Compositions and methods for genome editing by insertion of donor polynucleotides

Abstract

The present disclosure provides donor polynucleotides, genome editing systems, methods and kits for correcting or inducing mutations in gDNA.

Description

[0001] Related information

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 691,573, filed on June 28, 2018. The entire contents of the above-referenced provisional patent application are incorporated herein by reference. Background Art

[0003] Genome editing therapies using programmable and / or engineered nucleases in combination with designed exogenous DNA repair template molecules have been developed for the treatment of intractable diseases such as viral infections (Lin et al. (2014) Mol Ther Nucleic Acids 3:e186), enzyme deficiencies (Yin et al. (2016) Nat Biotechnol 34(3):328-333), and hereditary myopathies (Long et al. (2014) Science 345:1184–1188; Long et al. (2016) Science 351:400–403; Tabebordbar et al. (2016) Science 351:407–411). Therapeutic methods for targeting the genome often rely on the homology-directed repair (HDR) pathway, which enables accurate genomic repair of introduced double-strand breaks (DSBs) using exogenous single-stranded or double-stranded DNA repair templates (e.g., donor polynucleotides), but is often highly suppressed in non-dividing cells (e.g., G1 phase cells) (Orthwein et al. (2015) Nature 528(7582):422-6). Despite the rapid progress in this field of biomedical research and the potential for clinical application, the targeted integration of transgenes or other polynucleotides for in vivo therapeutic purposes remains challenging because current methods are particularly inefficient for non-dividing cells that make up most adult tissues.

[0004] DSBs induced within the genome by an external source (e.g., a modified nuclease) can be repaired in cells by double-strand break repair (DSBR) mechanisms, such as the HDR pathway and the non-homologous end joining (NHEJ) DNA repair pathway. The canonical HDR pathway is known to function in dividing cells (e.g., cells in S phase) because it requires homologous sister chromatids for execution, whereas the NHEJ pathway can function in both dividing and non-dividing cells and is independent of the cell cycle (Iyama & Wilson (2013) DNA Repair 12(8), 620–636). In contrast to the HDR pathway, the NHEJ repair pathway is often used in genome editing methods that do not employ an exogenous DNA repair template (e.g., a donor polynucleotide) and are directed toward the formation of random insertions or deletions ('indels') of one or more nucleotides at the site of the DSB, resulting in disruption of the translational reading frame of the coding sequence or the binding site of a trans-acting factor, for example, in a promoter or enhancer.

[0005] Because genetic abnormalities are widely recognized as the primary etiological basis of numerous diseases, the inability or low efficiency of therapeutically targeted genome editing poses a technical barrier to developing treatments for a wide range of genetic disorders. Therefore, new methods are needed to therapeutically target the genome, particularly in non-dividing cells. Summary of the Invention

[0006] Pre-mRNA (pre-mRNA) splicing accurately is crucial for correct protein expression.Vertebrate gene architecture is often made up of relatively long introns and shorter internal exons.Unbound by theory, in contrast to spanning introns, the exon-limited model assumes that splice sites span exon pairing, and in pre-mRNA with large introns, the splicing mechanism searches for a pair of closely spaced splice sites (that is, 3' splice sites upstream and 5' splice sites downstream) (Berget (1995) J Biol Chem 270:2411-2414) in the exon polarity. A pair of splice sites in relatively short (50 to 300 nucleotides) regions around the exon are identified by the splicing mechanism, reducing the frequency of the hidden intron splice sites randomly distributed in the identification intron, therefore increasing the accuracy of splicing. Many pathogenic mutations are known to be located within exons (eg, protein-coding mutations) or introns (eg, splicing signal mutations), which can lead to abnormalities in protein production or function, ultimately causing or contributing to disease.

[0007] The present disclosure is based, at least in part, on the discovery that mutations (e.g., deleterious or pathogenic mutations) can be corrected or induced in genomic DNA molecules (gDNA) by genome editing compositions and methods, particularly by using donor polynucleotides as described herein, which result in desired changes in the nucleotide sequence of the gDNA and regulate exon definition, thereby resulting in the inclusion of desired changes in RNA transcripts (e.g., pre-mRNA) transcribed from the edited gDNA. Accordingly, the present disclosure provides donor polynucleotides that, when used to repair double-strand breaks (DSBs) introduced into gDNA by site-directed nucleases (e.g., Cas nucleases), correct or induce mutations and regulate exon definition by incorporating one or more splicing signals near the corrected or induced mutations. In some embodiments, the donor polynucleotides provided by the present disclosure are designed to comprise a nucleotide sequence comprising the desired change to correct a mutation in a target nucleic acid (e.g., genomic DNA), and one or more splicing signals that act to regulate splice site recognition. In some embodiments, the donor polynucleotides provided by the present disclosure are designed to comprise a nucleotide sequence comprising a desired change and one or more splicing signals that act to regulate exon definition, directing the cell's splicing machinery to incorporate the desired change into a transcript (e.g., pre-mRNA).

[0008] The present disclosure also provides methods for correcting or inducing mutations (e.g., pathogenic mutations) in gDNA by inserting linear, double-stranded DNA donor polynucleotides into CRISPR / Cas9-mediated DSBs generated near a mutation, including cells, in vitro and in vivo methods. Although actively dividing cells can use both HDR and NHEJ approaches to repair DNA damage, non-dividing cells predominantly use NHEJ approaches. The use of CRISPR / Cas9 allows the introduction of site-specific DNA breaks, which can be repaired by the NHEJ approach so that exogenous DNA polynucleotides are connected or inserted into the genomic DNA of dividing cells or non-dividing cells. In some embodiments, the donor polynucleotides of the present disclosure include such nucleotide sequences that both correct or induce mutations and include one or more splicing signals, thereby promoting the occurrence of desired RNA processing events by establishing exon limits on desired exons (e.g., exons comprising pathogenic mutations) and / or destroying exon limits on undesirable exons (e.g., exons comprising pathogenic mutations). Also provided herein are compositions, systems, and kits for performing such methods. Also provided are cells produced by such methods.

[0009] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising:

[0010] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and a nucleotide sequence that comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0011] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0012] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300 or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0013] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising:

[0014] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and a nucleotide sequence that comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0015] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0016] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0017] In some embodiments, the mutation is a substitution, missense, nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the mutation is a substitution mutation. In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the mutation is an insertion mutation. In some embodiments, the mutation is a deletion mutation. In some embodiments, the mutation is a frameshift mutation.

[0018] In some embodiments, the mutation is in an exon. In some embodiments, the mutation is a substitution, insertion, or deletion, and the mutation is in an intron. In some embodiments, the mutation is a substitution mutation located in an intron. In some embodiments, the mutation is an insertion mutation located in an intron. In some embodiments, the mutation is a deletion mutation located in an intron.

