprint 1 region includes one or more ribonucleotides or an RNA region. In some embodiments, the ligation sprint 1 region includes one or more deoxyribonucleotides or a DNA region. In some embodiments, the ligation sprint 1 region includes a ribonucleotide and a deoxyribonucleotide. In some embodiments, the ligation sprint 1 region includes at least one ribonucleoside and at least one deoxyribonucleoside.

[0073] In some embodiments, the LS1 region of the guide polynucleotide forms a DNA-RNA (DR) loop upon association with the target nucleic acid. The DR loop provides a structural framework and stability for the formation of a complex between the target nucleic acid, the guide polynucleotide, and the engineered protein provided herein. In some embodiments, LS1 forms a DR loop upon association between the target nucleic acid and the engineered protein provided herein.

[0074] In some embodiments, the LS1 region, upon association with the target nucleic acid, forms a DNA(D) loop. The D loop is formed after cleavage of the target DNA by nickase. The D loop is a DNA structure in which the two strands of a double-stranded DNA molecule are separated over a certain section and held apart by a third DNA strand. In some embodiments, the third DNA strand in the D loop is the LS2 or LS1 region of a guide polynucleotide.

[0075] In some embodiments, the LS1 region forms an R-loop upon association with the target nucleic acid. In some embodiments, the LS1 region forms an RNA(R)-loop upon association with the target nucleic acid and the manipulated protein provided herein. The R-loop may be formed between the complementary strand of the target DNA, the RNA portion of the LS1 region of the guide polynucleotide, and the leading strand of the target DNA after cleavage of the target nucleic acid by nickase.

[0076] In some embodiments, the ligation sprint 1 region hybridizes to the leading strand of DNA when the target nucleic acid is cleaved by the engineered protein construct provided herein. In some embodiments, a ribonucleoside, ribonucleotide, or RNA region within the ligation sprint 1 region hybridizes to the leading strand. In some embodiments, the LS1 region is 3' to the LS2 region. In some embodiments, the LS1 region is located within the protein-binding region.

[0077] In some embodiments, the ligation sprint 1 region includes a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 1:1 to a maximum of 20:1. In the place embodiment, the ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4: 1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9 :4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9:20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12:1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 14:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15,This includes ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratios of 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9, 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:1. In some embodiments, the ligation sprint 1 region contains at least about 3 nucleic acid bases to a maximum of about 20 nucleic acid bases.

[0078] The interaction between a donor nucleic acid, a guide polynucleotide provided herein, and a manipulated protein provided herein is shown in Figure 1A. An exemplary guide polynucleotide structure is shown in Figure 1B. Thus, in some embodiments, the guide polynucleotide provided herein includes, in the 5'-3' direction: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region comprising a secondary structure that binds to a nickase region; (iii) a ligation sprint 2 region complementary to the donor nucleic acid, the target nucleic acid, and at least one mismatched nucleic acid base compared to the target nucleic acid; and (iv) a ligation sprint 1 region comprising a DNA nucleoside and an RNA nucleoside, wherein the ligation sprint 1 region is complementary to the target nucleic acid. In some embodiments, upon introduction into cells or a cell-free system, the composition or system provided herein incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid. In some embodiments, the newly inserted nucleic acid comprises a single-stranded DNA sequence that is substantially complementary to the complementary strand (including the PAM sequence) and also contains at least one mismatched nucleic acid base relative to the complementary strand. In some embodiments, the ligation sprint 1 region forms an R-loop upon association with the target nucleic acid and DNA ligase.

[0079] In some embodiments, the guide polynucleotide includes a sequence or a portion of a sequence in Table 3 or Table 7. In some embodiments, the guide polynucleotide includes a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs. 45-57 or SEQ ID NOs. 102-123.

[0080] The guide polynucleotides provided herein may be generated, for example, by phosphoramidite chemosynthesis, in vitro transcription (IVT) technology, M13 bacteriophage methods, DNA isolation, RNA isolation, tagmentation, and combinations thereof. Furthermore, guide polynucleotides may be generated and expressed by transfecting or transfecting cells with plasmid DNA or a vector containing a polynucleotide encoding an expression cassette.

[0081] In some embodiments, the guide polynucleotide includes a 5' cap. The free 5' hydroxyl group can be septicumed by acetylation. In some embodiments, the guide polynucleotide includes a 3' polyadenylated tail. In some embodiments, the guide polynucleotide includes a polynucleotide linker or spacer region.

[0082] The engineered proteins that interact with guide polynucleotides provided herein may be used in the systems and compositions provided herein for nucleic acid insertion. Engineered proteins useful for nucleic acid editing and targeted cleavage of specific target sequences are described in further detail below.

[0083] Manipulated proteins Provided herein are engineered proteins that specifically bind to a target nucleic acid using a guide polynucleotide to hybridize to a target sequence, or polynucleotides that encode the engineered proteins provided herein. The engineered proteins also ligate nucleic acid donor oligonucleotides for integration into the target nucleic acid. The engineered proteins provided herein may be fused to one or more additional protein constructs for specific applications. In some embodiments, specific applications are DNA ligation or nucleic acid cleavage. Multimerization of the engineered protein constructs provided herein may be achieved by direct fusion with another engineered protein construct. In some embodiments, each protein construct is operably linked by a linker polypeptide.

[0084] Systems, compositions, and engineered proteins comprising a nuclease or nickase region are provided herein. A nuclease is an enzyme that cleaves nucleic acids. For example, a nuclease can cause single-strand or double-strand breaks in a target nucleic acid. Nucleases and nickases target specific nucleic acid sequences by binding to a guide polynucleotide that hybridizes to the target nucleic acid. Various types of nucleases may be used in the systems and compositions provided herein.

[0085] Systems, compositions, and engineered proteins comprising nickase or a nickase region are provided herein. In some embodiments, the engineered protein comprises nickase. In some embodiments, the engineered protein provided herein comprises an engineered protein construct comprising a nickase region. Nickase is an enzyme that cleaves one strand of double-stranded DNA.

[0086] In some embodiments, the nickases provided herein cleave one strand of a DNA double helix to produce a nicked DNA molecule. Exemplary amino acid sequences of nucleases and nickases, but not limited to, include, for example: NCBI Gene ID:1238121:NP_858382.1 [Conjugation-transmitted nickase / helicase TraI (plasmid) [Shigella flexneri 2a str.301]] [ka] [ka] UniProt:Q8YS92[DNA niccasse - Nostoc sp. (PCC 7120 strains / SAG 25.82 strains / UTEX 2576 strains)]: [ka] Streptococcus pyogenes Cas9: [ka] Streptococcus pyogenes Cas9 H840A nickase (bold / underlined indicates amino acid substitutions): [ka] [ka] Streptococcus pyogenes Cas9 R221K,N394K,H840A nickase (bold / underlined indicates amino acid substitutions): [ka] Staphylococcus aureus Cas9 N580A nickase (bold / underlined indicates amino acid substitutions): [ka] [ka] and Francisella novicida Cas9 H969A niccase (bold / underlined indicates amino acid substitutions): [ka] In some embodiments, the systems or manipulated proteins provided herein include a sequence that is at least 85% identical to any one of SEQ ID NOs. 67-73. In some embodiments, the systems or manipulated proteins provided herein include a sequence that is at least 90% identical to any one of SEQ ID NOs. 67-73. In some embodiments, the systems or manipulated proteins provided herein include a sequence that is at least 95% identical to any one of SEQ ID NOs. 67-73. In some embodiments, the systems or manipulated proteins provided herein include a sequence that is at least 99% identical to any one of SEQ ID NOs. 67-73. In some embodiments, the systems or manipulated proteins provided herein include any one of SEQ ID NOs. 67-73.

[0087] In some embodiments, the manipulated protein comprises a Cas protein or a fragment thereof. In some embodiments, the manipulated protein comprises a Cas protein or a fragment thereof comprising at least one amino acid substitution compared to a reference amino acid sequence (e.g., wild-type sequence) for the Cas protein or (of) the fragment thereof. In some embodiments, Cas is catalytically dead Cas or partially dead Cas. For example, partially dead Cas may include nickase. In some embodiments, catalytically dead Cas or partially dead Cas may be Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12a (Cpf1), Cas 12b, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, C The Cas proteins are selected from the group consisting of sf1, Csf2, CsO, Csf4, c2c1, c2c3, Cas9HiFi, xCas9, CasX, CasY, CasRX, SpCas9-VQR, SpCas9-VRQR, SpCas9-VRER, SaCas9-KKH, SpCas9-NG, SpCas9-NRRH, SpCas9-NRTH, SpCas9-NRCH, iSpyMac, St1Cas9 LMD9-LMG18311, St1Cas9 LMD9-CNRZ1066, St1Cas9-KQKL, their variants, fragments, mutants, or catalytically inactive derivatives thereof. In some embodiments, the Cas proteins or mutant Cas proteins provided herein are V-type Cas proteins.In some embodiments, the V-type Cas protein includes Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, its variants, its fragments, its variants, or its derivatives. In some embodiments, the Cas protein is a chimeric Cas protein containing domains derived from different Cas proteins provided herein.

[0088] In some embodiments, the Cas protein is derived from bacteria. In some embodiments, the bacteria are from the genera Streptococcus, Francisella, Lactococcus, Lactobacillus, Pseudomonas, Geobacillus, Clostridium, Streptomyces, Actinoplanes, Synechococcus, Corynebacterium, Haloferax, Haloarcula, Methanococcus, Neisseria, Campylobacter, or Staphylococcus.

[0089] In some embodiments, the Cas protein is derived from a bacterium, specifically Streptococcus pyogenes (e.g., SpCas9). In some embodiments, the Cas protein is Streptococcus pyogenes Cas9 (SpCas9), which contains a sequence that is at least 90% identical to sequence number 69. In some embodiments, the Cas protein is Streptococcus pyogenes Cas9, which contains a sequence that is at least 95% identical to sequence number 69. In some embodiments, the Cas protein is Streptococcus pyogenes Cas9, which contains a sequence that is at least 99% identical to sequence number 69. In some embodiments, the Cas protein is Streptococcus pyogenes Cas9, which contains a sequence that is identical to sequence number 69. In some embodiments, Streptococcus pyogenes Cas9 includes mutations. In some embodiments, Streptococcus pyogenes Cas9 contains mutations, the mutations comprising one or more amino acid substitutions.

[0090] In some embodiments, the nuclease or nickase region of a protein construct binds to a guide polynucleotide provided herein. In some embodiments, the nuclease or nickase region of a protein construct binds to a protein-binding region of a guide polynucleotide provided herein. In some embodiments, the nuclease or nickase region of a protein construct binds to a target nucleic acid to form a complex. In some embodiments, the complex further includes a portion of the guide polynucleotide sequence. For example, the protein-binding region and target-directing region of the guide polynucleotide form a complex between the nuclease or nickase region of the manipulated protein provided herein and the target nucleic acid via the target-directing sequence.

[0091] Binding to a nuclease, nickase, or fragment thereof is mediated by the complete or partial complementarity of the target-directing region of the guide polynucleotide provided herein. In some embodiments, the engineered protein construct (e.g., nickase or nuclease) binds to the guide polynucleotide provided herein, which binds to or adjacent to the PAM sequence. Enzymes and proteins from different bacterial species can recognize different sequence motifs or PAMs. In some embodiments, the engineered protein construct binds to a guide polynucleotide that binds to a target nucleic acid of up to 5 nucleotide bases, 10 nucleotide bases, 15 nucleotide bases, or 20 nucleotide bases of the PAM sequence.

[0092] Upon binding to a target nucleic acid via a guide sequence, the nucleases and nickases provided herein specifically cleave the target nucleic acid at the distal or proximal end of the PAM. In some embodiments, the nuclease or nickase region of the protein construct generates a single-strand break in the target nucleic acid. In some embodiments, the nickase region generates a double-strand break in the target nucleic acid. In some embodiments, the nucleases and nickases provided herein generate blunt ends when cleaving the target nucleic acid. In some embodiments, the nucleases provided herein improve gene editing efficiency by generating staggered ends when cleaving the target nucleic acid, enabling a higher integration rate of the synthesized DNA. In some embodiments, the nucleases and nickases provided herein cleave the non-target strand (NTS). In some embodiments, the nucleases and nickases provided herein cleave the complementary strand.

[0093] Systems, compositions, and engineered proteins comprising DNA ligases or functional fragments thereof are provided herein. In some embodiments, the engineered protein comprises a DNA ligase or a DNA ligase region. In some embodiments, the DNA ligase region is operably ligated to a nuclease or nickase region, or a nickase region, provided herein. In some embodiments, the DNA ligase is an ATP-dependent ligase. In some embodiments, the DNA ligase is a bacterial, eukaryotic, insect, or plant DNA ligase. In some embodiments, the DNA ligase is selected from the group consisting of E. coli DNA ligase, Taq DNA ligase, T4 DNA ligase, human DNA ligase I, human DNA ligase III, and human DNA ligase IV. In some embodiments, the DNA ligase is T4 DNA ligase. Exemplary DNA ligase sequences are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0094] In some embodiments, a DNA ligase, a DNA ligase region, or a DNA ligase fragment binds to a ligation sprint 1 region of a guide polynucleotide. In some embodiments, the DNA ligase does not bind to the target nucleic acid sequence. In some embodiments, the DNA ligase attaches the 5' end of the donor nucleic acid to the 3' end of the leading strand. The donor nucleic acid may substitute at least a portion of the target sequence in the double-stranded target nucleic acid, thereby editing the double-stranded target nucleic acid.

[0095] In some embodiments, the engineered proteins provided herein further comprise one or more additional protein regions or protein constructs, which include nucleases, acetylases, acetyltransferases, ATPases, Argonaute proteins, base editors, Cas polypeptides, catalytically dead Cas polypeptides, deacetylases, deaminases, decapping proteins, endonucleases, exonucleases, helicases, ligases, meganucleases, methylases, methyltransferases, nickases, polymerases, proteases, recombinases, restriction enzymes, ribonucleoproteins (RNPs), autocleavage protein sequences, splicing factors, transcription activators, transcription activator-like effector nucleases (TALENs), transcriptional repressors, transposases, zinc fingers, or any combination thereof. In some embodiments, the compositions and systems provided herein comprise additional engineered proteins. In some embodiments, additional manipulated proteins include nucleases, acetylases, acetyltransferases, ATPases, Argonaute proteins, base editors, Cas polypeptides, catalytically dead Cas polypeptides, deacetylases, deaminases, decapsulating proteins, endonucleases, exonucleases, helicases, ligases, meganucleases, methylases, methyltransferases, nickases, polymerases, proteases, recombinases, restriction enzymes, ribonucleoproteins (RNPs), autocleavage protein sequences, splicing factors, transcription activators, transcription activator-like effector nucleases (TALENs), transcriptional repressors, transposases, zinc fingers, or any combination thereof.

[0096] In some embodiments, the engineered proteins provided herein include protein constructs that regulate transcription. In some embodiments, the engineered proteins provided herein include transcriptional repressor protein constructs. In some embodiments, the engineered proteins provided herein include zinc finger protein constructs. In some embodiments, the zinc finger protein construct includes a Kruppel-associated box (KRAB) protein or a functional fragment thereof. In some embodiments, the engineered protein further includes a KRAB domain that binds to a transcriptional corepressor protein. In some embodiments, the engineered proteins provided herein include SUMO protein constructs.

[0097] In some embodiments, the engineered proteins provided herein include transcription activator protein constructs. Transcription activator protein constructs recruit transcription factors from host cells to target nucleic acids for the regulation of target nucleic acid expression. Exemplary transcription activators include VP64, VP16, VP160, VP48, VP96, p65, Rta, VPR, hsf1, and p300. In some embodiments, the engineered proteins provided herein include one or more VP16 protein constructs. In some embodiments, the engineered proteins provided herein include one or more VP64 protein constructs. In some embodiments, the engineered proteins provided herein include one or more VPR protein constructs. In some embodiments, the engineered proteins provided herein include one or more SunTag protein constructs. In some embodiments, the engineered proteins provided herein include VP64, p65, and HSF1 (SunTag-p65-heat shock factor 1 or SPH). In some embodiments, the manipulated proteins provided herein include a CREB-binding protein (CPB). The CBP may be used to recruit transcriptional mechanisms and may function as a histone acetyltransferase (HAT) that alters chromatin structure.

[0098] In some embodiments, the manipulated protein provided herein further comprises an aptamer. In some embodiments, the aptamer binds to one or more MS2 proteins.

[0099] In some embodiments, the engineered proteins provided herein include self-cleaving protein sequences. Non-limiting examples of self-cleaving protein sequences include E2A, P2A, and T2A. In some embodiments, the engineered proteins provided herein further include antibiotic resistance protein constructs or antibiotic resistance selection markers. Non-limiting examples of antibiotic resistance proteins and antibiotic resistance selection markers include aminoglycoside acetyltransferases, rifampin ADP-ribosyltransferases, dihydrofolate reductases, multidrug and toxic compound efflux transporters, antibiotic resistance ATP-binding cassette family F (ARE ABC-F) proteins, β-lactamases, blastocydin-S deaminases, penicillin-binding proteins (PBPs), and puromycin-N-acetyltransferases. In some embodiments, the ARE ABC-F proteins are MsrE, Erm, Vga, Lsa, Sal, or OptrA.

[0100] In some embodiments, the manipulated proteins provided herein include a base editor. In some embodiments, the base editor is selected from the group consisting of adenine base editors, adenosine base editors, cytidine deaminases, cytosine-to-guanine base editors, and deaminase dimers. In some embodiments, the cytidine deaminase is activation-inducible deaminase (AID), APOBEC deaminase, APOBEC3G, APOBEC1, cytidine deaminase 1 (CDA1), their functional fragments, or derivatives thereof. In some embodiments, the adenosine base editor is ecTadA, saTadA, their functional fragments, or derivatives thereof. In some embodiments, the manipulated proteins provided herein further include uracil-DNA glycosylase, uracil-DNA glycosylase inhibitors, their functional fragments, or derivatives thereof.

[0101] In some embodiments, the systems, compositions, or engineered proteins provided herein further include nuclear localization sequences (NLS). The NLS directs the protein toward the nucleus of a cell, thereby localizing the engineered proteins provided herein in close proximity to target nucleic acids within the nucleus of a cell. In some embodiments, the systems, compositions, or engineered proteins include more than one nuclear localization sequence (NLS). In some embodiments, the NLS is derived from monkey vacuolated virus 40 (SV40). In some embodiments, the NLS includes a monosegmental SV40 NLS. In some embodiments, the NLS includes a bisegmental SV40 NLS. In some embodiments, the NLS includes PKKKRKV (SEQ ID NO: 74) or KRTADGSEFEPKKKRKV (SEQ ID NO: 75). In some embodiments, the NLS includes a nucleoplasmin sequence. In some embodiments, the nucleoplasmin sequence includes KRPAATKKAGQAKKKK (SEQ ID NO: 76).