[0019] In some embodiments, the mutation is near a splice signal in the gDNA. In some embodiments, the mutation is near a 3' splice site in the gDNA. In some embodiments, the mutation is near a 5' splice site in the gDNA. In some embodiments, the mutation is in a splice signal in the gDNA. In some embodiments, the mutation is in a 3' splice site in the gDNA. In some embodiments, the mutation is in a 5' splice site in the gDNA. In some embodiments, the mutation is in a polypyrimidine tract. In some embodiments, the mutation is in a branch point sequence. In some embodiments, the mutation is a protein-coding mutation. In some embodiments, the mutation is associated with or causes a disease.

[0020] In some embodiments, the donor polynucleotides provided by the present disclosure comprise an intron sequence. In some embodiments, the intron sequence corrects the mutation.

[0021] In some embodiments, the donor polynucleotides provided by the present disclosure comprise exonic sequences. In some embodiments, the exonic sequences correct for mutations.

[0022] In some embodiments, the donor polynucleotides provided by the present disclosure comprise one or more splicing signals selected from the group consisting of:

[0023] (a) natural or enhanced 3' splice site;

[0024] (b) native or enhanced 5' splice site;

[0025] (c) Polypyrimidine tablets;

[0026] (d) branching points;

[0027] (e) exonic splicing enhancer (ESE);

[0028] (f) intronic splicing enhancer (ISE);

[0029] (g) exonic splicing silencer (ESS);

[0030] (h) intronic splicing silencers (ISSs); and

[0031] (i) A combination of any one of (a) to (h).

[0032] In some embodiments, one or more splicing signals are natural or enhanced 3' splice sites. In some embodiments, one or more splicing signals are natural 3' splice sites. In some embodiments, one or more splicing signals are enhanced 3' splice sites. In some embodiments, one or more splicing signals are natural or enhanced 5' splice sites. In some embodiments, one or more splicing signals are natural 5' splice sites. In some embodiments, one or more splicing signals are enhanced 3' splice sites. In some embodiments, one or more splicing signals are polypyrimidine fragments. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are nucleotide sequences comprising branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESEs). In some embodiments, one or more splicing signals are intron splicing enhancers (ISEs). In some embodiments, one or more splicing signals are exon splicing silencers (ESSs). In some embodiments, one or more splicing signals are intron splicing silencers (ISSs). In some embodiments, the one or more splicing signals are a combination comprising a native or enhanced 3' splice site and a polypyrimidine tract. In some embodiments, the one or more splicing signals are a combination comprising a native or enhanced 3' splice site, a polypyrimidine tract, and a branch point.

[0033] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell, and one or more splicing signals that control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide includes a first splicing signal comprising a branch point sequence, wherein the donor polynucleotide comprises:

[0034] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first branch point sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0035] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second branch point sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0036] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, the nucleotide sequence comprising the first splicing signal conforms to the branch point consensus sequence on either chain, wherein the nucleotide sequences of the first branch point sequence and the second branch point sequence are complementary.

[0037] In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0038] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell, and one or more splicing signals that control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide includes a first splicing signal comprising a branch point sequence, wherein the donor polynucleotide comprises:

[0039] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first branch point sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0040] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second branch point sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0041] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, the nucleotide sequence comprising the first splicing signal conforms to the branch point consensus sequence on either chain, wherein the nucleotide sequences of the first branch point sequence and the second branch point sequence are complementary.

[0042] In some embodiments, the branch point consensus sequence is YTNAY (SEQ ID NO: 49), wherein Y is a nucleotide comprising a cytosine (C) or thymine (T) nucleobase, and wherein N is a nucleotide comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C). In some embodiments, Y comprises cytosine (C). In some embodiments, Y comprises thymine (T). In some embodiments, N comprises adenine (A). In some embodiments, N comprises guanine (G). In some embodiments, N comprises thymine (T). In some embodiments, N comprises cytosine (C).

[0043] In some embodiments, the first branch point sequence is TATTAAC (SEQ ID NO: 50).

[0044] In some embodiments, the second branch point sequence is GTTAATA (SEQ ID NO: 51).

[0045] In some embodiments, the second branch point sequence is TACTGAC (SEQ ID NO: 52).

[0046] In some embodiments, the donor polynucleotide comprises a second splicing signal comprising a polypyrimidine tract, wherein the first strand comprises a first polypyrimidine tract downstream of the first branch point sequence; and the second strand comprises a second polypyrimidine tract downstream of the second branch point sequence. In some embodiments, the nucleotide sequence comprising the first polypyrimidine tract and the second polypyrimidine tract each comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), and wherein the nucleotide sequence is about 100%, about 90%-100%, or about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 80%-90% pyrimidine nucleobases.

[0047] In some embodiments, the nucleotide sequence comprising a polypyrimidine stretch is TTTTTTTCT (SEQ ID NO: 53). In some embodiments, the nucleotide sequence comprising a polypyrimidine stretch is TTTTTTTCTTTTTT (SEQ ID NO: 54). In some embodiments, the nucleotide sequence comprising a polypyrimidine stretch is CTTCTTCTCTTCTTCC (SEQ ID NO: 55).

[0048] In some embodiments, the first branch point sequence and the first polypyrimidine tract are adjacent to each other. In some embodiments, the second branch point sequence and the second polypyrimidine tract are adjacent to each other.

[0049] In some embodiments, the donor polynucleotide comprises a third splice signal, wherein the third splice signal comprises a 3' splice site, wherein the first strand comprises a nucleotide sequence comprising a first 3' splice site downstream of the first polypyrimidine tract; and wherein the second strand comprises a nucleotide sequence comprising a second 3' splice site downstream of the second polypyrimidine tract. In some embodiments, the first 3' splice site and the second 3' splice site comprise the nucleotide sequence YAG, and wherein Y is a nucleotide comprising a nucleobase selected from thymine (T) and cytosine (C). In some embodiments, Y comprises thymine (T). In some embodiments, Y comprises cytosine (C).

[0050] In some embodiments, the donor polynucleotide comprises a coding sequence, wherein the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first nucleotide sequence that corrects the mutation in the gDNA comprises the first coding sequence, wherein the second nucleotide sequence that corrects the mutation in the gDNA comprises the second coding sequence, wherein the first coding sequence is located downstream of a first 3' splice site, and wherein the second coding sequence is located downstream of a second 3' splice site. In some embodiments, the nucleotide sequence comprising the first coding sequence and the second coding sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C). In some embodiments, the coding sequences comprising (i) and (ii) are non-identical or non-complementary to reduce self-annealing.

[0051] In some embodiments, the donor polynucleotide comprises one or more delimiter sequences comprising a nucleotide sequence selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or 1 nucleotide in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-40 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-30 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-20 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-15 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-10 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 30 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 20 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 10 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 9 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 8 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 7 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 6 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 5 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 4 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 3 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 2 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1 nucleotide in length.