[0102] In some embodiments, the systems, compositions, or manipulated proteins provided herein further include linkers. A linker is a molecular entity that can directly or indirectly link two parts of a composition. For example, a linker can link a first protein construct to a second protein construct, and a second protein construct to a third protein construct, and so on. Linkers may be configured as needed for specific purposes. For example, a linker may be configured for improved stability or to achieve an optimal length between two amino acid sequences. In some embodiments, a linker may be configured to allow the multimerization of a nuclease or nickase provided herein by a DNA ligase, for example, from a monomer to a dimer, trimer, tetramer, pentamer, or higher-order multimeric complex, while maintaining biological activity. In some embodiments, the biological activity includes cleavage of a target nucleic acid or a set of target nucleic acids. In some embodiments, the linker is (GS) n (Sequence ID 229) or (GGS) nA linker may be selected from (Sequence ID 230), where n is 1, 2, 3, 4, or 5. Other exemplary linkers are provided in Table 2. [Table 2]

[0103] In some embodiments, the linker is configured to facilitate the expression and purification of the nuclease or nickases provided herein. In some embodiments, the engineered proteins provided herein include cleavable or non-cleavable linkers between different protein constructs or domains of the engineered protein. For example, the linker may be a polypeptide linker, for example, having an amino acid length of 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, 50 or more. Generally, the number of additional elements in a manipulated protein depends on many factors, including, for example, the size or molecular weight of the protein for delivery to cells, or the translocation of the protein to the desired cell type.

[0104] Synthesized protein constructs and polynucleotides encoding synthesized proteins are provided herein, the synthesized protein construct comprising synthesized nickase and DNA ligase or a functional fragment thereof. In some embodiments, the synthesized protein construct further comprises a linker between the nickase region or nuclease region and the DNA ligase or a functional fragment thereof. In some embodiments, the linker comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs. 77-88. In some embodiments, the linker comprises an amino acid sequence that is at least 99% identical to any one of SEQ ID NOs. 77-88. In some embodiments,

[0105] Combination composition A composition is provided herein comprising (a) a donor nucleic acid, (b) a polynucleotide encoding a nickase or a variant thereof, (c) a polynucleotide encoding a protein construct comprising a nickase or a variant thereof and a DNA ligase or a functional fragment thereof, and (d) a guide polynucleotide as described herein. In some embodiments, the nickase is an engineered Cas protein or a functional variant thereof. In some embodiments, the engineered Cas protein or a functional variant thereof comprises at least one amino acid substitution at position 840 corresponding to SEQ ID NO: 69. In some embodiments, the engineered Cas protein or a functional variant thereof further comprises at least one amino acid substitution at position 221, position 394, or a combination thereof corresponding to SEQ ID NO: 69. In some embodiments, the engineered Cas protein or a functional variant thereof comprises the amino acid substitutions R221K, N394K, H840A, or any combination thereof. In some embodiments, the engineered Cas protein or a functional variant thereof comprises the amino acid substitution H840A. In some embodiments, the manipulated Cas protein or its functional variant includes the R221K amino acid substitution. In some embodiments, the manipulated Cas protein or its functional variant includes the N394K amino acid substitution. In some embodiments, the manipulated Cas protein or its functional variant includes a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70 or SEQ ID NO: 71. In some embodiments, the manipulated Cas protein or its functional variant includes the sequence of SEQ ID NO: 70 or SEQ ID NO: 71. In some embodiments, the DNA ligase or functional fragment includes E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, their variants, or combinations thereof.In some embodiments, the DNA ligase or its functional fragment includes Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV. In some embodiments, the DNA ligase or its functional fragment contains sequences that are at least 97%, at least 98%, at least 99%, or 100% identical to sequences of SEQ ID NOs. 58-66. In some embodiments, the DNA ligase or its functional fragment contains sequences of SEQ ID NOs. 58-66. In some embodiments, the DNA ligase or its functional fragment includes Chlorella virus DNA ligase containing sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to sequence of SEQ ID NO. 58. In some embodiments, the DNA ligase or its functional fragment includes Chlorella virus DNA ligase containing the sequence of SEQ ID NO. 58.

[0106] An engineered fusion protein is provided herein, comprising (a) a DNA ligase or a functional fragment thereof, and (b) an engineered nickase comprising three amino acid substitutions at positions corresponding to amino acid positions 221, 394, and 840 of a nuclease comprising the sequence of SEQ ID NO: 69. In some embodiments, the substitutions result in enhanced nickase activity compared to an otherwise equivalent engineered nickase. In some embodiments, the engineered nickase comprises the amino acid substitutions R221K, N394K, and H840A. In some embodiments, the engineered nickase or a functional variant thereof comprises a sequence 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71. In some embodiments, the engineered nickase or a functional variant thereof comprises the sequence of SEQ ID NO: 71. In some embodiments, the DNA ligase or functional fragment includes E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants thereof, or combinations thereof. In some embodiments, the DNA ligase or its functional fragment includes Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV. In some embodiments, the DNA ligase or its functional fragment includes sequences that are 97%, 98%, 99%, or 100% identical to sequence numbers 58-66. In some embodiments, the DNA ligase or its functional fragment includes sequences of sequence numbers 58-66. In some embodiments, the DNA ligase or its functional fragment comprises a Chlorella virus DNA ligase containing a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 58.In some embodiments, the DNA ligase or its functional fragment comprises a Chlorella virus DNA ligase containing a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 58. In some embodiments, the DNA ligase or its functional fragment comprises a Chlorella virus DNA ligase containing the sequence of SEQ ID NO: 58. In some embodiments, the manipulated fusion protein further comprises a linker, a nuclear localization sequence (NLS), or additional protein constructs. In some embodiments, additional protein constructs include cell-targeting moieties, receptor-targeting moieties, regulatory elements, nucleases, acetylases, acetyltransferases, ATPases, Argonaut proteins, base editors, Cas polypeptides, catalytically inactive Cas polypeptides, deacetylases, deaminases, decapping proteins, endonucleases, exonucleases, helicases, ligases, meganucleases, methylases, methyltransferases, nickases, polymerases, proteases, recombinases, restriction enzymes, ribonucleoproteins (RNPs), autocleavage protein sequences, splicing factors, transcription activators, transcription activator-like effector nucleases (TALENs), transcriptional repressors, transposases, zinc fingers, or any combination thereof.

[0107] An engineered fusion protein having an amino acid sequence at least 90% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein having an amino acid sequence at least 95% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein having an amino acid sequence at least 96% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein having an amino acid sequence at least 97% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein having an amino acid sequence at least 98% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein having an amino acid sequence at least 99% identical to any one of SEQ ID NOs. 89-101 is provided herein. An engineered fusion protein is provided herein, comprising an amino acid sequence at least 95% identical to any one of sequence numbers 89-101, wherein the DNA ligase region comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acids. An engineered fusion protein is provided herein, comprising an amino acid sequence at least 95% identical to any one of sequence numbers 89-101, wherein the engineered nickase region comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acids. An engineered fusion protein comprising the amino acid sequences of sequence numbers 89-101 is provided herein.

[0108] Nucleic acids and polynucleotides encoding any one of the manipulated fusion proteins provided herein are provided herein. In some embodiments, the polynucleotide encoding the manipulated fusion protein includes DNA. In some embodiments, the polynucleotide encoding the manipulated fusion protein includes RNA. Vectors comprising any of the nucleic acids or polynucleotides provided herein, or a plurality of nucleic acids or polynucleotides provided herein, are provided herein. In some embodiments, the nucleic acids, vectors, or viral vectors provided herein further include guide polynucleotides provided herein. In some embodiments, the nucleic acids, vectors, or viral vectors provided herein further include DNA encoding guide polynucleotides provided herein.

[0109] target nucleic acid The guide polynucleotides provided herein may include some degree of complementarity with the target polynucleotide sequence or its chain. The target-directed region of the guide polynucleotide provided herein may include at least partial sequence complementarity with the target polynucleotide. The target-directed sequence may have sequence complementarity with the target nucleic acid to a degree sufficient for the guide polynucleotide to hybridize with the target polynucleotide. In some cases, the target-directed sequence includes 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with the target polynucleotide. Sequence complementarity may be determined by using alignment methods known in the art, for example, sequence alignment may be performed using publicly available software such as BLAST, Align, or ClustalW2. In some embodiments, the target nucleic acid provided herein includes a gene or polynucleotide containing DNA. In some embodiments, the gene or polynucleotide includes a mammalian gene or polynucleotide. In some embodiments, the gene or polynucleotide includes a human gene or polynucleotide. In some embodiments, the target nucleic acid provided herein includes DNA. In some embodiments, the target nucleic acid provided herein includes single-stranded DNA. In some embodiments, the target nucleic acid provided herein includes double-stranded DNA. In some embodiments, the target nucleic acid provided herein includes RNA. In some embodiments, the target nucleic acid provided herein includes single-stranded RNA. In some embodiments, the target nucleic acid provided herein includes double-stranded RNA.

[0110] In some embodiments, the target nucleic acid or complementary strand provided herein comprises a protospacer adjacent motif ("PAM"), where the PAM is a short T-rich sequence. In some embodiments, cleavage of the target nucleic acid by the manipulated protein provided herein occurs downstream or at the 3' end of the PAM sequence. In some embodiments, cleavage of the target nucleic acid occurs upstream or at the 5' end of the PAM sequence. In some embodiments, the PAM is an NGG PAM sequence. In some embodiments, the PAM comprises NGAN, NGNG, NGAG, NGCG, where N is A, G, C, or T. In some embodiments, the PAM is a T-rich PAM. In some embodiments, the PAM has a nucleotide sequence (T)XN, where X is the number of thymines (e.g., 1 to 10), and N is A, G, C, or T. In certain embodiments, X is equal to 2, and therefore the PAM is TTN. In some embodiments, X is 3, and therefore the PAM is TTTN. In some embodiments, the nuclease or nickase provided herein recognizes a sequence motif TTTN and instructs it to cleave a target nucleic acid sequence 1 to 24 bp downstream of that sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24).

[0111] The target nucleic acid sequence may originate from any cell or organism, but is not limited to this. Determining a suitable sequence for the guide polynucleotide to bind to the target nucleic acid provided herein depends on the sequence of the desired target and the structure of the nuclease or nickase provided herein. Methods for designing target-directed regions of guide polynucleotides for gene editing are known in the art and include, for example, the use of software and databases such as Breaking-Cas, Cas-OFFinder, CRISPR-DT, CHOPCHOP, CCTOP, CRISPick, or CRISPOR.

[0112] In some embodiments, the target nucleic acid includes double-stranded DNA (dsDNA). In the case of dsDNA, the polynucleotide and the nickase region of the engineered protein provided herein bind to the target nucleic acid, generating a single-strand break in the dsDNA. The break can occur on the bottom or top strand of the double-stranded DNA. The break in the dsDNA generates a DNA replication fork, forming a leading strand and a complementary strand (also called the lagging strand). The guide polynucleotide binds to both strands of the target DNA via different regions of the guide polynucleotide. For example, the ligation sprint 1 region binds to the leading strand of the DNA after the dsDNA is broken by the nickase or nickase region of the engineered polynucleotide. The target-directing region of the guide polynucleotide binds to the complementary strand containing RNA and a PAM sequence. The ligation sprint 2 region mediates hybridization with the ligation donor nucleic acid. Once the ligation sprint 1 region binds to the leading strand and the donor nucleic acid hybridizes to the ligation sprint 2 region, the DNA ligase of the manipulated protein catalyzes the formation of a phosphodiester bond between the 3' hydroxyl end of the leading strand and the 5' phosphate end of the donor nucleic acid. The DNA ligase does not bind to the complementary strand containing the PAM sequence on the target nucleic acid. Once the DNA ligase completes ligation, the guide polynucleotide dissociates from the complementary strand of the nicked target nucleic acid, and the donor nucleic acid is incorporated into the target nucleic acid. In some cases, because the new sequence is different from the complementary target DNA strand, a DNA bulge is formed in the ligated DNA sequence at the site of the DNA editing. Additional DNA mismatch repair proteins recognize the bulge, which can facilitate the correction of the abnormal sequence in the target nucleic acid (compared to the wild-type reference sequence) and modify the target nucleic acid. In some embodiments, the target nucleic acid can be repaired by DNA mismatch repair (MMR) proteins, homologous recombination repair (HDR) proteins, or non-homologous end joining (NHEJ) repair proteins. In some embodiments, MMR proteins, HDR proteins, and / or NHEJ proteins are endogenous to the cell.In some embodiments, MMR proteins, HDR proteins, and / or NHEJ proteins are introduced into cells or cell-free systems. In some cases, when a nickase cleaves the bottom strand of DNA (e.g., the complementary strand) away from the editing site, the donor nucleic acid or LS2 region can be incorporated into the genome.

[0113] (2) Delivery vehicles and vectors A composition comprising a guide polynucleotide and a delivery vehicle provided herein is provided herein. A composition comprising an engineered protein and a delivery vehicle provided herein is provided herein. A system and one or more delivery vehicles provided herein are provided herein. The compositions and cells provided herein can be delivered to a target cell, tissue, organ or object by any suitable means.

[0114] The engineered proteins, guide polynucleotides, and any polynucleotides encoding the engineered proteins or guide polynucleotides provided herein may be mixed with a delivery vehicle that enables the delivery of the system to a target nucleic acid sequence within a cell, tissue, or object. The polynucleotides and sets of polynucleotides encoding the engineered proteins and / or guide polynucleotides provided herein include DNA, RNA, or both DNA and RNA. In some embodiments, a certain RNA encodes the engineered protein or guide polynucleotide provided herein. For example, RNA delivery of a protein may improve the expression of the engineered protein in human cells. In some embodiments, the polynucleotides provided herein include self-replicating RNA or viral RNA.

[0115] In some embodiments, the delivery vehicle is a liposome. Liposomes are composed of phospholipids that disperse in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 angstroms, containing an aqueous solution in their core. Liposomes interact with cells through a variety of mechanisms, including: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface by either nonspecific weak hydrophobic or electrostatic forces, or specific interactions with cell surface components; fusion with the cell plasma membrane by insertion of the liposome's lipid bilayer into the plasma membrane, accompanied by simultaneous release of liposome contents into the cytoplasm; and the movement of lipids from liposomes to the cell membrane or intracellular membrane, or vice versa, without any association of liposome contents. By changing the liposome formulation, it is possible to change which mechanisms are activated, but more than one can be activated simultaneously. Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles can also be used as delivery vehicles.

[0116] In some embodiments, the delivery vehicle is a phospholipid. Depending on the molar ratio of lipid to water, phospholipids can form various structures other than liposomes when dispersed in water. At low ratios, liposomes are formed. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar substances, but at high temperatures, they undergo a phase transition that significantly alters their permeability. The phase transition involves a change from a tightly packed, regular structure known as the gel state to a loosely packed, irregular structure known as the fluid state. This occurs at a characteristic phase transition temperature and results in increased permeability to ions, sugars, and drugs.

[0117] In some embodiments, the delivery vehicle is nanoparticles. Nanoparticle carriers provided herein that specifically target tissues may also be used as pharmaceutically acceptable carriers. In some embodiments, the nanoparticles are gold nanoparticles, platinum nanoparticles, iron oxide nanoparticles, lipid nanoparticles, selenium nanoparticles, tumor-targeting glycol chitosan nanoparticles (CNPs), cathepsin B-sensitive nanoparticles, hyaluronic acid nanoparticles, paramagnetic nanoparticles, or polymer nanoparticles. In some embodiments, the delivery vehicle is lipid nanoparticles.

[0118] A composition comprising lipid nanoparticles, comprising a system provided herein, a set of polynucleotides encoding a system provided herein, a vector provided herein, an engineered protein provided herein, a guide polynucleotide or a portion thereof, or any composition provided herein, is provided herein. In some embodiments, the lipid nanoparticles are solid lipid nanoparticles (SLNs) or nanostructured lipid carriers (NLCs).

[0119] Lipid nanoparticles provided herein may include cationic lipids, ionized lipids, lipid mixtures, zwitterionic lipids, and / or phospholipids. In some embodiments, the lipid nanoparticles include cationic lipids selected from the group consisting of 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), and ethylphosphatidylcholine (ePC). In some embodiments, the lipid nanoparticles include 2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide (DOGS;Transfectam), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), DC-cholesterol and N4-cholesteryl-spermine (GL67), 9Z,12Z-octadecadienoic acid, and 3-[4,4-bis(octyloxy)-1-oxy Sobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP01), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecyloxyhexyl (undecoxyhexyl))amino]octanoate (SM-102), [(4-hydroxybutyl)azandiyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)nonanamide)nonadecanedioate (Lipid A9) comprises an ionized lipid selected from the group consisting of 5-(dimethylamino)-pentanoic acid, (6Z)-1,2-di-(4Z)-4-decen-1-yl-6-dodecen-1-yl ester (Lipid CL1), and 7-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]heptyl 2-octyldecanoate (Lipid 5). In some embodiments, the lipid nanoparticles contain phosphatidylcholine.In some embodiments, the lipid nanoparticles include cholesterol or cholesterol analogs. In some embodiments, the lipid nanoparticles include cholesterol analogs selected from the group consisting of β-sitosterol, vitamin D3, vitamin D2, calcipotriol, stigmasterol, betulin, lupeol, ursolic acid, oleanolic acid, stigmastanol, campesterol, fucosterol, brassicasterol, and ergosterol. In some embodiments, the lipid nanoparticles include polyethylene glycol (PEG). In some embodiments, the PEG is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG). 2000 -DMG) or 1,2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG 2000 -DSG). In some embodiments, the lipid nanoparticles include N-acetylgalactosamine (GalNAc). In some embodiments, GalNAc is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[tris-GalNAc-GABA-(polyethylene glycol)-2000](Tri-GalNAc-PEG2000-DSPE).

[0120] In some embodiments, the composition containing lipid nanoparticles is in the form of an emulsion. In some embodiments, the composition containing lipid nanoparticles is in the form of a liquid. In some embodiments, the composition containing lipid nanoparticles is in the form of a gel. In some embodiments, the composition containing lipid nanoparticles is in the form of a solid. In some embodiments, the system provided herein, a set of polynucleotides encoding the system provided herein, a vector provided herein, an engineered protein provided herein, or a guide polynucleotide or a portion thereof is encapsulated by lipid nanoparticles. In some embodiments, the system provided herein, a set of polynucleotides encoding the system provided herein, a vector provided herein, an engineered protein provided herein, or a guide polynucleotide or a portion thereof forms a complex with lipid nanoparticles.

[0121] The compositions provided herein may be delivered to a cell line using a vector comprising, for example, a polynucleotide sequence encoding the system provided herein, a polynucleotide sequence encoding a guide polynucleotide, a polynucleotide sequence encoding the engineered protein, or a polynucleotide sequence encoding the composition. In some embodiments, the systems described herein may be delivered without a viral vector. Any vector system may be used, including but not limited to plasmid vectors, viral vectors, and oncolytic viral vectors. Furthermore, any of these vectors may contain one or more transcription factors, transgenes, or molecular tags.

[0122] In some embodiments, the vectors provided herein are viral vectors. Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno-associated virus vectors (AAVs), adenovirus vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, picornavirus vectors, and oncolytic virus vectors.

[0123] In some embodiments, the viral vector contains AAV. AAV may have one or more of the AAV wild-type genes, either entirely or partially deleted. For example, the rep and / or cap genes of AAV may be entirely or partially deleted but still retain a functional flanking ITR sequence. The functional ITR sequence is necessary for the rescue, replication, and packaging of the AAV virion. The ITR does not need to be a wild-type nucleotide sequence and may be modified, for example, by nucleotide insertion, deletion, or substitution, as long as the sequence provides functional rescue, replication, and packaging. A recombinant AAV vector (rAAV) contains an infectious replication-deficient virus, comprising an AAV protein shell in which the AAV ITR encapsulates the desired heterogeneous nucleotide sequence flanked on both sides. The rAAV vector is produced in a suitable host cell containing the AAV vector and AAV helper and accessory functions. In this way, the host cell is enabled to encode the AAV polypeptide necessary to package the AAV vector (containing the recombinant nucleotide sequence of the choice) into infectious recombinant virion particles for subsequent gene delivery. In some embodiments, the AAV or rAAV provided herein includes serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or any combination thereof. In some embodiments, the delivery vehicle includes a hybrid AAV-lipid nanoparticle delivery system.

[0124] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), Visna-Maedi virus (VMV) virus, canine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and monkey immunodeficiency virus (SIV), or fragments, derivatives, or variants thereof.

[0125] Conventional virus-based and non-virus-based gene delivery methods may be used to introduce polynucleotides encoding compositions provided herein, polynucleotides encoding systems, polynucleotides encoding guide polynucleotides, or polynucleotides encoding engineered proteins into cells and target tissues. Exemplary non-viral vector delivery systems may include DNA plasmids, naked nucleic acids, and nucleic acids complexed with a delivery vehicle such as liposomes or poloxamers. Viral vector delivery systems may also include DNA viruses and RNA viruses that have either an episomatic genome or an integrated genome after delivery to cells.

[0126] Nonviral methods of nucleic acid delivery include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, bioristic methods, virosomes, liposomes, immunoliposomes, polycation:nucleic acid conjugates or lipid:nucleic acid conjugates, naked DNA, mRNA, artificial virions, and drug-enhanced DNA incorporation. For example, sonoporation using the Sonitron 2000 system (Rich-Mar) can also be used for nucleic acid delivery. Additional exemplary nucleic acid delivery systems include those offered by Lonza Nucleofactor Technologies (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.), and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., TRANSFECTAM® and LIPOFECTIN®).

[0127] The compositions and systems provided herein may be delivered to cells (ex vivo) or to target tissues (in vivo). Additional delivery methods include packaging the polynucleotides to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are delivered specifically to target tissues using bispecific antibodies, where one arm of the antibody is specific to the target tissue and the other arm is specific to the EDV. The antibody brings the EDV to the surface of the target cells, and the EDV is then delivered into the cells by endocytosis.

[0128] Vectors, including viral and non-viral vectors, containing nucleic acids encoding the nucleic acid editing systems provided herein, may also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mRNA may be administered. Administration is by any of the routes commonly used to introduce molecules to ultimately come into contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. One or more routes may be used to administer a particular composition.

[0129] In some embodiments, the compositions, manipulated proteins, guide polynucleotides, or systems provided herein can be shuttle-transported to the cell nucleus. For example, a vector may include a nuclear localization sequence (NLS). The vectors or any compositions provided herein can also be transported by proteins or protein complexes. In some embodiments, the compositions or systems provided herein can be introduced into cells or target tissues by minicircle vectors.

[0130] In some embodiments, the vectors or polynucleotides provided herein may be pre-complexed with the engineered proteins provided herein before electroporation into cells. The engineered proteins that may be used for shuttle transport may be nickase or catalytically dead Cas proteins. The nucleases that may be used for shuttle transport may be nuclease-competent proteins. In some embodiments, the engineered proteins provided herein may be pre-mixed with the guide polynucleotides provided herein and any additional elements (such as transgenes or other engineered proteins).

[0131] Cells may be transfected with a mutant adeno-associated virus vector or a chimeric adeno-associated virus vector encoding a system or composition provided herein. For example, the AAV vector concentration may range from about 0.5 nanograms to a maximum of 50 micrograms.

[0132] The systems or compositions provided herein may also be introduced into cells via electroporation technology. The amount of polynucleotides that can be introduced into cells by electroporation can be varied to optimize transfection efficiency and / or cell viability. In some embodiments, less than about 100 picograms of nucleic acid may be added to each cell sample, which may contain one or more cells being electroporated. In some embodiments, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 Micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms of nucleic acid may be added to each cell sample. For example, 1 microgram of the nucleic acid, polynucleotide, vector, or composition provided herein may be added to each cell sample for electroporation.In some embodiments, the amount of nucleic acid required for optimal transfection efficiency and / or cell viability may be cell type-specific. In some embodiments, the amount of nucleic acid used for each sample may directly correspond to transfection efficiency and / or cell viability. The transfection efficiency into cells using the nucleic acid delivery platforms described herein, for example either nucleofection or electroporation, can be greater than or equal to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.9%.

[0133] Viral particles such as AAV can be used to deliver viral vectors comprising a gene of interest or viral vectors comprising a transgene to cells ex vivo or in vivo. In some embodiments, the mutant adeno-associated viral vector or chimeric adeno-associated viral vector as disclosed herein can be measured as pfu (plaque forming units). In some embodiments, the pfu of the recombinant virus or mutant adeno-associated viral vector or chimeric adeno-associated viral vector of the compositions and methods of the present disclosure is about 10 8 to about 5×10 10 pfu. In some embodiments, the recombinant virus of the present disclosure is at least about 1×10 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9, 9×10 9 , 1 x 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , and 5×10 10 It is a pfu. In some embodiments, the recombinant virus of the present disclosure is up to about 1 × 10⁻¹⁶ 8 , 2×10 8 , 3 x 10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1 x 10 9 , 2×10 9 , 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1 x 10 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , and 5×10 10 It is a pfu. In some embodiments, the mutant adeno-associated virus vector or chimeric adeno-associated virus vector of the Disclosure may be measured as a vector genome. In some embodiments, the recombinant virus of the Disclosure is 1 × 10⁻¹⁶ 10 ~3×10 12 Vector genome, or 1 × 10⁶ 9 ~3×10 13 Vector genome, or 1 × 10⁶ 8 ~3×10 14 Vector genome, or at least about 1 × 10⁻¹⁶ 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 1011 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1×10 18 vector genomes, or 1×10 8 to 3×10 14 vector genomes, or up to about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , and 1×10 18 vector genomes.

[0134] In some embodiments, the mutant adeno-associated virus vector or chimeric adeno-associated virus vector of the present disclosure can be measured using a multiplicity of infection (MOI). In some embodiments, the MOI may refer to the ratio or multiple of vector or viral genomes to the cells to which the nucleic acid may be delivered. In some embodiments, the MOI may refer to the ratio or multiple of vector or viral genomes to the cells to which the nucleic acid may be delivered. In some embodiments, the MOI is 1×10 6 GC / mL. In some embodiments, the MOI is 1×10 5 GC / mL to 1×10 7 GC / mL. In some embodiments, the MOI is 1×10 4 GC / mL to 1×10 8It may be GC / mL. In some embodiments, the recombinant virus of this disclosure is at least about 1 × 10⁶ 1 GC / mL, 1 × 10 2 GC / mL, 1 × 10 3 GC / mL, 1 × 10 4 GC / mL, 1 × 10 5 GC / mL, 1 × 10 6 GC / mL, 1 × 10 7 GC / mL, 1 × 10 8 GC / mL, 1 × 10 9 GC / mL, 1 × 10 10 GC / mL, 1 × 10 11 GC / mL, 1 × 10 12 GC / mL, 1 × 10 13 GC / mL, 1 × 10 14 GC / mL, 1 × 10 15 GC / mL, 1 × 10 16 GC / mL, 1 × 10 17 GC / mL, and 1 × 10⁻⁶ 18 The MOI is GC / mL. In some embodiments, the mutant adeno-associated virus or chimeric adeno-associated virus of this disclosure is approximately 1 × 10⁻⁶. 8 GC / mL ~ approx. 3×10 14 Is the GC / mL MOI, or is it at most about 1 × 10⁻⁶? 1 GC / mL, 1 × 10 2 GC / mL, 1 × 10 3 GC / mL, 1 × 10 4 GC / mL, 1 × 10 5 GC / mL, 1 × 10 6 GC / mL, 1 × 10 7 GC / mL, 1 × 10 8 GC / mL, 1 × 10 9 GC / mL, 1 × 10 10 GC / mL, 1 × 10 11 GC / mL, 1 × 10 12 GC / mL, 1 × 10 13 GC / mL, 1 × 10 14 GC / mL, 1 × 10 15 GC / mL, 1 × 10 16 GC / mL, 1 × 10 17 GC / mL, and 1 × 10⁻⁶ 18 It is GC / mL MOI.

[0135] In some embodiments, nonviral vectors or nucleic acids may be delivered without using mutant adeno-associated virus vectors or chimeric adeno-associated virus vectors and may be measured according to the amount of nucleic acid. Generally, any suitable amount of nucleic acid may be used with the compositions and methods of this disclosure. In some embodiments, the nucleic acid may be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg The dosage may be 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. In some embodiments, nucleic acids are present in amounts up to approximately 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, The dosage may be 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.

[0136] The proteins, vectors, plasmids, compositions, systems, engineered proteins, and guide polynucleotides provided herein may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, rapid DNA coating pellets, viral infection, and protoplast fusion. Methods used to construct any embodiment of the compositions provided herein include genetic engineering, recombinant engineering, and synthetic techniques.

[0137] Polynucleotides encoding engineered proteins, polynucleotides encoding guide polynucleotides, or polynucleotides encoding compositions provided herein can be delivered to cells by electroporation. For example, electroporation using the NEON® Transfection System (ThermoFisher Scientific) or Lonza Nucleofactor technologies is also used for the delivery of nucleic acids and proteins to cells. For example, engineered proteins provided herein can be purified and complexed with appropriate guide polynucleotides for delivery to cells. Electroporation parameters can be adjusted to optimize delivery efficiency and / or cell viability. Electroporation devices may have multiple electrical waveform pulse settings, such as exponential decay, time constant, and square wave. Every cell type has an intrinsic optimal electric field strength (E), which depends on the applied pulse parameters, such as voltage, capacitance, and resistance. Applying the optimal electric field strength induces increased electrical permeability via the induction of transmembrane voltage, thereby enabling nucleic acids to pass through the cell membrane. In some embodiments, the electroporation pulse voltage, electroporation pulse width, pulse count, cell density, and tip type may be adjusted to optimize transfection efficiency and / or cell viability.

[0138] (3) Cells and cell-free systems Cells comprising a system, guide polynucleotide, composition, or engineered protein provided herein are provided herein. In some embodiments, polynucleotides encoding engineered proteins provided herein and polynucleotides encoding guide polynucleotides provided herein are administered to cells or populations of cells.

[0139] The compositions, polynucleotides, engineered proteins, guide polynucleotides, and systems provided herein can be delivered to any suitable cell. In some embodiments, the compositions, polynucleotides, engineered proteins, guide polynucleotides, and systems provided herein modulate the genes, proteins, and / or functional phenotype of a cell.

[0140] Suitable cells may include, but are not limited to, eukaryotic and prokaryotic cells and / or eukaryotic and prokaryotic cell lines. Suitable cells may, for example, be human primary cells. Primary cells may be taken directly from living tissue (i.e., biopsy material) and established for in vitro growth, having undergone very little population doubling, and therefore represent more of the major functional components and characteristics of the tissue from which they originate compared to continuous tumorigenic cell lines or artificially immortalized cell lines. Primary cells may be obtained from a variety of sources, such as organs, vascular structures, buffy coat, whole blood, apheresis, plasma, bone marrow, tumors, cell banks, cryopreservation banks, or blood samples. Primary cells may be stem cells.

[0141] Suitable cells that may come into contact with the compositions, polynucleotides, manipulated proteins, guide polynucleotides, or systems provided herein include epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, leukocytes, lymphocytes (B, NK, and T), macrophages, monocytes, mononuclear cells, cardiomyocytes, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, pancreatic islet cells, blood cells, hematopoietic progenitor cells, osteocytes, bone progenitor cells, neural stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, hepatic stellate cells, aortic smooth muscle cells, cardiomyocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, This includes, but is not limited to, parathyroid cells, parotid gland cells, tumor cells, glial cells, astrocytes, erythrocytes, leukocytes, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, cardiac cells, pacemaker cells, spleen cells, antigen-presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, spermatid cells, testicular cells, germ cells, egg cells, Leydig cells, peritubular cells, Sertoli cells, luteal cells, cervical cells, endometrial cells, mammary gland cells, follicular cells, mucinous cells, ciliated cells, non-keratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, dopaminergic cells, squamous epithelial cells, osteocytes, osteoblasts, osteoclasts, dopaminergic cells, embryonic stem cells, fibroblasts, and fetal fibroblasts. Furthermore, one or more cells may include, but are not limited to, pancreatic alpha cells, pancreatic beta cells, pancreatic delta cells, pancreatic f cells (also called pancreatic polypeptide cells or PP cells), or pancreatic epsilon cells, such as islet cells and / or cell clusters.

[0142] Suitable cells also include stem cells, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, and mesenchymal stem cells. Suitable cells may include any number of primary cells, such as human cells, non-human cells, and / or mouse cells. Suitable cells may be progenitor cells. Suitable cells may originate from the subject being treated. For example, the subject being treated may be a subject with a disease, a subject requiring treatment, or an immunocompromised subject. Suitable cells may originate from a human donor.

[0143] In some embodiments, cells are genetically modified by methods, systems, and compositions provided herein. The cells provided herein may be administered to subjects in need, for example, subjects having a disease or condition requiring treatment.

[0144] Methods for obtaining suitable cells, such as primary human cells, may include cell selection. In some embodiments, cells may include markers that can be used to select them. For example, such markers may include GFP, resistance genes (e.g., genes that confer antibiotic resistance), cell surface markers, and endogenous tags. Cells may be selected using any endogenous marker. Suitable cells may be selected using any technique. Such techniques may include flow cytometry and / or magnetic columns. The selected cells may also be expanded to a large number.

[0145] Delivery vehicles for in vivo and ex vivo use may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is determined in part by the specific composition to be administered, e.g., polynucleotides, proteins, vectors, or cells, and by the specific method used to administer the composition.

[0146] Cell-free systems comprising systems, compositions, guide polynucleotides, or engineered proteins are provided herein. The cell-free systems contain sufficient components for a synthetic reaction and, in some cases, retain the biological activity of DNA ligases and nickases when stored at room temperature for a certain period. In some embodiments, the cell-free systems comprise a set of reagents capable of providing or supporting a biosynthetic reaction. Non-limiting examples of biosynthetic reactions include in vitro DNA replication, transcription, translation, or combinations of reactions in the absence of cells. Cell-free systems may be prepared using enzymes, coenzymes, and other intracellular components that have been isolated or purified from eukaryotic or prokaryotic cells, including recombinant cells, or as extracts or fractions of such cells. Cell-free systems may originate from a variety of sources, including but not limited to eukaryotic and prokaryotic cells, bacteria (including, but not limited to, E. coli, thermophilic bacteria), wheat germ, rabbit reticulocytes, mouse L cells, Ehrlich ascites cancer cells, HeLa cells, CHO cells, and budding yeast. In some embodiments, the cell-free system is freeze-dried. In some embodiments, the cell-free system further includes a scaffold.

[0147] (4) Pharmaceutical compositions, medications, and administrations Pharmaceutical compositions comprising a composition, guide polynucleotide, engineered protein, or polynucleotide provided herein, and a pharmaceutically acceptable diluent, carrier, or excipient are provided herein. Pharmaceutical compositions comprising a vector provided herein, and a pharmaceutically acceptable diluent, carrier, or excipient are provided herein.

[0148] In some embodiments, the compositions provided herein (e.g., vectors comprising the systems or compositions provided herein) are combined with pharmaceutically acceptable salts, excipients, and / or carriers to form pharmaceutical compositions. The pharmaceutical salts, excipients, and carriers may be selected based on the route of administration, the location of the target tissue (issue), and the time course of drug delivery. Pharmaceutically acceptable carriers or excipients may include solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are suitable for pharmaceutical administration.

[0149] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid, or gas (aerosol). Injectable preparations, such as sterile aqueous or sterile oily suspensions for injection, can be formulated according to known techniques using a suitable dispersant or wetting agent and a suspending agent. Sterile injectable preparations can also be sterile injectable solutions, sterile suspensions, or sterile emulsions in non-toxic, parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that can be used include water, Ringer's solution, United States Pharmacopeia (USP), and isotonic sodium chloride solution. Furthermore, sterile fixatives have conventionally been used as solvents or suspension media. For this purpose, any non-irritating fixative, including synthetic monoglycerides or synthetic diglycerides, can be used. Additionally, fatty acids such as oleic acid are used in the preparation of injectable preparations. Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media before use.

[0150] The compositions and systems provided herein may be formulated in dosing unit forms for ease of administration and uniformity of dosage. A unit dosage form is a physically distinct unit of the composition provided herein that is suitable for the target being treated. For any composition provided herein, the therapeutically effective dose may first be estimated in either a cell culture assay or an animal model (such as a mouse, rabbit, dog, pig, or non-human primate). The animal model may also be used to achieve the desired concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans. The therapeutic efficacy and toxicity of the compositions provided herein may be determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., ED 50 (The dose is therapeutically effective in 50% of the population) and LD 50 (This can be determined by whether the dose is lethal to 50% of the population.) The dose-to-toxicity ratio is the therapeutic index, which is the ratio LD50. 50 / ED 50 It can be expressed as follows. Pharmaceutical compositions exhibiting a large therapeutic index may be useful in some embodiments. Data obtained from cell culture assays and animal studies may be used when formulating a range of doses for human use.