[0052] In some embodiments, one or more delimiter sequences are located between a first branch point sequence and a second branch point sequence. In some embodiments, one or more delimiter sequences are located between a first branch point sequence and a first polypyrimidine stretch. In some embodiments, one or more delimiter sequences are located between a second branch point sequence and a second polypyrimidine stretch.

[0053] In some embodiments, the present disclosure provides donor polynucleotides configured for bidirectional insertion into a DSB, wherein the first and second splice signals, optionally the third splice signal, and the coding sequence constitute the sense strand when the donor polynucleotide is inserted into the DSB in either orientation, thereby correcting the mutation and providing one or more splice signals to control processing of pre-mRNA transcribed from the gDNA.

[0054] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell, and one or more splicing signals that control processing of a pre-mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises:

[0055] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first 5′ splice site sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0056] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second 5′ splice site sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0057] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first coding sequence is located upstream of a first 5' splice site, and wherein the second coding sequence is located upstream of a second 5' splice site, and wherein the coding sequences in the first and second strands are non-complementary (or comprise one, two, three, four or more mismatches) to reduce self-annealing. In some embodiments, the donor polynucleotide comprises a delimiter sequence located between the first 5' splice site and the second 5' splice site, which includes a nucleotide sequence comprising a nucleobase selected from the group consisting of: adenine (A), guanine (G), thymine (T), and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or 1 nucleotides in length. In some embodiments, the donor polynucleotide is configured for bidirectional insertion into a DSB, wherein when the donor polynucleotide is inserted into the DSB in a first orientation, the first 5' splice site and the first coding sequence constitute the sense strand, thereby correcting the mutation and providing splicing signals to control the processing of the pre-mRNA transcribed from the gDNA, and wherein when the donor polynucleotide is inserted into the DSB in a second orientation, the second 5' splice site and the second coding sequence constitute the sense strand, thereby correcting the mutation and providing one or more splicing signals to control the processing of the pre-mRNA transcribed from the gDNA. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length.In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0058] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell, and one or more splicing signals that control processing of a pre-mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises:

[0059] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first 5′ splice site sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0060] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second 5′ splice site sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0061] Wherein the length of the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, the first chain comprises a first coding sequence, wherein the second chain comprises a second coding sequence, wherein the first coding sequence is located upstream of the first 5' splice site, and wherein the second coding sequence is located upstream of the second 5' splice site, and wherein the coding sequences in the first chain and the second chain are non-complementary (or comprise one, two, three, four or more mismatches) to reduce self-annealing. In some embodiments, the donor polynucleotide comprises a delimiter sequence located between the first 5' splice site and the second 5' splice site, which includes a nucleotide sequence comprising a nucleobase selected from the group consisting of: adenine (A), guanine (G), thymine (T), and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or 1 nucleotides in length. In some embodiments, the donor polynucleotide is configured for bidirectional insertion into a DSB, wherein when the donor polynucleotide is inserted into the DSB in a first orientation, the first 5' splice site and the first coding sequence constitute the sense strand, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, and wherein when the donor polynucleotide is inserted into the DSB in a second orientation, the second 5' splice site and the second coding sequence constitute the sense strand, thereby correcting the mutation and providing one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA.

[0062] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0063] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0064] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0065] (a) natural or enhanced 3' splice site;

[0066] (b) Polypyrimidine tablets;

[0067] (c) branching points;

[0068] (d) exonic splicing enhancer (ESE);

[0069] (e) intronic splicing enhancer (ISE);

[0070] (f) exonic splicing silencer (ESS);

[0071] (g) intronic splicing silencers (ISSs); and

[0072] (h) A combination of any one of (a) to (g),

[0073] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, one or more splicing signals are natural or enhanced 3' splice sites. In some embodiments, one or more splicing signals are polypyrimidine pieces. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISSs). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0074] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0075] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0076] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0077] (a) natural or enhanced 3' splice site;

[0078] (b) Polypyrimidine tablets;

[0079] (c) branching points;

[0080] (d) exonic splicing enhancer (ESE);

[0081] (e) intronic splicing enhancer (ISE);

[0082] (f) exonic splicing silencer (ESS);

[0083] (g) intronic splicing silencers (ISSs); and

[0084] (h) A combination of any one of (a) to (g),

[0085] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by a site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, one or more splicing signals are natural or enhanced 3' splice sites. In some embodiments, one or more splicing signals are polypyrimidine pieces. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0086] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0087] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA (gDNA) molecule in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 5' splice site, wherein the first strand comprises intron sequences and exon sequences, wherein the exon sequences correct the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0088] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0089] wherein the one or more splicing signals are selected from:

[0090] (a) natural or enhanced 5' splice site;

[0091] (b) exonic splicing enhancer (ESE);

[0092] (c) intronic splicing enhancer (ISE);

[0093] (d) exonic splicing silencer (ESS);

[0094] (e) intronic splicing silencers (ISSs); and

[0095] (f) A combination of any one of (a) to (e),

[0096] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, one or more splicing signals are natural or enhanced 5' splice sites. In some embodiments, one or more splicing signals are polypyrimidine pieces. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISSs). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0097] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0098] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA (gDNA) molecule in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 5' splice site, wherein the first strand comprises intron sequences and exon sequences, wherein the exon sequences correct the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0099] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0100] wherein the one or more splicing signals are selected from:

[0101] (a) natural or enhanced 5' splice site;

[0102] (b) exonic splicing enhancer (ESE);

[0103] (c) intronic splicing enhancer (ISE);

[0104] (d) exonic splicing silencer (ESS);

[0105] (e) intronic splicing silencers (ISSs); and

[0106] (f) A combination of any one of (a) to (e),

[0107] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by the site-directed nuclease, thereby correcting the mutation. In some embodiments, one or more splicing signals are natural or enhanced 5' splice sites. In some embodiments, one or more splicing signals are polypyrimidine pieces. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0108] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0109] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 3' splice site; and

[0110] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0111] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300 or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0112] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0113] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 3' splice site; and

[0114] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0115] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0116] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0117] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site and a polypyrimidine tract; and

[0118] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0119] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300 or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0120] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0121] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site and a polypyrimidine tract; and

[0122] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0123] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0124] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0125] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site, a polypyrimidine tract, and a branch point; and

[0126] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0127] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300 or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0128] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0129] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site, a polypyrimidine tract, and a branch point; and

[0130] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0131] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0132] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0133] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 3′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0134] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0135] wherein the one or more splicing signals are selected from:

[0136] (a) Polypyrimidine tablets;

[0137] (b) branching points;

[0138] (c) exonic splicing enhancer (ESE);

[0139] (d) intronic splicing enhancer (ISE);

[0140] (e) exonic splicing silencer (ESS);

[0141] (f) intronic splicing silencers (ISSs); and

[0142] (g) A combination of any one of (a) to (f),

[0143] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, one or more splicing signals are polypyrimidine fragments. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISSs). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0144] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0145] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 3′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0146] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0147] wherein the one or more splicing signals are selected from:

[0148] (a) Polypyrimidine tablets;

[0149] (b) branching points;

[0150] (c) exonic splicing enhancer (ESE);

[0151] (d) intronic splicing enhancer (ISE);

[0152] (e) exonic splicing silencer (ESS);

[0153] (f) intronic splicing silencers (ISSs); and

[0154] (g) A combination of any one of (a) to (f),

[0155] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, one or more splicing signals are polypyrimidine pieces. In some embodiments, one or more splicing signals are branch points. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0156] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0157] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the donor polynucleotide comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 5' splice site; and

[0158] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0159] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300 or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0160] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0161] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the donor polynucleotide comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 5' splice site; and

[0162] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0163] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by a site-directed nuclease, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0164] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0165] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 5′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0166] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0167] (a) Exonic splicing enhancer (ESE);

[0168] (b) intronic splicing enhancer (ISE);

[0169] (c) exonic splicing silencer (ESS);

[0170] (d) intronic splicing silencers (ISSs); and

[0171] (e) A combination of any one of (a) to (d),

[0172] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the donor polynucleotide is inserted into the double-stranded DNA break (DSB) by the non-homologous end joining (NHEJ) DNA repair pathway, and the DSB is introduced into the gDNA at a position close to the mutation by the site-directed nuclease, thereby correcting the mutation. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISS). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0173] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0174] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 5′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0175] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0176] (a) Exonic splicing enhancer (ESE);

[0177] (b) intronic splicing enhancer (ISE);

[0178] (c) exonic splicing silencer (ESS);

[0179] (d) intronic splicing silencers (ISSs); and

[0180] (e) A combination of any one of (a) to (d),

[0181] Wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by the site-directed nuclease, thereby correcting the mutation. In some embodiments, one or more splicing signals are exon splicing enhancers (ESE). In some embodiments, one or more splicing signals are intron splicing enhancers (ISE). In some embodiments, one or more splicing signals are exon splicing silencers (ESS). In some embodiments, one or more splicing signals are intron splicing silencers (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0182] In some embodiments, insertion of the donor polynucleotide into the DSB results in exon formation in the gDNA comprising an exonic sequence. In some embodiments, one or more splicing signals direct the inclusion of an exon comprising an exonic sequence that corrects a mutation in the mRNA.

[0183] In some embodiments, insertion of the donor polynucleotide results in the formation of an intron comprising an intron sequence, wherein the intron sequence corrects the mutation.

[0184] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the first strand comprises the formula:

[0185] 5'-[B] a -[S1] b -[P] c -[S2] d -XE-3', where

[0186] (i) B, if present, is a branchpoint sequence comprising a nucleotide sequence that conforms to the branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a is an integer whose value indicates the number of nucleotides comprised by B, wherein a=0 or 5-7;

[0187] (ii) P is a polypyrimidine tract comprising a nucleotide sequence comprising nucleobases selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by P, wherein c = 9-20, wherein the nucleotide sequence comprising P is about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases;

[0188] (iii) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0189] (iv) X is a nucleotide sequence comprising a 3' splice site; and

[0190] (v) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0191] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, B (if present), P, X (if present), and E (if present) comprise the sense strand, wherein B (if present), P, and X (if present) comprise one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0192] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B is present. In some embodiments, B is absent. In some embodiments, a = 0. In some embodiments, a = 5-7. In some embodiments, a = 5. In some embodiments, a = 6. In some embodiments, a = 7. In some embodiments, the nucleotide sequence constituting P is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence constituting P is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence constituting P is about 80%-90% pyrimidine nucleobases. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13. In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7.In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0193] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the first strand comprises the formula:

[0194] 5'-[B] a -[S1] b -[P] c -[S2] d -XE-3', where

[0195] (i) B, if present, is a branchpoint sequence comprising a nucleotide sequence that conforms to the branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a is an integer whose value indicates the number of nucleotides comprised by B, wherein a=0 or 5-7;

[0196] (ii) P is a polypyrimidine tract comprising a nucleotide sequence comprising nucleobases selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by P, wherein c = 9-20, wherein the nucleotide sequence comprising P is about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases;

[0197] (iii) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0198] (iv) X is a nucleotide sequence comprising a 3' splice site; and

[0199] (v) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0200] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, B (if present), P, X (if present), and E (if present) comprise the sense strand, wherein B (if present), P, and X (if present) comprise one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0201] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B is present. In some embodiments, B is absent. In some embodiments, a = 0. In some embodiments, a = 5-7. In some embodiments, a = 5. In some embodiments, a = 6. In some embodiments, a = 7. In some embodiments, the nucleotide sequence constituting P is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence constituting P is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence constituting P is about 80%-90% pyrimidine nucleobases. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13. In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7.In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0202] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA,

[0203] wherein the first chain comprises the formula:

[0204] 5'-EYI-3', where

[0205] (i) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0206] (ii) Y is a nucleotide sequence comprising a 5' splice site; and

[0207] (iii) I, if present, comprises an intron sequence comprising nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C),

[0208] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, E (if present), Y, and I (if present) comprise the sense strand, wherein Y comprises one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0209] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, E is present. In some embodiments, E is not present. In some embodiments, I is present. In some embodiments, I is not present. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0210] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA,

[0211] wherein the first chain comprises the formula:

[0212] 5'-EYI-3', where

[0213] (i) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0214] (ii) Y is a nucleotide sequence comprising a 5' splice site; and

[0215] (iii) I, if present, comprises an intron sequence comprising nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C),

[0216] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, E (if present), Y, and I (if present) comprise the sense strand, wherein Y comprises one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0217] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, E is present. In some embodiments, E is not present. In some embodiments, I is present. In some embodiments, I is not present. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0218] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0219] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0220] (ii) a second strand comprising, from 5' to 3', a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0221] 5'-[P1] a -[S1] b -[B] c -[S2] d -[P2] e -3', where

[0222] (a) B comprises a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by B, wherein c = 5-7;

[0223] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and e are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a=9-20 and e=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, and wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide; and

[0224] (c) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0225] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B and P2 constitute the sense strand, and B and P2 comprise the first splicing signal and the second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B and P1 constitute the sense strand, and B and P2 comprise the first splicing signal and the second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0226] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, a = 9. In some embodiments, a = 10. In some embodiments, a = 11. In some embodiments, a = 12. In some embodiments, a = 13. In some embodiments, a = 14. In some embodiments, a = 15. In some embodiments, a = 16. In some embodiments, a = 17. In some embodiments, a = 18. In some embodiments, a = 19. In some embodiments, a = 20. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13.In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0227] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0228] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0229] (ii) a second strand comprising, from 5' to 3', a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0230] 5'-[P1] a -[S1] b -[B] c -[S2] d -[P2] e -3', where