[0151] Pharmaceutical compositions for administering a composition or system to a subject in need thereof are provided herein. In some embodiments, the pharmaceutical composition is a treatment for a disease or condition provided herein. In some embodiments, the pharmaceutical composition provided herein is a form that enables the composition provided herein to be administered to a subject. In some embodiments, the pharmaceutical composition is formulated for intratumoral delivery. In some embodiments, the administration of the pharmaceutical composition provided herein is local or systemic. In some embodiments, the pharmaceutical composition provided herein is formulated for / for use in administration via an intratumoral route, subcutaneous route, intradermal route, intramuscular route, inhalation, intravenous route, intraperitoneal route or intracranial route. In some embodiments, the administration is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administration is at least about 5 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours (1 day), at least about 48 hours (2 days), at least about 72 hours (3 days), at least about 96 hours (4 days), at least about 120 hours (5 days), at least about 144 hours (6 days), at least about 168 hours (7 days), at least about 336 hours (14 days), at least about 504 hours (21 days), at least about 672 hours (28 days), and up to 744 hours (31 days). In some embodiments, the administration is every 744 hours (once a month) or once a year (365 days).

[0152] Vectors can be delivered in vivo by administration to individual subjects, typically by systemic administration, such as intravenous, intraperitoneal, intramuscular, subdermal, or intracranial injection. Vectors can also be delivered by topical application, as described below. Alternatively, vectors can be delivered ex vivo to cells (e.g., cells explanted from individual subjects, such as lymphocytes, T cells, bone marrow aspirates, or tissue biopsies), followed by re-implantation of the cells into the subjects, usually after selection of the vector-incorporated cells. Before or after selection, cells can be expanded in cell culture or a bioreactor.

[0153] Cells expressing the systems or compositions provided herein are provided herein. In some embodiments, cells are contacted in vitro or ex vivo with nucleic acids encoding the systems or compositions provided herein. In some embodiments, cells are contacted in vitro or ex vivo with vectors encoding the manipulated proteins, systems, or compositions provided herein.

[0154] (5) Scaffolding and systems This specification provides scaffolds comprising any composition, system, or guide polynucleotide provided herein. Scaffolds provided herein may be useful in the detection of newly edited nucleic acids or in the identification of target nucleic acids provided herein. In some embodiments, the compositions provided herein are immobilized on the scaffold. In some embodiments, the scaffold includes a surface. In some embodiments, the surface includes a solid, semi-solid, or gel surface. In some embodiments, the scaffold includes reaction chips, paper, quartz microfibers, cellulose ester mixtures, porous aluminum oxide, patterned surfaces, tubes, wells, or matrices. In some embodiments, the scaffold includes a patterned surface suitable for immobilizing molecules in a regular pattern. In some embodiments, a patterned surface refers to the arrangement of various regions within or on the exposed layer of the scaffold. In some embodiments, the scaffold includes an array of wells or an array of depressions on its surface. The composition and geometric shape of the scaffold may vary depending on its application. In some embodiments, the scaffold is a planar structure, such as a slide, chip, microchip, and / or array. Thus, the surface of the scaffold may be in the form of a planar layer. In some embodiments, the scaffold comprises one or more surfaces of a flow cell. A flow cell is a type of chamber containing a solid surface through which one or more fluid reagents can flow. In some embodiments, the scaffold or its surface is non-planar (e.g., the inner or outer surface of a tube or vessel). In some embodiments, the scaffold comprises microspheres or beads. Microspheres, beads, or particles can be made of a variety of materials, including but not limited to plastics, ceramics, glass, and polystyrene. In some embodiments, the microspheres are magnetic microspheres or magnetic beads. Alternatively or additionally, the beads may be porous. Bead sizes range from nanometers (nm) (e.g., about 100 nm) to millimeters (e.g., about 1 mm).

[0155] In some embodiments, the scaffold comprises a set of engineered proteins, a set of polynucleotides provided herein, a set of systems provided herein, or any combination thereof. A system further comprising the scaffold is provided herein, where the scaffold comprises a surface. In some embodiments, the scaffold further comprises a cell-free system provided herein. In some embodiments, the system and scaffold provided herein further comprises reagents for nucleic acid amplification. In some embodiments, the system and scaffold provided herein further comprises reagents for DNA replication. In some embodiments, the system provided herein further comprises (a) the scaffold provided herein, (b) a reporter molecule, and (c) a detector. In some embodiments, when a target nucleic acid forms a complex with the scaffold, a guide polynucleotide provided herein, or an engineered protein provided herein, the reporter molecule generates a detectable signal that is detected by the detector. In some embodiments, the reporter molecule is selected from the group consisting of fluorophores, dyes, polypeptides, antibodies, nucleic acids, and any combination thereof. In some embodiments, the detectable signal is a calorimetry signal, a potentiometry signal, an amperometry signal, a light signal, or a piezoelectric signal. Reporter molecules can be used to identify cells containing novel nucleic acids edited by the manipulated proteins provided herein. Reporter molecules can also be used, for example, in cell sorting, nucleic acid isolation, nucleic acid sequencing, or immunochemical techniques.

[0156] (6) Kit This specification provides kits comprising systems, compositions, polynucleotides, vectors, guide polynucleotides, or engineered mRNA or engineered proteins provided herein, and packaging and its materials. In some embodiments, the kit further comprises a scaffold provided herein. In some embodiments, the kit further comprises a cell-free system. In some embodiments, the kit further comprises a population of cells. In some embodiments, the cells are stored in a cryopreservation medium. In some embodiments, the cryopreservation medium comprises dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium comprises a buffer, an isotonic agent, or an apoptosis inhibitor. Non-limiting examples of buffer elements include citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris. Non-limiting examples of isotonic agents include, for example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, histidine, and Tris. Additional isotonic agents include sodium chloride, potassium chloride, boric acid, sodium borate, mannitol, glycerin, propylene glycol, polyethylene glycol, maltose, sucrose, erythritol, arabitol, xylitol, sorbitol, trehalose, and glucose. Apoptosis inhibitors may include, for example, Rho-related kinase (ROCK) inhibitors, catalase, and zVAD-fmk. In some embodiments, the kit includes reagents. In some embodiments, the reagents include sugars and sugar derivatives. In some embodiments, the sugar derivatives include sodium carboxymethylcellulose or cellulose acetate. In some embodiments, the reagents include surfactants, glycols, polyols, esters, buffers, alginic acid, and / or organic solvents.

[0157] In some embodiments, formulations of the compositions described herein are prepared in a single container for administration to cells, cell-free systems, or subjects. In some embodiments, formulations of the compositions provided herein are prepared in two containers for administration, separating the guide polynucleotide or the polynucleotide encoding the guide polynucleotide and / or the polynucleotide encoding the manipulated protein provided herein. As used herein, “container” includes vessels, vials, ampoules, tubes, cups, boxes, bottles, flasks, jars, dishes, wells in single-well devices or multi-well devices, reservoirs, tanks, or other devices in which the compositions disclosed herein are placed, stored, and / or transported and can be accessed for removal of their contents. Examples of such containers include glass and / or plastic, sealed or resealable tubes and ampoules, which include those having rubber septa or other sealing means suitable for removal of contents using needles and syringes. In some embodiments, the containers are RNase-free.

[0158] Provided herein is a kit comprising a first container containing a donor nucleic acid, a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, and a second container containing an engineered protein or a polynucleotide encoding an engineered protein, wherein the guide polynucleotide comprises (i) a target-directing region having complementarity with the target nucleic acid, (ii) a protein-binding region (the protein-binding region comprising a secondary structure that binds to a nuclease or nickase), (iii) a ligation sprint 2 region having complementarity with the donor nucleic acid, the target nucleic acid, and at least one mismatched nucleic acid base to the target nucleic acid, and (iv) a ligation sprint 1 region (the ligation sprint 1 region comprising DNA nucleotides and RNA nucleotides), and the engineered protein comprises a nickase operably linked to a DNA ligase. In some embodiments, the kit provided herein further comprises reagents for nucleic acid amplification, transcription, translation, or nucleic acid isolation. In some embodiments, the kit provided herein further comprises a reporter molecule provided herein.

[0159] (7) Method for determining gene editing activity This specification provides methods for determining the gene editing activity, efficiency, and selectivity of the engineered proteins, compositions, or systems provided herein with respect to a target nucleic acid sequence. In some embodiments, the target nucleic acid is cleaved by a nuclease or nickase provided herein when a guide polynucleotide binds to the target nucleic acid sequence. In some embodiments, the target nucleic acid is DNA. In some embodiments, the nickase region of the engineered protein provided herein cleaves the DNA, resulting in a single-strand break. In some embodiments, the nickase region of the engineered protein provided herein cleaves the target DNA via staggered single-strand or double-strand DNA breaks. The ability of the nuclease or nickase provided herein to recognize a PAM sequence can be determined by an in vitro selection assay.

[0160] The activity of the systems provided herein can be assayed using cells expressing a reporter protein or cells containing a reporter gene. For example, a reporter gene may be engineered to contain an interferant such as a stop codon, frameshift mutation, spacer, linker, or transcription terminator, and the system may then be used to remove the interferant, and the resulting functional reporter protein may be detected. Similarly, a reporter gene may be introduced into a target nucleic acid for target detection. In some embodiments, a reporter gene may be designed such that a specific sequence modification is required to restore the functionality of the reporter protein. In other embodiments, a reporter gene may be designed such that any insertion or deletion resulting in a frameshift of one or two bases in the target nucleic acid may be sufficient to restore the functionality of the reporter protein. Examples of reporter proteins encoded by a reporter gene include colorimetric enzymes, metabolic enzymes, fluorescent proteins, enzymes and transporters associated with antibiotic resistance, and luminescent enzymes. Examples of such reporter proteins include β-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, red fluorescent protein, and firefly luciferase and sea urchin luciferase. Various detection methods can be used for various reporter proteins. For example, reporter proteins may affect cell viability, cell growth, fluorescence, luminescence, or the expression of a detectable product. In some embodiments, reporter proteins may be detected using colorimetric assays. In some embodiments, the reporter protein may be a fluorescent protein, and DNA editing may be assayed by measuring the degree of fluorescence in treated cells, or the number of treated cells with at least a threshold level of fluorescence. In some embodiments, the transcription level of the reporter gene may be evaluated. In other embodiments, the reporter gene may be evaluated by sequencing.

[0161] The integration of donor nucleic acids ligated by DNA ligase into target nucleic acids can be measured using any technique, for example, by denatured urea-polyacrylamide gel electrophoresis, PAGE gel electrophoresis, flow cytometry, surveyor crease assay, tracking of indels by decomposition (TIDE), junction PCR, droplet digital PCR, or any combination thereof. In other embodiments, transgene integration can be measured by PCR or droplet digital PCR. TIDE analysis can also be performed on manipulated cells. Ex vivo cell transfection can also be used for diagnostics, research, or gene therapy. In some embodiments, transfected cells are reinjected into a host organism. In some embodiments, cells are isolated from a target organism, transfected with nucleic acids, and reinjected into the target.

[0162] The amount of genetically modified cells that may be necessary for therapeutic efficacy in a target may vary depending on cell viability and the efficiency with which the cells are genetically modified. For example, the efficiency with which a transgene is incorporated into one or more cells may determine the number of cells administered to the target. In some embodiments, the product (e.g., multiplication) of the viability of genetically modified cells and the efficiency of transgene integration may correspond to a therapeutic aliquot of cells available for administration to the target. In some embodiments, an increase in the viability of genetically modified cells may correspond to a decrease in the amount of cells required for administration to be therapeutically effective in the target. In some embodiments, an increase in the efficiency with which a transgene is incorporated into one or more cells may correspond to a decrease in the amount of cells required for administration to be therapeutically effective in the target. In some embodiments, determining the amount of cells required for therapeutic efficacy may involve determining a function corresponding to changes in cell viability over time. In some embodiments, determining the amount of cells required for therapeutic efficacy may involve determining a function with respect to a time-dependent variable corresponding to changes in the efficiency with which a transgene can be incorporated into one or more cells. The variables may include, for example, cell culture time, electroporation time, and cell stimulation time.

[0163] Non-homologous end joining (NHEJ) and homologous recombination repair (HDR) can be quantified using various methods. For example, the percentage of NHEJ, HDR, or a combination of both can be determined by co-delivering gene editing molecules (e.g., guide polynucleotides and engineered proteins provided herein) to cells together with donor nucleic acids encoding promoter-free tags or promoter-free markers. In some embodiments, the target marker is GFP of a polynucleotide encoding green fluorescent protein (GFP). After a certain duration (e.g., about 72 hours to about 96 hours), flow cytometry is performed to determine the total number of cells (N TotalThis can be performed to quantify the number of tag-positive and tag / GFP-negative cells. In tag-negative cells, next-generation sequencing may be performed to identify cells without mutations and cells with mutations. HDR efficiency and NHEJ efficiency can be calculated from this assay.

[0164] Additional assays for determining the gene editing efficiency of the systems or compositions provided herein may include, but are not limited to, RT-PCR, nucleic acid sequencing, T7 endonuclease 1 (T7E1) mismatch detection assays, tracking of indels by decomposition (TIDE) assays, and indel detection by amplicon analysis (IDAA) assays. Indel patterns induced at target sites of programmable nucleases or programmable nickases can also be determined by PCR amplification of the respective regions and subsequent next-generation sequencing. Cell surface markers may be targeted to obtain a quantitative single-cell view of gene editing efficiency, and signal loss resulting from indel formation may be quantified by flow cytometry. Single-cell sequencing may be used to assess genome editing efficiency.

[0165] (8)Applications Methods for modifying a target nucleic acid, or for generating changes in a target nucleic acid or gene within a cell, are provided herein. In some embodiments, the method comprises administering a system, a composition, or a cell provided herein to a cell, tissue, or subject, the administration of which generates a change in the target nucleic acid. In some embodiments, the number of changes in the target nucleic acid is at least one change. In some embodiments, the percentage of change in the target nucleic acid molecule is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%. In some embodiments, the method for modifying a target nucleic acid further comprises mutations in the target nucleic acid sequence. In some embodiments, the alteration or modification of the target nucleic acid includes insertions, deletions, substitutions, copy number changes, point mutations, frameshift mutations, missense mutations, nonsense mutations, mutations at stop codons, epigenetic marks, or any combination thereof.

[0166] In some embodiments, the alteration is generated in the target nucleic acid to form an edited nucleic acid. In some embodiments, the edited nucleic acid restores the expression of the wild-type protein encoded by its gene compared to an equivalent cell or population of cells that has not been in contact with either the system or composition provided herein.

[0167] Methods for ex vivo modification of cells are provided herein. In some embodiments, the method involves contacting cells with a composition provided herein under conditions that allow for cleavage of a target nucleic acid molecule by a nuclease or nickase, thereby modifying the cells. Methods for ex vivo modification of cells are provided herein. In some embodiments, the method involves contacting cells with a composition provided herein under conditions that allow for DNA ligation of a new nucleic acid sequence for incorporation into a target nucleic acid, thereby modifying the target nucleic acid and the cells.

[0168] Methods for ex vivo modification of cells are further provided herein, the methods comprising contacting cells with a ribonucleoprotein (RNP) complex, the RNP complex comprising (i) an engineered protein provided herein and (ii) a polynucleotide that binds to a target nucleic acid, and upon contact of cells with the RNP complex, the engineered protein cleaves the target nucleic acid molecule and ligates a new nucleic acid, thereby modifying the cells. In some embodiments, the cells are immune cells or stem cells. In some embodiments, the immune cells are leukocytes, lymphocytes, natural killer cells, dendritic cells, macrophages, myeloid cells, T cells, B cells, stem cells, induced-pluripotent stem cell-derived cells, cancer cells, or endothelial cells. In some embodiments, the stem cells are embryonic stem cells, induced pluripotent stem cells (iPSCs), or adult stem cells.

[0169] The methods for modifying target nucleic acids provided herein may be used in agricultural applications, such as the production of improved plant species. The methods for modifying target nucleic acids provided herein may also be used in biomedical applications, drug screening platforms, and therapeutic treatments. For example, the compositions and systems provided herein may be used to generate cell therapies for the treatment of diseases or disorders.

[0170] Provided herein is a method for modifying nucleic acids, the method comprising: a cell or cell-free system; (a) a donor nucleic acid; (b) a guide polynucleotide or polynucleotide encoding a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region having complementarity with the target nucleic acid; (ii) a protein-binding region (the protein-binding region comprising a secondary structure that binds to nickase); (iii) a ligation sprint 2 region having complementarity with the donor nucleic acid, the target nucleic acid, and at least one mismatch nucleic acid base for the target nucleic acid; and (iv) a ligation sprint 1 region (the ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide); The method further comprises (c) contacting a manipulated protein or a polynucleotide encoding a manipulated protein (the manipulated protein includes (i) a nickase region and (ii) a DNA ligase region), wherein the guide polynucleotide complexes with the manipulated protein via a protein-binding region, the target-directing sequence complexes with the complementary strand of the target nucleic acid, the nickase region of the manipulated protein causes a cleavage in the target nucleic acid to generate a leading strand, the ligation sprint 1 region complexes with the leading strand, the ligation sprint 2 region complexes with the donor nucleic acid, and the DNA ligase region catalyzes the formation of a phosphodiester bond between the 3' hydroxyl end of the leading strand and the 5' phosphate end of the donor nucleic acid, thereby inserting the new nucleic acid. In some embodiments, the target-directing region dissociates from the complementary strand. In some embodiments, the new nucleic acid is incorporated into the target nucleic acid by hybridizing with the complementary strand. In some embodiments, donor nucleic acid incorporation recruits DNA repair proteins to the target nucleic acid to edit its nucleic acid bases.

[0171] Provided herein are methods for detecting nucleic acids in a test sample, the methods comprising (a) immobilizing a guide polynucleotide on a scaffold provided herein, and (b) contacting the guide polynucleotide with (i) a test sample and (ii) an engineered protein provided herein. In some embodiments, the test sample comprises a target nucleic acid capable of binding to the guide polynucleotide. In some embodiments, a complex is formed between the guide polynucleotide, the engineered protein provided herein, and the target nucleic acid. In some embodiments, once the complex is formed, the engineered protein (e.g., a nickase region) cleaves the target nucleic acid. In some embodiments, the method further comprises detecting a signal indicating cleavage of the target nucleic acid molecule, or detecting a new nucleic acid chain containing a reporter molecule, thereby detecting the target nucleic acid in the sample.

[0172] In some embodiments, the method further includes amplifying the target nucleic acid before detection. In some embodiments, amplification includes polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification (NEAR), multiple substitution amplification (MDA), rolling circle amplification (RCA), improved multiple substitution amplification (EVIDA), simple method amplifying RNA targets (SMART), single-primer isothermal amplification (SPIA), ligase chain reaction (LCR), transcription-mediated amplification (TMA), ramification amplification method (RAM), or any combination thereof. In some embodiments, the method further includes performing an endonuclease mismatch detection assay, immunoassay, gel electrophoresis, plasmid interference assay, nucleic acid sequencing, or any combination thereof. In some embodiments, detection includes heat detection, potential difference detection, current detection, optical detection, piezoelectric detection, or any combination thereof.