[0231] (a) B comprises a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by B, wherein c = 5-7;

[0232] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and e are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a=9-20 and e=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, and wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide; and

[0233] (c) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0234] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B and P2 constitute the sense strand, and B and P2 comprise a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B and P1 constitute the sense strand, and B and P2 comprise a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA,

[0235] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, a = 9. In some embodiments, a = 10. In some embodiments, a = 11. In some embodiments, a = 12. In some embodiments, a = 13. In some embodiments, a = 14. In some embodiments, a = 15. In some embodiments, a = 16. In some embodiments, a = 17. In some embodiments, a = 18. In some embodiments, a = 19. In some embodiments, a = 20. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13.In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0236] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0237] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0238] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0239] 5'-[P1] a -[S1] b -[B1] c -[S2] d -[B2] e -[S3] f -[P2] g -3', where

[0240] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c and e are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein c = 0 or 5-7, wherein e = 5-7;

[0241] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a = 9-20 and g = 9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide; and

[0242] (c) S1, S2 and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b, d and f are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and f = 0 or 1-20, and wherein d = 0 or 1-40,

[0243] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2 and P2 constitute the sense strand, and B2 and P2 comprise a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1 and P1 constitute the sense strand, and B1 and P1 provide a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0244] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, c = 0. In some embodiments, c = 5-7. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, a = 9. In some embodiments, a = 10. In some embodiments, a = 11. In some embodiments, a = 12. In some embodiments, a = 13. In some embodiments, a = 14. In some embodiments, a = 15. In some embodiments, a = 16. In some embodiments, a = 17. In some embodiments, a = 18. In some embodiments, a = 19. In some embodiments, a = 20. In some embodiments, g = 9. In some embodiments, g = 10. In some embodiments, g = 11. In some embodiments, g = 12. In some embodiments, g = 13. In some embodiments, g = 14. In some embodiments, g = 15. In some embodiments, g = 16. In some embodiments, g = 17. In some embodiments, g = 18. In some embodiments, g = 19. In some embodiments, g = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4.In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, f = 0. In some embodiments, f = 1. In some embodiments, f = 2. In some embodiments, f = 3. In some embodiments, f = 4. In some embodiments, f = 5. In some embodiments, f = 6. In some embodiments, f = 7. In some embodiments, f = 8. In some embodiments, f = 9. In some embodiments, f = 10. In some embodiments, f = 11. In some embodiments, f = 12. In some embodiments, f = 13. In some embodiments, f = 14. In some embodiments, f = 15. In some embodiments, f = 16. In some embodiments, f = 17. In some embodiments, f = 18. In some embodiments, f = 19. In some embodiments, f = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, d = 21. In some embodiments, d = 22. In some embodiments, d = 23. In some embodiments, d = 24. In some embodiments, d = 25. In some embodiments, d = 26.In some embodiments, d = 27. In some embodiments, d = 28. In some embodiments, d = 29. In some embodiments, d = 30. In some embodiments, d = 31. In some embodiments, d = 32. In some embodiments, d = 33. In some embodiments, d = 34. In some embodiments, d = 35. In some embodiments, d = 36. In some embodiments, d = 37. In some embodiments, d = 38. In some embodiments, d = 39. In some embodiments, d = 40. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0245] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0246] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0247] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0248] 5'-[P1] a -[S1] b -[B1] c -[S2] d -[B2] e -[S3] f -[P2] g -3', where

[0249] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c and e are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein c = 0 or 5-7, wherein e = 5-7;

[0250] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a = 9-20 and g = 9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide; and

[0251] (c) S1, S2 and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b, d and f are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and f = 0 or 1-20, and wherein d = 0 or 1-40,

[0252] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2 and P2 constitute the sense strand, and B2 and P2 comprise a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1 and P1 constitute the sense strand, and B1 and P1 provide a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA,

[0253] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, c = 0. In some embodiments, c = 5-7. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, a = 9. In some embodiments, a = 10. In some embodiments, a = 11. In some embodiments, a = 12. In some embodiments, a = 13. In some embodiments, a = 14. In some embodiments, a = 15. In some embodiments, a = 16. In some embodiments, a = 17. In some embodiments, a = 18. In some embodiments, a = 19. In some embodiments, a = 20. In some embodiments, g = 9. In some embodiments, g = 10. In some embodiments, g = 11. In some embodiments, g = 12. In some embodiments, g = 13. In some embodiments, g = 14. In some embodiments, g = 15. In some embodiments, g = 16. In some embodiments, g = 17. In some embodiments, g = 18. In some embodiments, g = 19. In some embodiments, g = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4.In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, f = 0. In some embodiments, f = 1. In some embodiments, f = 2. In some embodiments, f = 3. In some embodiments, f = 4. In some embodiments, f = 5. In some embodiments, f = 6. In some embodiments, f = 7. In some embodiments, f = 8. In some embodiments, f = 9. In some embodiments, f = 10. In some embodiments, f = 11. In some embodiments, f = 12. In some embodiments, f = 13. In some embodiments, f = 14. In some embodiments, f = 15. In some embodiments, f = 16. In some embodiments, f = 17. In some embodiments, f = 18. In some embodiments, f = 19. In some embodiments, f = 20. In some embodiments, d = 0. In some embodiments, d = 1. In some embodiments, d = 2. In some embodiments, d = 3. In some embodiments, d = 4. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, d = 8. In some embodiments, d = 9. In some embodiments, d = 10. In some embodiments, d = 11. In some embodiments, d = 12. In some embodiments, d = 13. In some embodiments, d = 14. In some embodiments, d = 15. In some embodiments, d = 16. In some embodiments, d = 17. In some embodiments, d = 18. In some embodiments, d = 19. In some embodiments, d = 20. In some embodiments, d = 21. In some embodiments, d = 22. In some embodiments, d = 23. In some embodiments, d = 24. In some embodiments, d = 25. In some embodiments, d = 26.In some embodiments, d = 27. In some embodiments, d = 28. In some embodiments, d = 29. In some embodiments, d = 30. In some embodiments, d = 31. In some embodiments, d = 32. In some embodiments, d = 33. In some embodiments, d = 34. In some embodiments, d = 35. In some embodiments, d = 36. In some embodiments, d = 37. In some embodiments, d = 38. In some embodiments, d = 39. In some embodiments, d = 40. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0254] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0255] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0256] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0257] 5'-[E1] a -X1-[P1] b -[S1] c -[B] d -[S2] e -[P2] f -X2-[E2] g -3', where

[0258] (a) B is a branchpoint sequence comprising a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein d is an integer whose value indicates the number of nucleotides comprised by B, wherein d = 5-7;