[0173] Methods for treating a disease or condition in a subject requiring treatment of the disease or condition are provided herein. In some embodiments, the subject has, is suspected of having, or is diagnosed with a disease or condition. In some embodiments, the method comprises administering a system, composition, vector, pharmaceutical composition, or polynucleotide provided herein to the subject. In some embodiments, the administration is topical or systemic. In some embodiments, the administration is intranasal, subcutaneous, intravenous, inhalation, intramuscular, intratumoral, peritumoral, intrathecal, vaginal, or intradermal. In some embodiments, the method further comprises administering a therapeutic agent to the subject.

[0174] The use of compositions and systems provided herein in the manufacture of pharmaceuticals is also provided. The use of compositions provided herein in the manufacture of pharmaceuticals for the therapeutic and / or prophylactic treatment of diseases or conditions described herein is also provided. In some embodiments, the disease or condition is caused by a mutated disease-associated gene. In some embodiments, the disease-associated gene is any gene associated with an increased risk of having or developing the disease. In some embodiments, the disease-associated gene is any gene or polynucleotide that produces a transcript or translation product at an abnormal level or in an abnormal form in cells derived from diseased tissue compared to non-disease control tissue or cells. In some embodiments, the disease-associated gene is a gene that becomes expressed at an abnormally high level. In some embodiments, the disease-associated gene is a gene that becomes expressed at an abnormally low level, and the altered expression correlates with the onset and / or progression of the disease. In some embodiments, the disease-associated gene is a gene with a mutation or genetic mutation that is the cause of the disease or is in linkage disequilibrium with a gene that is the cause of the disease. The transcript or translation product may be known or unknown and may be at normal or abnormal levels.

[0175] In some embodiments, the disease or condition is caused by mutations associated with DNA repeat instability and mutations associated with neurological disorders. Certain aspects of tandem repeat sequences have been found to be the cause of more than 20 human diseases. This system can be used to correct these defects of genomic instability. In some embodiments, the disease or condition is a neurological disorder, cardiovascular disease, cancer, respiratory disease, diabetes, obesity, eye disease, hearing loss, blindness, or a rare genetic disorder or rare genetic disorder. In some embodiments, the disease or condition is age-related macular degeneration, schizophrenia, triplet repeat disease, or fragile X syndrome. In some embodiments, the disease or condition is a secretase-related disorder. In some embodiments, the disease or condition is a prion-related disorder. In some embodiments, the disease or condition is ALS. In some embodiments, the disease or condition is drug addiction. In some embodiments, the disease or condition is autism. In some embodiments, the disease or condition is Alzheimer's disease. In some embodiments, the disease or condition is inflammation. In some embodiments, the disease or condition is Parkinson's disease. Further examples of diseases and conditions treatable with the systems and compositions provided herein include, but are not limited to, Ecardi-Goutier syndrome, Alexander disease, Alan Herndon-Dudley syndrome, POLG-related disorders, α-mannosidosis (types II and III), Alström syndrome, Angelman syndrome, ataxia telangiectasia, neuronal ceroid lipofuscinosis, β-thalassemia, bilateral optic atrophy and (infant) optic atrophy type 1, retinoblastoma (bilateral), Canavan disease, and cerebrooculofacioskeletal syndrome.Syndrome type 1 (COFS1), cerebral tendon xanthomatous disease, Cornelia de Lange syndrome, MAPT-related disorders, hereditary prion diseases, Dravet syndrome, early-onset familial Alzheimer's disease, Friedreich's ataxia (FRDA), Flins syndrome, fucosidosis, Fukuyama-type congenital muscular dystrophy, galactosialidosis, Gaucher disease, organic acid metabolism disorders, hemophagocytic lymphohistiocytosis, progeria syndrome, mucolipidosis type II, infant free sialic acid storage, PLA2G6-associated neurodegeneration, Javer-Lange-Nielsen syndrome, junctional epidermolysis bullosa, Huntington's disease, Krabbe disease (infant form), mitochondrial DNA-associated Lie syndrome and NARP, Lesch-Nyhan syndrome, LIS1-associated lissencephaly, Lowe syndrome, maple syrup urine disease, MECP2 duplication syndrome, ATP7A-associated copper transport disorder, LAMA2-associated muscular dystrophy, arylsulfatase A deficiency, mucopolysaccharidosis type I, mucopolysaccharidosis type II or muco Polysaccharidosis type III, peroxisome dysplasia (Zellweger syndrome spectrum), neurodegenerative disease with intracranial iron accumulation, acid sphingomyelinase deficiency, Niemann-Pick disease type C, glycine encephalopathy, ARX-related disorders, urea cycle disorders, COL1A1 / 2-related osteogenesis imperfecta, mitochondrial DNA depletion syndrome, PLP1-related disorders, Perry syndrome, Phelan-McDermid syndrome, glycogen storage disease type II (Pompe disease) (infant form), MAPT-related disorders, MECP Collagen-related disorders, rhizoid chondrodysplasia type 1, Roberts syndrome, Sandhoff disease, Schindler disease type 1, adenosine deaminase deficiency, Smith-Lemle-Oppitz syndrome, spinal muscular atrophy, infantile-onset spinocerebellar degeneration, hexosaminidase A deficiency, thanatophoric dysplasia type 1, collagen-related disorders type VI, Usher syndrome type I, congenital muscular dystrophy, Wolf-Hirschhorn syndrome, lysosomal acid lipase deficiency, and xeroderma pigmentosum. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0176] Exemplary Embodiments A composition is provided herein, comprising (a) a donor nucleic acid; (b) a polynucleotide encoding a protein construct comprising a nickase or a variant thereof and a DNA ligase or a functional fragment thereof; (c) a guide polynucleotide, comprising (i) a target-directing region having complementarity with a target nucleic acid, (ii) a protein-binding region comprising a secondary structure that binds to an engineered protein construct comprising a nickase region; (iii) a ligation sprint 2 region, wherein the ligation sprint region has complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint region comprises at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region, wherein the ligation sprint 1 region comprises a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region has complementarity with the target nucleic acid, comprising a guide polynucleotide. Compositions are provided herein in which a ligation sprint 1 region forms a DNA-RNA (DR) loop upon association with a target nucleic acid and DNA ligase. Compositions are also provided herein in which a ligase sprint 1 region comprises an RNA-to-DNA ratio of 1:1 to a maximum of 20:1. The ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3 :16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15,8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9:20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 1 1:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12:1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 14:13, 15:1 , 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18 Compositions comprising a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9, 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:1 are provided herein. Compositions comprising a ligation sprint 2 region comprising at least about 5 nucleotides to a maximum of 10,000 nucleotides are provided herein. Compositions comprising a ligation sprint 2 region comprising at least about 7 nucleotides to a maximum of 1,000 nucleotides are provided herein. Compositions comprising a ligation sprint 1 region comprising at least about 5 nucleotides to a maximum of 20 nucleotides are provided herein. A composition is provided herein in which the ligation sprint 2 region comprises the reverse complementary sequence of a non-coding polynucleotide sequence or a variant thereof.A composition comprising a reverse complementary sequence of a sequence encoding the coding region of a polynucleotide sequence or a variant thereof is provided herein. A composition comprising a ligation sprint 2 region comprising a reverse complementary sequence of a sequence encoding an exon or intron is provided herein. A composition comprising a ligation sprint 2 region comprising a complementary sequence of a sequence encoding a non-coding polynucleotide sequence or a variant thereof is provided herein. A composition comprising a ligation sprint 2 region comprising a complementary sequence of a sequence encoding the coding region of a polynucleotide sequence or a variant thereof is provided herein. A composition comprising a ligation sprint 2 region comprising a sequence comprising a sequence encoding an exon or intron is provided herein. A composition comprising a ligation sprint 2 region comprising a sequence comprising at least one nucleic acid base that is complementary or mismatched with the sequence encoding a splice acceptor site is provided herein. A composition comprising a nickase being an engineered Cas protein or a functional variant thereof is provided herein. A composition is provided herein in which an engineered Cas protein or a functional variant thereof comprises at least one amino acid substitution at position 840 corresponding to SEQ ID NO: 69. A composition is provided herein in which an engineered Cas protein or a functional variant thereof further comprises at least one amino acid substitution at position 221, position 394, or a combination thereof corresponding to SEQ ID NO: 69. A composition is provided herein in which an engineered Cas protein or a functional variant thereof comprises the amino acid substitutions R221K, N394K, H840A, or any combination thereof. A composition is provided herein in which an engineered Cas protein or a functional variant thereof comprises a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70 or SEQ ID NO: 71. A composition is provided herein in which an engineered Cas protein or a functional variant thereof comprises the sequence of SEQ ID NO: 70 or SEQ ID NO: 71. DNA ligases or functional fragments include E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase,Compositions comprising human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants, or combinations thereof are provided herein. Compositions comprising a DNA ligase or functional fragment thereof comprising Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV are provided herein. Compositions comprising a DNA ligase or functional fragment thereof comprising sequences 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to sequence numbers 58-66 are provided herein. Compositions comprising a DNA ligase or functional fragment thereof comprising sequences of sequence numbers 58-66 are provided herein. Compositions comprising a protein construct further comprising a linker, a nuclear localization sequence (NLS), or combinations thereof are provided herein. Compositions comprising a DNA ligase or functional fragment thereof linked to a nickasase or variant thereof are provided herein. Compositions are provided herein in which the protein constructs contain sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to sequence numbers 89-101.

[0177] Provided herein are engineered fusion proteins comprising (a) a DNA ligase or a functional fragment thereof and (b) an engineered nickase comprising three amino acid substitutions at positions 221, 394, and 840 of a nuclease comprising the sequence of SEQ ID NO: 69. Provided herein are engineered fusion proteins in which the substitutions result in enhanced nickase activity compared to otherwise equivalent engineered nickase. Provided herein are engineered fusion proteins in which the engineered nickase comprises the amino acid substitutions R221K, N394K, and H840A. Provided herein are engineered fusion proteins in which the engineered nickase or a functional variant thereof comprises a sequence 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71. Provided herein are engineered fusion proteins in which the engineered nickase or a functional variant thereof comprises the sequence of SEQ ID NO: 71. Engineered fusion proteins are provided herein in which the DNA ligase or functional fragment comprises E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants, or combinations thereof. Engineered fusion proteins are provided herein in which the DNA ligase or its functional fragment comprises Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV. Engineered fusion proteins are provided herein in which the DNA ligase or its functional fragment comprises a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 65. Engineered fusion proteins are provided herein in which a DNA ligase or a functional fragment thereof comprises the sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 65. Engineered fusion proteins are also provided herein in which the engineered fusion protein further comprises a linker, a nuclear localization sequence (NLS), or an additional protein construct.The following are provided herein: an engineered fusion protein having an amino acid sequence that is 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. The following are provided herein: an engineered fusion protein having an amino acid sequence that is The following engineered fusion proteins are provided herein, wherein the additional protein constructs include cell-targeting moieties, receptor-targeting moieties, regulatory elements, nucleases, acetylases, acetyltransferases, ATPases, Argonaut proteins, base editors, Cas polypeptides, catalytically inactive Cas polypeptides, deacetylases, deaminases, decapping proteins, endonucleases, exonucleases, helicases, ligases, meganucleases, methylases, methyltransferases, nickases, polymerases, proteases, recombinases, restriction enzymes, ribonucleoproteins (RNPs), self-cleaving protein sequences, splicing factors, transcription activators, transcription activator-like effector nucleases (TALENs), transcriptional repressors, transposases, zinc fingers, or any combination thereof.

[0178] Nucleic acids encoding the engineered fusion protein provided herein are provided herein. Nucleic acids encoding the engineered fusion protein, comprising RNA, are provided herein. Nucleic acids encoding the engineered fusion protein, comprising DNA, are provided herein.

[0179] Vectors comprising nucleic acids provided herein are provided herein. Vectors comprising viral vectors are provided herein. Vectors comprising lentiviral vectors, retroviral vectors, adeno-associated virus vectors (AAVs), adenovirus vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, picornavirus vectors, or oncolytic virus vectors are provided herein.

[0180] A system for modifying a target nucleic acid is provided herein, comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; (d) a guide polynucleotide, comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; (iii) a ligation sprint 2 region, the ligation sprint 2 region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint 2 region having at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region, the ligation sprint 1 region having complementarity with the target nucleic acid, and the guide polynucleotide, wherein when introduced into a cell or cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint region is A system is provided herein that comprises a guide polynucleotide comprising a ligation sprint 2 region having at least one change compared to a target nucleic acid, and (iv) a ligation sprint 1 region, the ligation sprint 1 region comprising one or more ribonucleotides or one or more deoxyribonucleotides, and the ligation sprint 1 region having complementarity with the target nucleic acid, wherein when introduced into a cell or cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and ligation A system is provided herein that comprises a guide polynucleotide, including a ligation sprint region having at least one change compared to the target nucleic acid, a ligation sprint 2 region, and (iv) a ligation sprint 1 region, the ligation sprint 1 region comprising one or more deoxyribonucleotides, and the ligation sprint 1 region having complementarity with the target nucleic acid, wherein when introduced into cells or a cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.A system for modifying a target nucleic acid, the system comprising: (a) a donor nucleic acid; (b) an engineered protein construct including a nickase region; (c) a DNA ligase or a functional fragment thereof; and (d) a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region complementary to the target nucleic acid; (ii) a protein-binding region including a secondary structure that binds to the engineered protein construct including the nickase region; and (iii) a ligation sprint 2 region, the ligation sprint region having complementarity with the donor nucleic acid and complementarity with the target nucleic acid, A system is provided herein which, when introduced into cells or a cell-free system, incorporates a donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid. The system comprises a ligation sprint region having at least one change compared to the target nucleic acid, a ligation sprint 2 region, and (iv) a ligation sprint 1 region, wherein the ligation sprint 1 region comprises a guide polynucleotide comprising a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region having complementarity with the target nucleic acid.

[0181] A system is further provided herein in which a ligation sprint 1 region forms a DNA-RNA (DR) loop upon association with a target nucleic acid and DNA ligase. A system is further provided herein in which a ligation sprint 1 region comprises a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 1:1 to a maximum of 20:1. A system is further provided herein in which a ligation sprint 1 region comprises a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 1:0 to a maximum of 20:0. Ligation sprint 1 regions: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:1 3, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9 ,7:10,7:11,7:12,7:13,7:15,8:1,8:3,8:5,8:7,8:9,8:11,8:13,8:15,9:1,9:2,9:4,9:5,9:7,9:8,9:10,9:11,9:13,9:15,9:17,9:19,9:20,10:1,10:3,10:7,10:9,10:11,10:13,10:15,10:17,10:19,11:1,11:2,11:3,11:4,1 1:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12:1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 14:13, 15:1, 15:2,15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18 Systems comprising ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratios of 1:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9, 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:1 are further provided herein. Systems in which a ligation sprint 1 region hybridizes to the leading strand when the target nucleic acid is cleaved by the engineered protein construct are further provided herein. Systems in which ribonucleotides and / or deoxyribonucleotides of a ligation sprint 1 region hybridize to the leading strand are further provided herein. A system in which the ligation sprint 2 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides is further provided herein. A system in which the ligation sprint 2 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides is further provided herein. A system in which the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 20 nucleotides is further provided herein. A system in which the ligation sprint 2 region comprises the reverse complementary sequence of a non-coding polynucleotide sequence or a variant thereof is further provided herein. A system in which the ligation sprint 2 region comprises the reverse complementary sequence of a sequence encoding the coding region of a polynucleotide sequence or a variant thereof is further provided herein. A system in which the ligation sprint 2 region comprises the reverse complementary sequence of a sequence encoding an exon or intron is further provided herein. A system in which the ligation sprint 2 region comprises the complementary sequence of a sequence encoding a non-coding polynucleotide sequence or a variant thereof,A system is further provided herein. A system is further provided herein in which the ligation sprint 2 region comprises a complementary sequence of a sequence encoding a coding region of a polynucleotide sequence or a variant thereof. A system is further provided herein in which the ligation sprint 2 region comprises a complementary sequence of a sequence encoding an exon or intron. A system is further provided herein in which the ligation sprint 2 region comprises a sequence comprising at least one nucleic acid base that is complementary or mismatched with the sequence encoding a splice acceptor site. A system is further provided herein in which a target-directed region hybridizes to the complementary strand of a target nucleic acid and a nickase region cleaves the target nucleic acid. A system is further provided herein in which a target-directed region hybridizes to the complementary strand of a target nucleic acid within at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, or at least about 20 nucleotides from the PAM sequence. Systems comprising manipulated protein constructs containing Cas proteins or mutant Cas proteins are further provided herein. Systems comprising Cas proteins or mutant Cas proteins being type V Cas proteins are further provided herein. Systems comprising type V Cas proteins including Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12g, Cas12h, Cas12i, Cas12j, and Cas12k are further provided herein. Systems comprising Cas proteins or mutant Cas proteins including Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, and Csn2 , Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Cs x14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, c2c1, c2c3, Cas9HiFi,Systems comprising xCas9, CasX, CasY, CasRX, SpCas9-VQR, SpCas9-VRQR, SpCas9-VRER, SaCas9-KKH, SpCas9-NG, SpCas9-NRRH, SpCas9-NRTH, SpCas9-NRCH, iSpyMac, St1Cas9 LMD9-LMG18311, St1Cas9 LMD9-CNRZ1066, St1Cas9-KQKL, their variants, or any combination thereof are further provided herein. Systems in which the Cas protein or variant Cas protein is a type II Cas protein are further provided herein. Systems in which the type II Cas protein comprises Cas9, Cas1, Cas2, or Csn2 are further provided herein. Systems in which the manipulated protein construct cleaves a target nucleic acid 5' upstream of the PAM sequence are further provided herein. A system is further provided herein in which an engineered protein construct cleaves a target nucleic acid to produce two single strands of DNA or two single strands of RNA double helix. A system is further provided herein in which an engineered protein construct generates a single-strand break in a target nucleic acid. A system is further provided herein in which a DNA ligase or a functional fragment thereof comprises E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants, or combinations thereof. DNA ligase or its functional fragment includes Archaeoglobus fulgidus DNA ligase, Pyrococcus furiosus DNA ligase, Methanothermobacter thermautotrophicus DNA ligase, Pyrococcus abyssi DNA ligase, Sulfolobus solfataricus DNA ligase, Haemophilus influenzae DNA ligase, or herpes simplex virus (HSV) DNA ligase.A system is further provided herein. A system is further provided wherein a ligation sprint 2 region hybridizes to a donor nucleic acid. A system is further provided herein in which the DNA ligase does not bind to the target nucleic acid. A system is further provided herein in which the DNA ligase attaches the donor nucleic acid to the leading strand. A system is further provided herein in which the new DNA strand has at least 50% to a maximum of 99.99% complementarity with the target nucleic acid sequence. A system is further provided herein in which the new DNA strand contains mismatched nucleic acid bases relative to the complementary strand of the target nucleic acid. A system is further provided herein in which the secondary structure of the protein-binding region of the guide polynucleotide includes a bulge, stem, loop, hairpin, fluctuating base pair, pseudoknot, or a combination thereof. A system is further provided herein in which the target nucleic acid includes DNA. A system is further provided herein in which the target nucleic acid includes RNA. A system is further provided herein in which the system further includes an additional protein construct, linker, or nuclear localization sequence (NLS). A system in which a protein construct and a DNA ligase are linked together by a polypeptide linker is further provided herein.