[0259] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein b and f are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and f=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide;

[0260] (c) E1 and E2 are each exonic sequences, each comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exonic sequence comprising E1 is in opposite orientation relative to the exonic sequence comprising E2 and is on opposite strands of the donor polynucleotide, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0261] (d) X1 and X2 are each a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence constituting X1 is in the opposite orientation relative to the nucleotide sequence constituting X2 and is on opposite strands; and

[0262] (e) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein c and e are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c = 0 or 1-20 and e = 0 or 1-20,

[0263] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B, P2, E2, and X2 constitute the sense strand, wherein B, P2, and X2 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B, P1, E1, and X1 constitute the sense strand, wherein B, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0264] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, f = 9. In some embodiments, f = 10. In some embodiments, f = 11. In some embodiments, f = 12. In some embodiments, f = 13. In some embodiments, f = 14. In some embodiments, f = 15. In some embodiments, f = 16. In some embodiments, f = 17. In some embodiments, f = 18. In some embodiments, f = 19. In some embodiments, f = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, c = 0. In some embodiments, c = 1. In some embodiments, c = 2. In some embodiments, c = 3. In some embodiments, c = 4. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, c = 8. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13.In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, e = 0. In some embodiments, e = 1. In some embodiments, e = 2. In some embodiments, e = 3. In some embodiments, e = 4. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, e = 8. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0265] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0266] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0267] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0268] 5'-[E1] a -X1-[P1] b -[S1] c -[B] d -[S2] e -[P2] f -X2-[E2] g -3', where

[0269] (a) B is a branchpoint sequence comprising a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein d is an integer whose value indicates the number of nucleotides comprised by B, wherein d = 5-7;

[0270] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein b and f are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and f=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide;

[0271] (c) E1 and E2 are each exonic sequences, each comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exonic sequence comprising E1 is in opposite orientation relative to the exonic sequence comprising E2 and is on opposite strands of the donor polynucleotide, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0272] (d) X1 and X2 are each a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence constituting X1 is in the opposite orientation relative to the nucleotide sequence constituting X2 and is on opposite strands; and

[0273] (e) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein c and e are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c = 0 or 1-20 and e = 0 or 1-20,

[0274] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B, P2, E2, and X2 constitute the sense strand, wherein B, P2, and X2 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B, P1, E1, and X1 constitute the sense strand, wherein B, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0275] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, f = 9. In some embodiments, f = 10. In some embodiments, f = 11. In some embodiments, f = 12. In some embodiments, f = 13. In some embodiments, f = 14. In some embodiments, f = 15. In some embodiments, f = 16. In some embodiments, f = 17. In some embodiments, f = 18. In some embodiments, f = 19. In some embodiments, f = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, c = 0. In some embodiments, c = 1. In some embodiments, c = 2. In some embodiments, c = 3. In some embodiments, c = 4. In some embodiments, c = 5. In some embodiments, c = 6. In some embodiments, c = 7. In some embodiments, c = 8. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13.In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, e = 0. In some embodiments, e = 1. In some embodiments, e = 2. In some embodiments, e = 3. In some embodiments, e = 4. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, e = 8. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0276] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0277] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0278] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0279] wherein the first and second chains each comprise the formula:

[0280] 5'-[E1] a -X1-[P1] b -[S1] c -[B1] d -[S2] e -[B2] f -[S3] g -[P2] h -X2-[E2] i -3', where

[0281] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein d and f are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein d and f = 5-7;

[0282] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b and h are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and h=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide;

[0283] (c) E1 and E2 are each an exon sequence, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and i are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence comprising E1 is in opposite orientation relative to the exon sequence comprising E2 and is on opposite strands, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0284] (d) X1 and X2 each comprise a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence of X1 is in an opposite orientation relative to the nucleotide sequence of X2 and is on opposite strands of the donor polynucleotide; and

[0285] (e) S1, S2, and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c, e, and g are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c = 0 or 1-20 and g = 0 or 1-20, wherein e = 0 or 1-40,

[0286] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2, P2, E2, and X2 constitute the sense strand, wherein B2, P2, and X2 each comprise a first splicing signal, a second splicing signal, and a third splicing signal, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in the second orientation, B1, P1, E1, and X1 constitute the sense strand, wherein B1, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of the pre-mRNA transcribed from the gDNA,

[0287] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, f = 5. In some embodiments, f = 6. In some embodiments, f = 7. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, h = 9. In some embodiments, h = 10. In some embodiments, h = 11. In some embodiments, h = 12. In some embodiments, h = 13. In some embodiments, h = 14. In some embodiments, h = 15. In some embodiments, h = 16. In some embodiments, h = 17. In some embodiments, h = 18. In some embodiments, h = 19. In some embodiments, h = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, c = 0. In some embodiments, c = 1. In some embodiments, c = 2. In some embodiments, c = 3. In some embodiments, c = 4. In some embodiments, c = 5. In some embodiments, c = 6.In some embodiments, c = 7. In some embodiments, c = 8. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13. In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, g = 0. In some embodiments, g = 1. In some embodiments, g = 2. In some embodiments, g = 3. In some embodiments, g = 4. In some embodiments, g = 5. In some embodiments, g = 6. In some embodiments, g = 7. In some embodiments, g = 8. In some embodiments, g = 9. In some embodiments, g = 10. In some embodiments, g = 11. In some embodiments, g = 12. In some embodiments, g = 13. In some embodiments, g = 14. In some embodiments, g = 15. In some embodiments, g = 16. In some embodiments, g = 17. In some embodiments, g = 18. In some embodiments, g = 19. In some embodiments, g = 20. In some embodiments, e = 0. In some embodiments, e = 1. In some embodiments, e = 2. In some embodiments, e = 3. In some embodiments, e = 4. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, e = 8. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, e = 21. In some embodiments, e = 22. In some embodiments, e = 23. In some embodiments, e = 24. In some embodiments, e = 25. In some embodiments, e = 26. In some embodiments, e = 27. In some embodiments, e = 28.In some embodiments, e = 29. In some embodiments, e = 30. In some embodiments, e = 31. In some embodiments, e = 32. In some embodiments, e = 33. In some embodiments, e = 34. In some embodiments, e = 35. In some embodiments, e = 36. In some embodiments, e = 37. In some embodiments, e = 38. In some embodiments, e = 39. In some embodiments, e = 40. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0288] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0289] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0290] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0291] wherein the first and second chains each comprise the formula:

[0292] 5'-[E1] a -X1-[P1] b -[S1] c -[B1] d -[S2] e -[B2] f -[S3] g -[P2] h -X2-[E2] i -3', where

[0293] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein d and f are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein d and f = 5-7;

[0294] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b and h are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and h=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide;

[0295] (c) E1 and E2 are each an exon sequence, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and i are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence comprising E1 is in opposite orientation relative to the exon sequence comprising E2 and is on opposite strands, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0296] (d) X1 and X2 each comprise a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence of X1 is in an opposite orientation relative to the nucleotide sequence of X2 and is on opposite strands of the donor polynucleotide; and

[0297] (e) S1, S2, and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c, e, and g are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c = 0 or 1-20 and g = 0 or 1-20, wherein e = 0 or 1-40,

[0298] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2, P2, E2, and X2 constitute the sense strand, wherein B2, P2, and X2 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1, P1, E1, and X1 constitute the sense strand, wherein B1, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0299] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, d = 5. In some embodiments, d = 6. In some embodiments, d = 7. In some embodiments, f = 5. In some embodiments, f = 6. In some embodiments, f = 7. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, h = 9. In some embodiments, h = 10. In some embodiments, h = 11. In some embodiments, h = 12. In some embodiments, h = 13. In some embodiments, h = 14. In some embodiments, h = 15. In some embodiments, h = 16. In some embodiments, h = 17. In some embodiments, h = 18. In some embodiments, h = 19. In some embodiments, h = 20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is approximately 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, c = 0. In some embodiments, c = 1. In some embodiments, c = 2. In some embodiments, c = 3. In some embodiments, c = 4. In some embodiments, c = 5. In some embodiments, c = 6.In some embodiments, c = 7. In some embodiments, c = 8. In some embodiments, c = 9. In some embodiments, c = 10. In some embodiments, c = 11. In some embodiments, c = 12. In some embodiments, c = 13. In some embodiments, c = 14. In some embodiments, c = 15. In some embodiments, c = 16. In some embodiments, c = 17. In some embodiments, c = 18. In some embodiments, c = 19. In some embodiments, c = 20. In some embodiments, g = 0. In some embodiments, g = 1. In some embodiments, g = 2. In some embodiments, g = 3. In some embodiments, g = 4. In some embodiments, g = 5. In some embodiments, g = 6. In some embodiments, g = 7. In some embodiments, g = 8. In some embodiments, g = 9. In some embodiments, g = 10. In some embodiments, g = 11. In some embodiments, g = 12. In some embodiments, g = 13. In some embodiments, g = 14. In some embodiments, g = 15. In some embodiments, g = 16. In some embodiments, g = 17. In some embodiments, g = 18. In some embodiments, g = 19. In some embodiments, g = 20. In some embodiments, e = 0. In some embodiments, e = 1. In some embodiments, e = 2. In some embodiments, e = 3. In some embodiments, e = 4. In some embodiments, e = 5. In some embodiments, e = 6. In some embodiments, e = 7. In some embodiments, e = 8. In some embodiments, e = 9. In some embodiments, e = 10. In some embodiments, e = 11. In some embodiments, e = 12. In some embodiments, e = 13. In some embodiments, e = 14. In some embodiments, e = 15. In some embodiments, e = 16. In some embodiments, e = 17. In some embodiments, e = 18. In some embodiments, e = 19. In some embodiments, e = 20. In some embodiments, e = 21. In some embodiments, e = 22. In some embodiments, e = 23. In some embodiments, e = 24. In some embodiments, e = 25. In some embodiments, e = 26. In some embodiments, e = 27. In some embodiments, e = 28.In some embodiments, e = 29. In some embodiments, e = 30. In some embodiments, e = 31. In some embodiments, e = 32. In some embodiments, e = 33. In some embodiments, e = 34. In some embodiments, e = 35. In some embodiments, e = 36. In some embodiments, e = 37. In some embodiments, e = 38. In some embodiments, e = 39. In some embodiments, e = 40. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0300] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0301] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0302] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0303] wherein the first and second chains each comprise the formula:

[0304] 5'-[E1] a -Y1-[S1] b -Y2-[E2] c -3', where

[0305] (a) E1 and E2 are each an exon sequence, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (T), wherein a and c are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence constituting E1 is in opposite orientation relative to the exon sequence constituting E2 and on opposite strands, and wherein the nucleotide sequences constituting E1 and E2 are not complementary;

[0306] (b) Y1 and Y2 each comprise a nucleotide sequence comprising a 5' splice site, wherein the nucleotide sequence of Y1 is in an opposite orientation relative to the nucleotide sequence of Y2 and is on opposite strands of the donor polynucleotide; and

[0307] (c) S1, if present, is a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein b is an integer whose value indicates the number of nucleotides comprised by the delimiter sequence, wherein b = 0 or 1-50,

[0308] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, E2 and Y2 constitute the sense strand, and E2 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, E1 and Y1 constitute the sense strand, and E1 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA,

[0309] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, b = 21. In some embodiments, b = 22. In some embodiments, b = 23. In some embodiments, b = 24. In some embodiments, b = 25. In some embodiments, b = 26. In some embodiments, b = 27. In some embodiments, b = 28. In some embodiments, b = 29. In some embodiments, b = 30. In some embodiments, b = 31. In some embodiments, b = 32. In some embodiments, b = 33. In some embodiments, b = 34. In some embodiments, b = 35. In some embodiments, b = 36. In some embodiments, b = 37. In some embodiments, b = 38. In some embodiments, b = 39. In some embodiments, b = 40. In some embodiments, b = 41. In some embodiments, b = 42. In some embodiments, b = 43. In some embodiments, b = 44. In some embodiments, b = 45. In some embodiments, b = 46. In some embodiments, b = 47. In some embodiments, b = 48. In some embodiments, b = 49. In some embodiments, b = 50. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length.In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides in length or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

[0310] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0311] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0312] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0313] wherein the first and second chains each comprise the formula:

[0314] 5'-[E1] a -Y1-[S1] b -Y2-[E2] c -3', where

[0315] (a) E1 and E2 are each an exon sequence, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (T), wherein a and c are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence constituting E1 is in opposite orientation relative to the exon sequence constituting E2 and on opposite strands, and wherein the nucleotide sequences constituting E1 and E2 are not complementary;

[0316] (b) Y1 and Y2 each comprise a nucleotide sequence comprising a 5' splice site, wherein the nucleotide sequence of Y1 is in an opposite orientation relative to the nucleotide sequence of Y2 and is on opposite strands of the donor polynucleotide; and

[0317] (c) S1, if present, is a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein b is an integer whose value indicates the number of nucleotides comprised by the delimiter sequence, wherein b = 0 or 1-50,

[0318] wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, E2 and Y2 constitute the sense strand, and E2 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, E1 and Y1 constitute the sense strand, and E1 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA,