[0182] A composition comprising a system provided herein, an engineered protein construct provided herein, a DNA ligase or functional fragment thereof provided herein, or a guide polynucleotide provided herein is provided herein.

[0183] A composition comprising a system and a delivery vehicle provided herein is provided herein.

[0184] Polynucleotides that encode a system provided herein, an engineered protein provided herein, or a guide polynucleotide provided herein are provided herein.

[0185] A set of polynucleotides encoding a system provided herein, an engineered protein provided herein, or a guide polynucleotide provided herein is provided herein.

[0186] Nanoparticles comprising a polynucleotide provided herein, a set of polynucleotides provided herein, a system provided herein, a composition provided herein, a cell provided herein, a vector provided herein, or any part thereof, are provided herein.

[0187] A vector comprising a polynucleotide provided herein, or a set of polynucleotides provided herein, is provided herein.

[0188] Cells comprising a system provided herein, an engineered protein provided herein, a composition provided herein, a vector provided herein, or a guide polynucleotide provided herein are provided herein.

[0189] A composition is provided herein, comprising a donor nucleic acid and a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region having complementarity with the target nucleic acid, (b) a protein-binding region comprising a secondary structure that binds to a protein, (c) a ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and (d) a ligation sprint 1 region, the guide polynucleotide, or a polynucleotide encoding a guide polynucleotide. A composition is provided herein, comprising a donor nucleic acid, a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region having complementarity with a target nucleic acid, (b) a protein-binding region comprising a secondary structure that binds to a protein, (c) a ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and (d) a ligation sprint 1 region comprising one or more ribonucleotides or one or more deoxyribonucleotides, the guide polynucleotide, or a polynucleotide encoding a guide polynucleotide. A composition comprising a donor nucleic acid and a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region complementary to the target nucleic acid, (b) a protein-binding region including a secondary structure that binds to a protein, and (c) a ligation sprint 2 region, the ligation sprint 2 region beingA composition is provided herein that comprises (d) a ligation sprint 2 region having complementarity with a donor nucleic acid, complementarity with a target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and a guide polynucleotide or a polynucleotide encoding a guide polynucleotide, comprising a ligation sprint 1 region having one or more deoxyribonucleotides. A composition is provided herein, comprising a donor nucleic acid and a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises, in a 5' to 3' order, (a) a target-directing region having complementarity with the target nucleic acid, (b) a protein-binding region comprising a secondary structure that binds to a protein, (c) a ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and (d) a ligation sprint 1 region comprising a ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide. A composition is provided herein, comprising a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the target nucleic acid comprises a double-stranded DNA (dsDNA) or single-stranded RNA (ssRNA) double helix that is cleaved by nickase. Compositions in which the protein-binding region binds to a nickase or endonuclease are further provided herein. Compositions in which the secondary structure comprises a bulge, stem, loop, hairpin, fluctuating base pair, pseudoknot, or a combination thereof are further provided herein. Compositions in which the ligation sprint 2 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides are further provided herein. Compositions in which the ligation sprint 2 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides are further provided herein. The ligation sprint 2 region,Compositions comprising at least one change that is a mismatched nucleic acid base are further provided herein. Compositions comprising A / C mismatch, A / T mismatch, A / G mismatch, and T / C mismatch, T / G mismatch, T / A mismatch, C / G mismatch, C / A mismatch, C / T mismatch, G / C mismatch, G / T mismatch, G / A mismatch, or combinations thereof with respect to a target nucleic acid are further provided herein. Compositions comprising the reverse complementary sequence of a non-coding region of a polynucleotide or a variant thereof as a donor nucleic acid are further provided herein. Compositions comprising the reverse complementary sequence of a sequence encoding a coding region of a polynucleotide or a variant thereof as a donor nucleic acid are further provided herein. Compositions comprising the reverse complementary sequence of a sequence encoding an exon or intron as a donor nucleic acid are further provided herein. Compositions are further provided herein in which the donor nucleic acid comprises a sequence containing at least one nucleic acid base that is complementary to or mismatched with the sequence encoding a splice acceptor site. Compositions are further provided herein in which the donor nucleic acid comprises a complementary sequence of a sequence encoding an intron or a variant thereof. Compositions are further provided herein in which the donor nucleic acid comprises a complementary sequence of a sequence encoding an exon or a variant thereof. Compositions are further provided herein in which the donor nucleic acid comprises complementary sequences of sequences encoding exons and introns. Compositions are further provided herein in which a ligation sprint 1 region forms a DNA-RNA (DR) loop when it associates with a target nucleic acid and DNA ligase. Compositions are further provided herein in which a ligase sprint 1 region comprises an RNA-to-DNA ratio of 1:1 to a maximum of 20:1. Compositions are further provided herein in which a ligase sprint 1 region comprises an RNA-to-DNA ratio of 1:0 to a maximum of 20:0. The ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19,3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4:15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9:20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12:1, 12:5, 1 2:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 14:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17 Compositions comprising ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratios of 8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9, 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18 or 20:1 are further provided herein. Ligation sprint 1 region,Further provided herein are compositions in which, upon cleavage of the target nucleic acid by nickase, hybridizes to the target nucleic acid, and nickase generates a leading strand and a complementary strand. Further provided herein are compositions in which ribonucleotides hybridize to the leading strand. Further provided herein are compositions in which the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides. Further provided herein are compositions in which the ligation sprint 1 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides. Further provided herein are compositions in which the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 20 nucleotides. Further provided herein are compositions in which the ligation sprint 1 region comprises a reverse complementary sequence encoding an intron or a variant thereof. Further provided herein are compositions in which the ligation sprint 1 region comprises a reverse complementary sequence of a sequence encoding an exon or a variant thereof. Compositions in which the ligation sprint 1 region comprises a reverse complementary sequence of a sequence encoding an exon and an intron are further provided herein. Compositions in which the ligation sprint 1 region comprises a sequence encoding a splice acceptor site or a nucleotide complementary to a splice acceptor site are further provided herein. Compositions in which the ligation sprint 1 region comprises a complementary sequence of a sequence encoding an intron or a variant thereof are further provided herein. Compositions in which the ligation sprint 1 region comprises a complementary sequence of a sequence encoding an exon or a variant thereof are further provided herein. Compositions in which the ligation sprint 1 region comprises a complementary sequence of a sequence encoding an exon and an intron are further provided herein. Compositions in which the ligation sprint 1 region comprises a mismatched nucleic acid base relative to a splice acceptor site in the sequence of a target nucleic acid are further provided herein. Compositions in which a ribonucleotide is located at the 3' end of the ligation sprint 1 region are further provided herein. Ligation sprint 1 region consists of 1 ribonucleotide, 2 ribonucleotides, 3 ribonucleotides,Compositions comprising four ribonucleotides, five ribonucleotides, or up to ten ribonucleotides are further provided herein. Compositions comprising a ligation sprint 1 region having a length of at least about five nucleotides to a maximum of ten nucleotides are further provided herein. Compositions comprising an engineered protein, or a polypeptide encoding an engineered protein, Further compositions are provided herein. Compositions comprising an engineered protein comprising (a) a nickase region and (b) a DNA ligase region are further provided herein. Compositions comprising a DNA ligase region binding to a ligation sprint 1 or ligation sprint 2 region of a guide polynucleotide are further provided herein. Compositions comprising a DNA ligase region ligating a donor nucleic acid to the 3' end of a leading strand are further provided herein. Compositions comprising a nickase region comprising a mutant Cas protein that generates a single-strand break in a target nucleic acid are further provided herein. The Cas proteins are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4 Compositions comprising Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, c2c1, c2c3, Cas9HiFi, xCas9, CasX, CasY, CasRX, variants, or any combination thereof are further provided herein. Compositions further comprising a delivery vehicle are further provided herein. Compositions comprising a delivery vehicle including vectors, lipids, nanoparticles, plasmids, viruses, liposomes, extracellular vesicles, emulsions, peptides, carbohydrates, polymers, chitosan, polyethyleneimine (PEI), poly(lactide-co-glycolide) (PLGA), poly-L-lysine (PLL), or combinations thereof are further provided herein.

[0190] A method for ligating a donor nucleic acid with a target nucleic acid, the method comprising: (a) a donor nucleic acid; (b) a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, the guide polynucleotide comprising: (i) a target-directing region having complementarity with the target nucleic acid; (ii) a protein-binding region comprising a secondary structure that binds to a nuclease or nickase; and (iii) a ligation sprint 2 region, the ligation sprint 2 region having complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and having at least one change compared to the target nucleic acid; and (iv) a ligation sprint 1 region comprising one or more deoxyribonucleotides or one or more ribonucleotides, the guide A method is provided herein that comprises (c) a polynucleotide or a polynucleotide encoding a guide polynucleotide, and (c) an engineered protein or a polynucleotide encoding an engineered protein, wherein the engineered protein comprises (i) a nickase region and (ii) a DNA ligase region, wherein the guide polynucleotide complexes with the engineered protein via a protein-binding region, a target-directed sequence complexes with a complementary strand, the nickase region of the engineered protein causes cleavage at the target nucleic acid to generate a leading strand and a complementary strand, a ligation sprint 1 region complexes with the leading strand, and the DNA ligase region attaches a donor nucleic acid to the leading strand, thereby ligating the donor nucleic acid to the target nucleic acid. A method is further provided herein that the target-directed strand dissociates from the complementary strand. A method is further provided herein that incorporates a new nucleic acid into the target nucleic acid by hybridizing with the complementary strand.

[0191] Methods are provided herein that include administering a system, composition, or cell provided herein to a subject, organ, tissue, or cell, wherein the administration modifies a gene in the subject, organ, tissue, or cell. Methods are further provided herein that include insertions, deletions, substitutions, copy number changes, point mutations, frameshift mutations, missense mutations, nonsense mutations, mutations in stop codons, epigenetic marks, or any combination thereof. Methods are further provided herein that are local or systemic in their administration. Methods are further provided herein that are administered intranasally, subcutaneously, intravenously, by inhalation, intramuscularly, intratumorally, peritumorally, intrathecally, vaginally, or intradermally. Methods are further provided herein that the subject is a mammal. Methods are further provided herein that the subject has, is suspected of having, or is diagnosed with a disease or condition. Methods are further provided herein for which the disease or condition includes a hereditary disease or hereditary condition. Methods are further provided herein for which the method further includes administering a therapeutic agent to a subject.

[0192] Methods are provided herein that include contacting cells or populations of cells with a system or composition provided herein to modify genes in the cells. Methods are further provided herein in which the contact is performed in vitro, in vivo, or ex vivo. Methods are further provided herein in which the gene alteration includes insertions, deletions, substitutions, copy number changes, point mutations, frameshift mutations, missense mutations, nonsense mutations, stop codon mutations, epigenetic marks, or any combination thereof. Methods are further provided herein in which the gene alteration restores the expression of a wild-type protein encoded by the gene compared to an equivalent cell or population of cells that has not been in contact with either the system or the composition. Methods are further provided herein in which the cells include eukaryotic cells or prokaryotic cells. Methods are further provided herein in which the cells include mammalian cells. Methods are further provided herein in which the population of cells includes a population of human leukocytes, a population of stem cells, or a population of bacteria.

[0193] A population of cells, which is produced by a method provided herein, is provided herein.

[0194] A kit is provided herein, comprising a system provided herein, a composition provided herein, a vector provided herein, a polynucleotide provided herein, or a cell provided herein, packaging, and materials thereof.

[0195] A kit is provided herein, comprising: a first container comprising a donor nucleic acid and a guide polynucleotide, or a polynucleotide encoding a guide polynucleotide, wherein the guide polynucleotide comprises (i) a target-directing region complementary to the target nucleic acid, (ii) a protein-binding region comprising a secondary structure that binds to a nuclease or nickase, (iii) a ligation sprint 2 region, wherein the region that hybridizes to the donor is complementary to the donor nucleic acid and complementary to the target nucleic acid, and the ligation sprint 2 region comprises at least one mismatch nucleic acid base for the target nucleic acid, and (iv) a ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide, and a second container comprising an engineered polypeptide comprising a nickase operably linked to a DNA ligase. Kits further comprising reagents for the amplification, transcription, translation, or isolation of nucleic acids are provided herein. Kits further comprising reporter molecules are also provided herein.

[0196] A scaffold is provided herein, comprising a system or composition provided herein, and a solid surface on which the system or composition is fixed.

[0197] Examples Example 1. Editing of double-stranded targets using a DNA ligase system. A DNA editing system comprising Cas9 nickase (H840A), DNA ligase, guide polynucleotide, and donor nucleic acid was constructed, and this system was evaluated with various double-stranded DNA targets (double-stranded DNA substrates). In particular, double-stranded DNA substrates, guide polynucleotides, and RNPs consisting of Cas9 H840A nickase, DNA ligase, and guide polynucleotide were constructed and evaluated as described below.

[0198] Double-stranded DNA substrate preparation: 5'-FAM-labeled double-stranded DNA (dsDNA) was obtained by annealing two oligos, oligo1 and oligo2, in a 1:1 ratio. In summary, these oligos were resuspended in nuclease-free water to a concentration of 100 μM. Subsequently, 10 μL of 5'-FAM-labeled oligo (oligo1) and 10 μL of oligo2 were mixed in PCR tubes with 10 μL of NEBuffer r3.1 (10×) and 70 μL of nuclease-free water, incubated at 95°C for 5 minutes, and slowly cooled at room temperature. Annealed FAM-labeled dsDNA was prepared at a concentration of 10 μM, or 10 picomoles / μL. The oligo sequences are listed in Table 3.

[0199] In vitro guide polynucleotide preparation: In summary, double-stranded DNA templates containing the T7 promoter for in vitro transcription were prepared by PCR using oligonucleotides (Oligo 3, Oligo 4, Oligo 5, and Oligo 6) (Q5® Hot Start High-Fidelity 2X Master Mix, NEB). DNA derived from the PCR reaction was purified using AMPure XP Beads (Beckman Coulter) and its concentration was measured using Nanodrop. 75 ng of the DNA template was used for the in vitro transcription reaction using HiScribe® T7 Quick High Yield RNA Synthesis Kit (NEB). Guide nucleic acid products were purified using Monarch® RNA Cleanup Kit (NEB) and their concentration was measured using Nanodrop (Thermo Fisher Scientific). The oligonucleotide sequences are listed in Table 3. In Table 3, A, G, C, and T are deoxyribonucleotides (DNA), and rA, rG, rC, and rU are ribonucleotides (RNA). [Table 3-1] [Table 3-2]

[0200] Preparation of synthetic guide polynucleotides: All synthetic guides listed in Table 4 were designed and chemically synthesized. The guides were purified by HPLC, and the mass of each guide polynucleotide was verified by mass spectrometry (LC-MS). In Table 4, A, C, G, and U are ribonucleotides (RNA), and dA, dC, dG, and dT are deoxyribonucleotides (DNA). [Table 4-1] [Table 4-2]

[0201] In vitro DNA nicking, DNA ligation by DNA ligase, and analysis: First, CRISPR ribonucleoprotein (RNP) was prepared by combining 61 picomoles of SpCas9 H840A nickingase with 80 picomoles of guide RNA and incubated at room temperature for 10 minutes to enable RNP complexation. Cas9 nicking and DNA ligase activity were tested in a total volume of 7 μL containing 1 μL of FAM dsDNA (10 picomoles), 100 picomoles of trans DNA editing oligo (with the DNA template incorporated into the guide polynucleotide sequence for cis-DNA writing), 0.7 μL of 10× T4 DNA ligase reaction buffer (NEB), 0.5 μL of DNA ligase, 2 μL of Cas9 nickingase-guide RNP, and the remaining volume of nuclease-free water to make a final volume of 7 μL. Ligation donor oligos of various lengths were provided in trans form at 100 picomoles per reaction. The reaction mixture was incubated at 37°C for 1 hour, and the sample was treated with 0.5 μL of proteinase K solution (20 mg / mL, Qiagen) and incubated at 56°C for 30 minutes. The sample was thermally inactivated at 95°C for 10 minutes, and the reaction product was combined with gel loading buffer II (2×, ThermoFisher Scientific), denatured at 95°C for 5 minutes, and separated on a denatured polyacrylamide gel (15% TBE-urea, 60°C, 150V) for 1 hour. The DNA product was visualized by FAM fluorescence signaling using the Life Technologies Gel Imaging system.

[0202] The editing products of targeted DNA editing using DNA ligase were analyzed on a denatured urea-polyacrylamide gel, as shown in Figure 2. Lanes 6-11 show that the system successfully inserted nucleotides of different lengths, including 15 nt (lane 6, lower band), 25 nt (lane 7, upper band), 30 nt (lane 8, upper band), 50 nt (lane 9, upper band), 70 nt (lane 10, upper band), and 90 nt (lane 11, upper band).

[0203] To determine the effect of the LS1 composition on the editing efficiency of the DNA editing system, the activity of DNA ligation editing by DNA ligases with DNA ligation sprints (LS) incorporated into various synthetic guide polynucleotides (Guide 1 to Guide 9 (SEQ ID NOs. 45 to 53)) was analyzed using a denatured urea polyacrylamide gel as shown in Figure 3. In particular, all of Guide 1 to Guide 9 have a ligation sprint 2 (LS2) with a 5'DDDDDDDDDDDDDDD3' structure (where D represents DNA), while LS1 consists entirely of DNA bases (Guide 1, Guide 2, and Guide 3 (SEQ ID NOs. 45 to 47)) or consists of both RNA and DNA (Guide 4, Guide 5, Guide 6, Guide 7, Guide 8, and Guide 9 (SEQ ID NOs. 48 to 53)). The LS1 in Guide 1, Guide 2, and Guide 3 have the structures 5'DDDDD3', 5'DDDDDD3', and 5'DDDDDDD3' in this order (D represents DNA and R represents RNA). The LS1 in Guide 4, Guide 5, and Guide 6, Guide 7, Guide 8, and Guide 9 have the structures 5'DDDDDRR3', 5'DDDDRRR3', 5'DDDRRRR3', 5'DDRRRRR3', 5'DRRRRRR3', and 5'RRRRRRR3' in this order. Lanes 3-11 contain ligation donor oligonucleotides (oligo 40) that add 15nt to nicked DNA to produce a 49nt edited product.

[0204] Figure 3 shows that the majority of nicked products were converted into ligation edit products. However, increasing the length of LS1 further maximizes efficiency. T4 DNA ligase activity for editing was significantly reduced when the number of DNA in LS1 was less than 3, suggesting that a minimum of 3 DNA nucleotides are required in LS1 for effective DNA editing by T4 DNA ligase.

[0205] Example 2. Plasmid cloning A backbone plasmid comprising a T7 RNA polymerase promoter, a 5' UTR sequence, and a 3' UTR sequence was constructed for cloning various editor constructs. Gene fragments containing various Cas enzymes and DNA ligases were assembled into the backbone plasmid to function as a template for in vitro transcription, utilizing ligation-based cloning or Gibson assembly. All plasmids were subjected to midi prep or maxi prep using the Qiagen Midi / Maxi Plus kit. The amino acid sequences of the produced engineered protein constructs are provided in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12]

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

[0206] Example 3. Culture and electroporation of HEK293T Frozen vials of the HEK293T (ATCC CRL-3216) cell line were thawed at 37°C and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% (v / v) fetal bovine serum (FBS) at 37°C with 5% CO2. Cells were grown to 90% confluence before subculturing. Cells were subculturised three times before each experiment. Before electroporation, 48-well plates were coated with poly-D-lysine for 1 hour, followed by three washes with PBS. After PBS washing, 400 μL of antibiotic-free complete medium was added to each well and incubated at 37°C with 5% CO2 before use. All electroporation was performed using the Neon NxT electroporation system. For electroporation, 1 μg of purified mRNA, 100 pmol of each legRNA and sprint, 100 pmol of donor DNA, and 100,000 cells were mixed in R buffer and electroporated using a 10 μl Neon tip. 1150 V, 20 ms, and 2-pulse electroporation settings were used for all electroporations. After electroporation, cells were added to a single well of a 48-well plate containing cell culture medium and incubated at 37°C with 5% CO2. Genomic DNA was extracted 48 hours after electroporation for NGS analysis.

[0207] Example 4. In vitro transcribed (IVT) mRNA Plasmids containing the target gene were completely digested and linearized using BsmBI (New England Biolabs) before use in in vitro transcription (IVT). The IVT reaction was performed using the NEB HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). In summary, the IVT reaction was performed at 37°C with the addition of CleanCap Reagent AG (Trilink Biotechnologies) accompanied by 100% substitution of UTP with N1-methylpseudoUTP (Trilink Biotechnologies). The IVT reaction was completed after 2 hours. After IVT, each reaction was incubated with DNase I (New England Biolabs) for 15 minutes. Subsequently, the RNA was purified using the Monarch RNA Cleanup Kit (New England Biolabs).

[0208] Example 5. Preparation and analysis of next-generation sequencing (NGS) libraries Specific genomic regions of interest were amplified using PCR primers containing Illumina-compatible adapter sequences (see Table 7). Following a standard PCR protocol using Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs), the resulting PCR products were purified using 0.7X Ampure XP Beads (Beckman Coulter). The purified PCR products were sent to Quintara Biosciences' AmpExpress service. Amplicon sequencing data were analyzed using CRISPResso. Edit efficiency was calculated from the allele frequency table file based on the number of sequencing reads containing the desired edit compared to the total number of sequencing reads in a given sample.

[0209] Example 6. Gene editing in HEK293 cells using DNA ligase and nicasse-modified proteins. To evaluate a gene editing system in human cells that includes Cas9 nickase, DNA ligase, guide polynucleotide, and donor nucleic acid, mRNA encoding the manipulated protein constructs in Table 5 was transcribed in vitro according to the method of Example 4, and synthetic guide RNA in Table 6 was prepared by chemical synthesis. The mRNA, synthetic guide RNA, and oligonucleotides were delivered to HEK293T cells as described in Example 3.

[0210] The engineered protein constructs (constructs 1-13, SEQ ID NOs: 89-101) were screened for their precise genome editing activity at the FANCF site 1, a genomic target site in HEK293T cells. The results suggest that DNA ligase genome editors facilitate precise genome editing in human cells. Of the 13 engineered protein constructs, the construct containing T4 DNA ligase, Chlorella DNA ligase, and human DNA ligase IV editor enabled precise introduction of 3bp substitution editing (+2 C>T; +4~5 TG>AC) at the genomic target FANCF site 1 in HEK293T cells (Figure 4).

[0211] To verify that sprint modifications and DNA donor modifications can enhance DNA ligase editing activity in human cells, various sprint modifications and DNA donor modifications (Table 7, SEQ ID NOs. 184-228) were tested, and ligase editing using a Chlorella DNA ligase editor construct (construct 10, SEQ ID NOs. 98) at the genomic target FANCF site 1 (gRVB_3) was tested to introduce 3bp substitution editing (+2 C>T; +4-5 TG>AC). The editing efficiency of gene editing systems using various combinations of sprints and DNA donors is shown in Figure 5. The results suggest that including modifications such as phosphorothioate binding (PS) and locating nucleic acid (LNA) (as exemplified by the combination of oRVB_61 and oRVB_97) also improved ligase editing activity by up to 21.5 times compared to combinations without such modifications (oRVB_62 / oRVB_95).

[0212] To verify the effect of RNA integration into guide RNA sprints in DNA ligase editing, a gene editing system containing a Chlorella DNA ligase editor construct (construct 10) and various ligase editing guide RNAs was tested at HEK site 3 and AAV site 1 in HEK293T cells. In vitro transcribed mRNA encoding the ligase editor, synthetic guide RNA, DNA sprint, and donor were delivered to HEK293T cells by electroporation as described in Example 4.

[0213] mRNA was delivered to HEK293T cells as described in Example 3. As shown in Figure 6, ligase editing guide RNAs (gRVB_5 / gRVB_6) with RNA inclusion within the sprint (oRVB_73 / oRVB_74) enhanced precise genome editing at HEK site 3 for attB site insertion by up to 36.9 times compared to guide RNA sprints without RNA inclusion (oRVB_81 / oRVB_82). Similarly, as shown in Figure 7, ligase editing guide RNAs (gRVB_1 / gRVB_2) with RNA inclusion within the sprint (oRVB_71 / oRVB_72) enhanced precise genome editing at AAV site 1 for attB site insertion by up to 10.5 times compared to guide RNA sprints without RNA inclusion (oRVB_79 / oRVB_80).

[0214] To investigate the effect of the amount of RNA incorporated into the sprint region of ligase editor guide RNA (legRNA), a gene editing system including a Chlorella DNA ligase editor construct (construction 10) and ligase editor guide RNA (legRNA) having sprint regions with varying degrees of RNA incorporation was tested on the ligase-editing-active genome target AAV site 1 in human cells. The desired editing efficiency refers to the efficiency of inserting the attB site into AAV site 1. In vitro transcribed mRNA encoding the ligase editor, synthetic guide RNA, and DNA donor were delivered to HEK293T cells by electroporation as described in Example 4. As shown in Figure 8, varying degrees of RNA incorporation into the sprint region of ligase editor guide RNA (legRNA) with a single guide RNA structure promoted ligase-editing activity at the genome target AAV site 1 in human cells. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0215] In Tables 6 and 7, A, C, G, and U are ribonucleotides (RNA), dA, dC, dG, and dT are deoxyribonucleotides (DNA), +A, +T, +C, and +G are loc nucleic acids (LNA), rA, rU, rC, and rG are ribonucleotides (RNA), * indicates a phosphorothioate (PS) bond, mA, mU, mC, and mG correspond to 2'-O-methyl RNA, / iMe-Dc / corresponds to 5'-methyl dC, / 3Me-dC / corresponds to 3'5-methyl dC, and / 5Phos / corresponds to 5' phosphorylation.

[0216] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided only as examples. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A composition, (a) Donor nucleic acid and (b) A polynucleotide encoding a protein construct comprising a nickasase or a variant thereof and a DNA ligase or a functional fragment thereof, (c) Guide polynucleotide, (i) A target-directed region having complementarity with the target nucleic acid, (ii) A protein-binding region comprising a secondary structure that binds to the manipulated protein construct comprising a nickase region, (iii) Ligation sprint 2 region, wherein the ligation sprint region is complementary to the donor nucleic acid and complementary to the target nucleic acid, and the ligation sprint region includes at least one change compared to the target nucleic acid, and (iv) Ligation sprint 1 region, wherein the ligation sprint 1 region comprises a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region is complementary to the target nucleic acid. Includes guide polynucleotides and A composition containing the following:

2. The composition according to claim 1, wherein the ligation sprint 1 region forms a DNA-RNA (DR) loop when it associates with the target nucleic acid and DNA ligase.

3. The composition according to claim 1, wherein the ligase sprint 1 region includes an RNA-to-DNA ratio of 1:1 to a maximum of 20:

1.

4. The ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4 :15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6 , 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9: 20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12: 1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 1 4:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9,The composition according to claim 3, comprising a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:

1.

5. The composition according to claim 1, wherein the ligation sprint 2 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides.

6. The composition according to claim 1, wherein the ligation sprint 2 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides.

7. The composition according to claim 1, wherein the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 20 nucleotides.

8. The composition according to claim 1, wherein the ligation sprint 2 region comprises a reverse complementary sequence of a non-coding polynucleotide sequence or a variant thereof.

9. The composition according to claim 1, wherein the ligation sprint 2 region includes an inverse complementary sequence of a sequence that encodes a coding region of a polynucleotide sequence or a variant thereof.

10. The composition according to claim 1, wherein the ligation sprint 2 region includes an inverse complementary sequence of a sequence encoding an exon or intron.

11. The composition according to claim 1, wherein the ligation sprint 2 region comprises a complementary sequence of a sequence encoding a non-coding polynucleotide sequence or a variant thereof.

12. The composition according to claim 1, wherein the ligation sprint 2 region includes a complementary sequence of a sequence encoding a coding region of a polynucleotide sequence or a variant thereof.

13. The composition according to claim 1, wherein the ligation sprint 2 region includes a complementary sequence of a sequence encoding an exon or intron.

14. The composition according to claim 1, wherein the ligation splint 2 region comprises a sequence containing at least one nucleic acid base that is complementary to or mismatched with the sequence encoding the splice acceptor site.

15. The composition according to claim 1, wherein the nickase is an engineered Cas protein or a functional variant thereof.

16. The composition according to claim 15, wherein the manipulated Cas protein or its functional variant comprises at least one amino acid substitution at position 840, corresponding to SEQ ID NO:

69.

17. The composition according to claim 16, wherein the manipulated Cas protein or its functional variant further comprises at least one amino acid substitution at position 221, position 394, or a combination thereof, corresponding to SEQ ID NO:

69.

18. The composition according to claim 16, wherein the manipulated Cas protein or its functional variant comprises amino acid substitutions of R221K, N394K, H840A, or any combination thereof.

19. The composition according to claim 15, wherein the manipulated Cas protein or its functional variant comprises a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70 or SEQ ID NO:

71.

20. The composition according to claim 15, wherein the manipulated Cas protein or a functional variant thereof comprises the sequence of SEQ ID NO: 70 or SEQ ID NO:

71.

21. The composition according to claim 1, wherein the DNA ligase or functional fragment comprises E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants, or combinations thereof.

22. The composition according to claim 19, wherein the DNA ligase or its functional fragment comprises Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV.

23. The composition according to claim 19, wherein the DNA ligase or its functional fragment contains a sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to sequence numbers 58-66.

24. The composition according to claim 19, wherein the DNA ligase or its functional fragment comprises the sequences of sequence numbers 58 to 66.

25. The composition according to claim 1, wherein the protein construct further comprises a linker, a nuclear localization sequence (NLS), or a combination thereof.

26. The composition according to claim 25, wherein the DNA ligase or a functional fragment thereof is linked to the nickase or a variant thereof.

27. The composition according to claim 25, wherein the protein construct contains sequences that are 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identical to sequence numbers 89-101.

28. A manipulated fusion protein, (a) DNA ligase or a functional fragment thereof, (b) Modified nicasse containing three amino acid substitutions at positions corresponding to amino acid positions 221, 394, and 840 of the nuclease containing the sequence of Sequence ID No. 69 and A manipulated fusion protein containing the above.

29. The engineered fusion protein according to claim 28, wherein the substitution results in enhanced nickase activity compared to an otherwise equivalent engineered nickase.

30. The manipulated fusion protein according to claim 28, wherein the manipulated nickase comprises amino acid substitutions R221K, N394K, and H840A.

31. The manipulated fusion protein according to claim 28, wherein the manipulated nickase or its functional variant comprises a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO:

71.

32. The manipulated fusion protein according to claim 28, wherein the manipulated nickase or a functional variant thereof comprises the sequence of SEQ ID NO:

71.

33. The manipulated fusion protein according to claim 32, wherein the DNA ligase or functional fragment comprises E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigas V, variants, or combinations thereof.

34. The manipulated fusion protein according to claim 32, wherein the DNA ligase or its functional fragment comprises Chlorella virus DNA ligase, T4 DNA ligase, or human DNA ligase IV.

35. The manipulated fusion protein according to claim 34, wherein the DNA ligase or its functional fragment comprises a sequence that is 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO:

65.

36. The manipulated fusion protein according to claim 34, wherein the DNA ligase or its functional fragment comprises the sequence of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO:

65.

37. The manipulated fusion protein according to any one of claims 28 to 36, further comprising a linker, a nuclear localization sequence (NLS), or an additional protein construct.

38. The manipulated fusion protein according to claim 37, comprising an amino acid sequence that is 97%, 98%, 99%, or 100% identical to any one of sequence numbers 89 to 101.

39. The manipulated fusion protein according to claim 37, comprising one amino acid sequence from sequence numbers 89 to 101.

40. The manipulated fusion protein according to claim 37, wherein the additional protein construct comprises a cell targeting moiety, a receptor targeting moiety, a regulatory element, a nuclease, an acetylase, an acetyltransferase, an ATPase, an argonaut protein, a base editor, a Cas polypeptide, a catalytically inactive Cas polypeptide, a deacetylase, a deaminase, a decapsulating protein, an endonuclease, an exonuclease, a helicase, a ligase, a meganuclease, a methylase, a methyltransferase, a nickase, a polymerase, a protease, a recombinase, a restriction enzyme, a ribonucleoprotein (RNP), a self-cleaving protein sequence, a splicing factor, a transcription activator, a transcription activator-like effector nuclease (TALEN), a transcription repressor, a transposase, a zinc finger, or any combination thereof.

41. A nucleic acid encoding an engineered fusion protein according to any one of claims 28 to 40.

42. The nucleic acid according to claim 41, wherein the nucleic acid encoding the manipulated fusion protein comprises RNA.

43. The nucleic acid according to claim 41, wherein the nucleic acid encoding the manipulated fusion protein comprises DNA.

44. A vector comprising the nucleic acid described in any one of claims 41 to 43.

45. The vector according to claim 44, comprising a viral vector.

46. The vector according to claim 45, wherein the viral vector includes a lentivirus vector, a retrovirus vector, an adeno-associated virus vector (AAV), an adenovirus vector, a herpes simplex virus vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, a picornavirus vector, or an oncolytic virus vector.

47. A system for modifying target nucleic acids, wherein the system is (a) Donor nucleic acid and (b) A polynucleotide encoding an engineered protein construct comprising a nickase region or a variant thereof and a DNA ligase or a functional fragment thereof, (c) Guide polynucleotide, (i) A target-directed region having complementarity with the target nucleic acid, (ii) A protein-binding region comprising a secondary structure that binds to the manipulated protein construct comprising a nickase region, (iii) Ligation sprint 2 region, wherein the ligation sprint region is complementary to the donor nucleic acid and complementary to the target nucleic acid, and the ligation sprint region includes at least one change compared to the target nucleic acid, and (iv) Ligation sprint 1 region, wherein the ligation sprint 1 region comprises a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region is complementary to the target nucleic acid. Includes guide polynucleotides and Includes, A system, upon introduction into a cell, nucleus, or cell-free system, wherein the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

48. The system according to claim 47, wherein the ligation sprint 1 region associates with the target nucleic acid and the DNA ligase to form a DNA-RNA (DR) loop.

49. The system according to claim 47, wherein the ligation sprint 1 region includes a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 1:1 to a maximum of 20:

1.

50. The ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4 :15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6 , 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9: 20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12: 1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 1 4:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9,The system according to claim 47, comprising a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:

1.

51. The system according to claim 47, wherein the ligation sprint 1 region hybridizes to the leading strand when the target nucleic acid is cleaved by the manipulated protein construct.

52. The system according to claim 47, wherein the ribonucleotides and / or deoxyribonucleotides of the ligation sprint 1 region hybridize to the leading strand.

53. The system according to claim 47, wherein the ligation sprint 2 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides.

54. The system according to claim 47, wherein the ligation sprint 2 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides.

55. The system according to claim 47, wherein the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 20 nucleotides.

56. The system according to claim 47, wherein the ligation sprint 2 region includes a reverse complementary sequence of a non-coding polynucleotide sequence or a variant thereof.

57. The system according to claim 47, wherein the ligation sprint 2 region includes an inverse complementary sequence of a sequence encoding a coding region of a polynucleotide sequence or a variant thereof.

58. The system according to claim 47, wherein the ligation sprint 2 region includes an inverse complementary sequence of a sequence encoding an exon or intron.

59. The system according to claim 47, wherein the ligation sprint 2 region includes a complementary sequence of a sequence encoding a non-coding polynucleotide sequence or a variant thereof.

60. The system according to claim 47, wherein the ligation sprint 2 region includes a complementary sequence of a sequence encoding a coding region of a polynucleotide sequence or a variant thereof.

61. The system according to claim 47, wherein the ligation sprint 2 region includes a complementary sequence of a sequence encoding an exon or intron.

62. The system according to claim 47, wherein the ligation splint 2 region includes a sequence comprising at least one nucleic acid base that is complementary to or mismatched with the sequence encoding the splice acceptor site.

63. The system according to claim 47, wherein the target-directing region hybridizes to the complementary strand of the target nucleic acid, and the nickase region cleaves the target nucleic acid.

64. The system according to claim 47, wherein the target-directing region hybridizes to the complementary strand of the target nucleic acid within at least about 5 nucleotides from the PAM sequence, at least about 10 nucleotides from the PAM sequence, at least about 15 nucleotides from the PAM sequence, or at least about 20 nucleotides from the PAM sequence.

65. The system according to claim 47, wherein the manipulated protein construct comprises a manipulated Cas protein.

66. The system according to claim 65, wherein the manipulated Cas protein is a V-type Cas protein.

67. The system according to claim 66, wherein the V-type Cas protein includes Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12g, Cas12h, Cas12i, Cas12j, and Cas12k.

68. The manipulated Cas protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Cs x17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, c2c1, c2c3, Cas9HiFi, xCas9, CasX, CasY, CasRX, Sp Cas9-VQR, SpCas9-VRQR, SpCas9-VRER, SaCas9-KKH, SpCas9-NG, SpCas9-NRRH, SpCas9-NRTH, SpCas9-NRCH, iSpyMac, St1Cas9 The system according to claim 66, comprising LMD9-LMG18311, St1Cas9 LMD9-CNRZ1066, St1Cas9-KQKL, their variants, or any combination thereof.

69. The system according to claim 66, wherein the manipulated Cas protein is a type II Cas protein.

70. The system according to claim 69, wherein the type II Cas protein comprises Cas9, Cas1, Cas2, or Csn2, or variants thereof.

71. The system according to any one of claims 47 to 70, wherein the manipulated protein construct cleaves the target nucleic acid 5' upstream of the PAM sequence.

72. The system according to any one of claims 47 to 71, wherein the manipulated protein construct cleaves the target nucleic acid to produce two single strands of DNA or two single strands of RNA double helix.

73. The system according to any one of claims 47 to 72, wherein the manipulated protein construct generates a single-strand break in the target nucleic acid.

74. The system according to any one of claims 47 to 73, wherein the DNA ligase or its functional fragment comprises E. coli DNA ligase, Taq DNA ligase, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, Chlorella virus DNA ligase, human DNA ligase I, human DNA ligase II, human DNA ligase III, human DNA ligase IV, hitrigase V, variants, or combinations thereof.

75. The system according to any one of claims 47 to 74, wherein the DNA ligase or functional fragment thereof comprises Archaeoglobus fulgidus DNA ligase, Archaeoglobus fulgidus DNA ligase, Pyrococcus furiosus DNA ligase, Methanothermobacter thermototropicus DNA ligase, Pyrococcus abyssi DNA ligase, Sulfolobus solfataricus DNA ligase, Haemophilus influenzae DNA ligase, or herpes simplex virus (HSV) DNA ligase.

76. The system according to any one of claims 47 to 75, wherein the ligation sprint 2 region hybridizes to the donor nucleic acid.

77. The system according to any one of claims 47 to 76, wherein the DNA ligase does not bind to the target nucleic acid.

78. The system according to any one of claims 47 to 77, wherein the DNA ligase attaches the donor nucleic acid to the leading strand.

79. The system according to claim 78, wherein the new DNA strand has at least 50% to a maximum of 99.99% complementarity with the target nucleic acid sequence.

80. The system according to claim 78, wherein the new DNA strand contains mismatched nucleic acid bases relative to the complementary strand of the target nucleic acid.

81. The system according to any one of claims 47 to 80, wherein the secondary structure of the protein-binding region of the guide polynucleotide includes a bulge, stem, loop, hairpin, fluctuating base pair, pseudoknot, or a combination thereof.

82. The system according to any one of claims 47 to 81, wherein the target nucleic acid comprises DNA.

83. The system according to any one of claims 47 to 82, wherein the target nucleic acid comprises RNA.

84. The system according to any one of claims 47 to 83, further comprising an additional protein construct, linker, or nuclear localization sequence (NLS).

85. The system according to any one of claims 47 to 84, wherein the nickase region or a variant thereof and the DNA ligase are linked together by a polypeptide linker.

86. The system according to claim 85, wherein the protein construct comprises the sequences of sequence numbers 89 to 101.

87. A system for modifying target nucleic acids, wherein the system is (a) Donor nucleic acid and (b) an engineered protein construct comprising a nickase region or a variant thereof and a DNA ligase or a functional fragment thereof, (c) Guide polynucleotide, (i) A target-directed region having complementarity with the target nucleic acid, (ii) A protein-binding region comprising a secondary structure that binds to the manipulated protein construct comprising a nickase region, (iii) Ligation sprint 2 region, wherein the ligation sprint region is complementary to the donor nucleic acid and complementary to the target nucleic acid, and the ligation sprint region includes at least one change compared to the target nucleic acid, and (iv) Ligation sprint 1 region, wherein the ligation sprint 1 region comprises a deoxyribonucleotide and a ribonucleotide, and the ligation sprint 1 region is complementary to the target nucleic acid. Includes guide polynucleotides, Includes, A system wherein, when the system is introduced into a cell, nucleus, or cell-free system, the system incorporates the donor nucleic acid into the target nucleic acid, thereby modifying the target nucleic acid.

88. A composition comprising the system and a delivery vehicle according to any one of claims 47 to 87.

89. A polynucleotide or a set of polynucleotides that encodes the system according to any one of claims 47 to 87.

90. A vector comprising the polynucleotide described in claim 89 or a set of polynucleotides described in claim 89.

91. Nanoparticles comprising the polynucleotide described in claim 89 or a set of polynucleotides described in claim 89.

92. A cell comprising the system according to any one of claims 47 to 87.

93. A composition, Donor nucleic acids and A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide is in the order 5' to 3', (a) A target-directed region having complementarity with the target nucleic acid, (b) A protein-binding region comprising a secondary structure that binds to a protein, (c) Ligation sprint 2 region, wherein the ligation sprint 2 region has complementarity with the donor nucleic acid, complementarity with the target nucleic acid, and has at least one change compared to the target nucleic acid or at least one nucleic acid strand thereof, and (d) Ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, A composition containing the following:

94. The composition according to claim 93, wherein the target nucleic acid comprises a double helix of double-stranded DNA (dsDNA) or single-stranded RNA (ssRNA) that is cleaved by nickase.

95. The composition according to claim 93, wherein the protein-binding region binds to a nicasse or endonuclease.

96. The composition according to claim 93, wherein the secondary structure comprises a bulge, a stem, a loop, a hairpin, a fluctuating base pair, a pseudoknot, or a combination thereof.

97. The composition according to claim 93, wherein the ligation sprint 2 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides.

98. The composition according to claim 93, wherein the ligation sprint 2 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides.

99. The composition according to claim 93, wherein the ligation sprint 2 region includes at least one change which is a mismatched nucleic acid base.

100. The composition according to claim 99, wherein the mismatched nucleic acid base includes A / C mismatch, A / T mismatch, A / G mismatch, and T / C mismatch, T / G mismatch, T / A mismatch, C / G mismatch, C / A mismatch, C / T mismatch, G / C mismatch, G / T mismatch, G / A mismatch, or a combination thereof with respect to the target nucleic acid.

101. The composition according to claim 93, wherein the donor nucleic acid comprises an inverse complementary sequence of a non-coding region of a polynucleotide or a variant thereof.

102. The composition according to claim 93, wherein the donor nucleic acid comprises a reverse complementary sequence of a sequence encoding a coding region of a polynucleotide or a variant thereof.

103. The composition according to claim 93, wherein the donor nucleic acid comprises a reverse complementary sequence of a sequence encoding an exon or an intron.

104. The composition according to claim 93, wherein the donor nucleic acid comprises a sequence comprising at least one nucleic acid base that is complementary to or mismatched with the sequence encoding the splice acceptor site.

105. The composition according to claim 93, wherein the donor nucleic acid comprises a complementary sequence of a sequence encoding an intron or a variant thereof.

106. The composition according to claim 93, wherein the donor nucleic acid comprises a complementary sequence of a sequence encoding an exon or a variant thereof.

107. The composition according to claim 93, wherein the donor nucleic acid comprises a complementary sequence of sequences encoding exons and introns.

108. The composition according to claim 93, wherein the ligation sprint 1 region forms a DNA-RNA (DR) loop when it associates with the target nucleic acid and DNA ligase.

109. The composition according to claim 93, wherein the ligase sprint 1 region includes an RNA-to-DNA ratio of 1:1 to a maximum of 20:

1.

110. The ligation sprint 1 region is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 2:1, 2:3, 2:5, 2:7, 2:9, 2:11, 2:13, 2:15, 2:17, 2:19, 3:1, 3:2, 3:4, 3:5, 3:7, 3:8, 3:10, 3:11, 3:13, 3:14, 3:15, 3:16, 3:17, 3:19, 4:1, 4:3, 4:5, 4:7, 4:9, 4:11, 4:13, 4 :15, 4:17, 4:19, 5:1, 5:2, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 5:16, 6:1, 6:5, 6:7, 6:9, 6:11, 6:13, 6:15, 7:1, 7:2, 7:3, 7:4, 7:5, 7:6 , 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 7:15, 8:1, 8:3, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:15, 9:17, 9:19, 9: 20, 10:1, 10:3, 10:7, 10:9, 10:11, 10:13, 10:15, 10:17, 10:19, 11:1, 11:2, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:15, 12: 1, 12:5, 12:7, 12:9, 12:11, 12:13, 13:1, 13:2, 13:3, 13:4, 13:5, 13:6, 13:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 14:1, 14:3, 14:5, 14:9, 14:11, 1 4:13, 15:1, 15:2, 15:4, 15:6, 15:8, 15:11, 15:13, 16:1, 16:3, 16:5, 16:7, 16:9, 16:11, 16:13, 16:15, 17:1, 17:2, 17:3, 17:4, 17:5, 17:6, 17:7, 17:8, 17:9, 17:10, 17:11, 17:12, 17:13, 17:14, 17:15, 17:16, 18:1, 18:5, 18:7, 18:11, 18:13, 18:17, 19:1, 19:2, 19:3, 19:4, 19:5, 19:6, 19:7, 19:8, 19:9,The composition according to claim 93, comprising a ribonucleic acid (RNA) to deoxyribonucleic acid (DNA) ratio of 19:10, 19:11, 19:12, 19:13, 19:14, 19:15, 19:16, 19:17, 19:18, or 20:

1.

111. The composition according to claim 93, wherein the ligation sprint 1 region hybridizes to the target nucleic acid when the target nucleic acid is cleaved by nickase, and the nickase generates a leading strand and a complementary strand.

112. The composition according to claim 111, wherein the ribonucleotide hybridizes to the leading chain.

113. The composition according to claim 93, wherein the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 10,000 nucleotides.

114. The composition according to claim 93, wherein the ligation sprint 1 region comprises at least about 7 nucleotides to a maximum of 1,000 nucleotides.

115. The composition according to claim 93, wherein the ligation sprint 1 region comprises at least about 5 nucleotides to a maximum of 20 nucleotides.

116. The composition according to claim 93, wherein the ligation sprint 1 region includes an inverse complementary sequence that codes for an intron or a variant thereof.

117. The composition according to claim 93, wherein the ligation sprint 1 region includes an inverse complementary sequence of a sequence encoding an exon or a variant thereof.

118. The composition according to claim 93, wherein the ligation sprint 1 region includes an inverse complementary sequence of sequences encoding exons and introns.

119. The composition according to claim 93, wherein the ligation splint 1 region includes a splice acceptor site or a sequence encoding a nucleotide complementary to the splice acceptor site.

120. The composition according to claim 93, wherein the ligation sprint 1 region includes a complementary sequence of a sequence encoding an intron or a variant thereof.

121. The composition according to claim 93, wherein the ligation sprint 1 region includes a complementary sequence of a sequence encoding an exon or a variant thereof.

122. The composition according to claim 93, wherein the ligation sprint 1 region includes complementary sequences of sequences encoding exons and introns.

123. The composition according to claim 93, wherein the ligation splint 1 region comprises a mismatched nucleic acid base for a splice acceptor site in the sequence of the target nucleic acid.

124. The composition according to claim 93, wherein the ribonucleotide is located at the 3' end of the ligation sprint 1 region.

125. The composition according to claim 93, wherein the ligation sprint 1 region comprises one ribonucleotide, two ribonucleotides, three ribonucleotides, four ribonucleotides, five ribonucleotides, or up to ten ribonucleotides.

126. The composition according to claim 93, wherein the ligation sprint 1 region is at least about 5 nucleotides to a maximum of 10 nucleotides in length.

127. The composition according to any one of claims 93 to 126, further comprising a manipulated protein or a polypeptide encoding the manipulated protein.

128. The manipulated protein (a) Niccase region and (b) DNA ligase region and The composition according to claim 127, comprising:

129. The composition according to claim 128, wherein the DNA ligase region is bound to the ligation sprint 1 region or the ligation sprint 2 region of the guide polynucleotide.

130. The composition according to claim 128, wherein the DNA ligase region ligates the donor nucleic acid to the 3' end of the leading strand.

131. The composition according to claim 128, wherein the nickase region comprises an engineered Cas protein that generates a single-strand break in a target nucleic acid.

132. The manipulated Cas proteins are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr 3. The composition according to claim 132, comprising Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, c2c1, c2c3, Cas9HiFi, xCas9, CasX, CasY, CasRX, variants, or any combination thereof.

133. The composition according to any one of claims 128 to 132, further comprising a delivery vehicle.

134. The composition according to claim 133, wherein the delivery vehicle comprises a vector, lipids, nanoparticles, plasmids, viruses, liposomes, extracellular vesicles, emulsions, peptides, carbohydrates, polymers, chitosan, polyethyleneimine (PEI), poly(lactidoglycolide) (PLGA), poly-L-lysine (PLL), or a combination thereof.

135. A cell comprising the composition according to any one of claims 128 to 134.

136. A method for ligating a donor nucleic acid with a target nucleic acid, wherein the method is Cells or cell-free systems, (a) Donor nucleic acid and (b) A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide is (i) A target-directed region having complementarity with the target nucleic acid, (ii) A protein-binding region comprising a secondary structure that binds to a nuclease, and (iii) Ligation sprint 2 region, wherein the ligation sprint 2 region is complementary to the donor nucleic acid and has complementarity with the target nucleic acid and at least one change compared to the target nucleic acid, (iv) Ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, (c) A manipulated protein, or a polynucleotide encoding the manipulated protein, wherein the manipulated protein is (i) Nuclease region, and (ii) DNA ligase region A manipulated protein, or a polynucleotide encoding the said manipulated protein, and Make contact Includes, The guide polynucleotide forms a complex with the manipulated protein via the protein-binding domain. The nuclease region of the manipulated protein undergoes cleavage in the target nucleic acid to generate a leading strand and a complementary strand. The target-directed sequence forms a complex with the complementary chain, The ligation sprint 1 region forms a complex with the leading chain, A method for attaching the donor nucleic acid to the leading strand using the DNA ligase region, thereby ligating the donor nucleic acid to the target nucleic acid.

137. The method according to claim 136, wherein the target-directed chain dissociates from the complementary chain.

138. The method according to claim 136, wherein the new nucleic acid is incorporated into the target nucleic acid by hybridizing with the complementary strand.

139. The method according to claim 136, wherein the incorporation of the new nucleic acid recruits a DNA repair protein that edits the nucleic acid bases of the target nucleic acid to the target nucleic acid.

140. It is a method, Administering the system according to any one of claims 47 to 87, the composition according to any one of claims 1 to 24 or 93 to 134, or the cells according to claim 92 or claim 135 to a subject, organ, tissue, or cell. A method comprising, wherein the administration modifies genes in the subject, the organ, the tissue, or the cells.

141. The method according to claim 140, wherein the change in the gene includes an insertion, deletion, substitution, copy number change, point mutation, frameshift mutation, missense mutation, nonsense mutation, mutation at a stop codon, epigenetic mark, or any combination thereof.

142. The method according to claim 140, wherein the administration is local or systemic.

143. The method according to claim 140, wherein the administration is performed by intranasal administration, subcutaneous administration, intravenous administration, inhalation, intramuscular administration, intratumoral administration, peritumoral administration, intrathecal administration, vaginal administration, or intradermal administration.

144. The method according to claim 140, wherein the subject is a mammal.

145. The method according to claim 140, wherein the subject has a disease or condition, is suspected to have a disease or condition, or is diagnosed with a disease or condition.

146. The method according to claim 145, wherein the disease or condition includes a hereditary disease or hereditary condition.

147. The method according to any one of claims 140 to 146, further comprising administering a therapeutic agent to the subject.

148. It is a method, Contacting cells or a population of cells with the system described in any one of claims 47 to 87 or the composition described in any one of claims 1 to 24 or 93 to 134, thereby causing a change in the target nucleic acid. Methods that include...

149. The method according to claim 148, wherein the contact is performed in vitro, in vivo, or ex vivo.

150. The method according to claim 148, wherein the change includes insertions, deletions, substitutions, copy number changes, point mutations, frameshift mutations, missense mutations, nonsense mutations, mutations in stop codons, epigenetic marks, or any combination thereof.

151. The method according to claim 148, wherein the change restores the expression of the wild-type protein encoded by the gene compared to an equivalent cell or population of cells that did not come into contact with either the system or the composition.

152. The method according to claim 148, wherein the cells include eukaryotic cells or prokaryotic cells.

153. The method according to claim 148, wherein the cells include mammalian cells.

154. The method according to claim 148, wherein the group of cells includes a group of human leukocytes, a group of stem cells, or a group of bacteria.

155. A population of cells prepared by the method described in any one of claims 148 to 154.

156. A system according to any one of claims 47 to 87, or a kit comprising a composition, packaging, and materials thereof according to any one of claims 1 to 24 or 93 to 134.

157. It's a kit, The first container, Donor nucleic acids and A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, wherein the guide polynucleotide is (i) A target-directed region having complementarity with the target nucleic acid, (ii) A protein-binding region comprising a secondary structure that binds to a nuclease, (iii) Ligation sprint 2 region, wherein the region that hybridizes to the donor has complementarity with the donor nucleic acid and complementarity with the target nucleic acid, and the ligation sprint 2 region contains at least one mismatched nucleic acid base relative to the target nucleic acid, and (iv) Ligation sprint 1 region comprising a deoxyribonucleotide and a ribonucleotide A guide polynucleotide, or a polynucleotide encoding the guide polynucleotide, and A first container containing, A second container comprising an engineered polypeptide containing nickase operably linked to a DNA ligase, and A kit that includes this.

158. The kit according to claim 157, further comprising reagents for nucleic acid amplification, transcription, translation, or nucleic acid isolation.

159. The kit according to claim 157 or claim 158, further comprising a reporter molecule.

160. It is scaffolding, A system according to any one of claims 47 to 87 or a composition according to any one of claims 1 to 24 or 93 to 134, A solid surface, wherein the system or composition is fixed to the solid surface and Scaffolding, including scaffolding.

161. The scaffolding according to claim 160, wherein the scaffolding comprises reaction chips, paper, quartz microfibers, a mixed ester of cellulose, porous aluminum oxide, a patterned surface, a tube, a well, or a matrix.

162. The scaffold according to claim 160 or 161, further comprising a reporter molecule that generates a detectable signal that is detected by a detector.

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