[0319] And wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, thereby correcting the mutation. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, b = 0. In some embodiments, b = 1. In some embodiments, b = 2. In some embodiments, b = 3. In some embodiments, b = 4. In some embodiments, b = 5. In some embodiments, b = 6. In some embodiments, b = 7. In some embodiments, b = 8. In some embodiments, b = 9. In some embodiments, b = 10. In some embodiments, b = 11. In some embodiments, b = 12. In some embodiments, b = 13. In some embodiments, b = 14. In some embodiments, b = 15. In some embodiments, b = 16. In some embodiments, b = 17. In some embodiments, b = 18. In some embodiments, b = 19. In some embodiments, b = 20. In some embodiments, b = 21. In some embodiments, b = 22. In some embodiments, b = 23. In some embodiments, b = 24. In some embodiments, b = 25. In some embodiments, b = 26. In some embodiments, b = 27. In some embodiments, b = 28. In some embodiments, b = 29. In some embodiments, b = 30. In some embodiments, b = 31. In some embodiments, b = 32. In some embodiments, b = 33. In some embodiments, b = 34. In some embodiments, b = 35. In some embodiments, b = 36. In some embodiments, b = 37. In some embodiments, b = 38. In some embodiments, b = 39. In some embodiments, b = 40. In some embodiments, b = 41. In some embodiments, b = 42. In some embodiments, b = 43. In some embodiments, b = 44. In some embodiments, b = 45. In some embodiments, b = 46. In some embodiments, b = 47. In some embodiments, b = 48. In some embodiments, b = 49. In some embodiments, b = 50.

[0320] In some embodiments, the exon sequence comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the 3' splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the 5' splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the polypyrimidine tract comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the branch point sequence comprising the sense strand corrects the mutation.

[0321] In some embodiments, the 5'-most nucleotide of the donor polynucleotide comprises a 5' phosphate group. In some embodiments, the length of the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides. In some embodiments, the length of the donor polynucleotide is about 50-60 nucleotides. In some embodiments, the length of the donor polynucleotide is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. In some embodiments, the length of the donor polynucleotide is 40 nucleotides. In some embodiments, the length of the donor polynucleotide is 41 nucleotides. In some embodiments, the length of the donor polynucleotide is 42 nucleotides. In some embodiments, the length of the donor polynucleotide is 43 nucleotides. In some embodiments, the length of the donor polynucleotide is 44 nucleotides. In some embodiments, the length of the donor polynucleotide is 45 nucleotides. In some embodiments, the length of the donor polynucleotide is 46 nucleotides. In some embodiments, the donor polynucleotide is 47 nucleotides in length. In some embodiments, the donor polynucleotide is 49 nucleotides in length. In some embodiments, the donor polynucleotide is 42 nucleotides in length. In some embodiments, the donor polynucleotide is 50 nucleotides in length. In some embodiments, the donor polynucleotide is 51 nucleotides in length. In some embodiments, the donor polynucleotide is 52 nucleotides in length. In some embodiments, the donor polynucleotide is 53 nucleotides in length. In some embodiments, the donor polynucleotide is 54 nucleotides in length. In some embodiments, the donor polynucleotide is 55 nucleotides in length. In some embodiments, the donor polynucleotide is 56 nucleotides in length. In some embodiments, the donor polynucleotide is 57 nucleotides in length. In some embodiments, the donor polynucleotide is 58 nucleotides in length. In some embodiments, the donor polynucleotide is 59 nucleotides in length. In some embodiments, the donor polynucleotide is 60 nucleotides in length.

[0322] In some embodiments, the donor polynucleotide comprises natural nucleotides. In some embodiments, the donor polynucleotide comprises naturally occurring nucleotides. In some embodiments, the donor polynucleotide comprises one or more non-natural and / or modified nucleotides. In some embodiments, one or more non-natural and / or modified nucleotides are 2'-O-methyl nucleotides. In some embodiments, the donor polynucleotide comprises one or more backbone modifications. In some embodiments, one or more backbone modifications are phosphorothioates. In some embodiments, the donor polynucleotide comprises two blunt ends. In some embodiments, the donor polynucleotide comprises one blunt end and includes an end comprising an overhang (e.g., a 5' or 3' overhang). In some embodiments, the nucleotide sequence of the donor polynucleotide comprises one or more nucleotides that prevent the site-directed nuclease from recognizing and cutting the donor polynucleotide.

[0323] In some embodiments, the disease is glycogen storage disease 1a (GSD1a). In some embodiments, the mutation is located in the human G6PC gene on human chromosome 17q21. In some embodiments, the mutation in the G6PC gene results in an R83C, R83H, or E110K amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an R83C amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an R83H amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an E110K amino acid substitution in the human G6PC protein.

[0324] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a nucleotide sequence that corrects a mutation in a genomic DNA molecule (gDNA) in a cell, the mutation causing glycogen storage disease 1a, wherein the mutation is located in the human G6PC gene on human chromosome 17q21 and results in the amino acid substitution R83C or R83H, the donor polynucleotide comprising:

[0325] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising an exon sequence that corrects the mutation, and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from a genomic DNA molecule, wherein the exon sequence comprises a codon encoding arginine (R) corresponding to the codon at position 83 in the G6PC gene, wherein the one or more splicing signals are a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

[0326] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0327] wherein the donor polynucleotide is about 40-70 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each chain of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, wherein the insertion of the donor polynucleotide forms an exon in the gDNA comprising an exon sequence that corrects the mutation, wherein the splicing signal directs the exon to be included in the mRNA, thereby correcting the mutation. In some embodiments, the branch point sequence comprises the nucleotide sequence TTCAT, wherein the polypyrimidine tract comprises the nucleotide sequence CTTGTTCTGTTTTTTT, wherein the 3' splice site comprises the nucleotide sequence TAG, and wherein the exon sequence comprises the nucleotide sequence GATTCTCTTTGGACAGCGCCCTTACT.

[0328] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 30 (CH34 54-0). In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 20 (CH32 50-0).

[0329] In some embodiments, the disease is Pompe disease. In some embodiments, the mutation is located in the human glucosidase alpha (GAA) gene on human chromosome 17q25.3. In some embodiments, the mutation is in the splicing signal of GAA, which results in an mRNA transcript of the activated GAA gene lacking exon 2 and / or one or more cryptic splice sites.

[0330] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating dsDNA molecule comprising a nucleotide sequence that corrects a mutation in a gDNA molecule in a cell, the mutation causing Pompe disease, wherein the mutation is in a splicing signal of GAA that results in an mRNA transcript of an activated GAA gene lacking exon 2 and / or one or more cryptic splice sites, the donor polynucleotide comprising:

[0331] (i) a first strand comprising, from 5′ to 3′, a first nucleotide sequence that corrects the mutation, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

[0332] (ii) a second strand comprising, from 5′ to 3′, a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence, wherein the second strand is complementary to the first strand,

[0333] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each strand of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into a cell in combination with a site-directed nuclease, the NHEJ DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease proximal to the location of the mutation, wherein the donor polynucleotide is configured for bidirectional insertion into the DSB break, wherein insertion in either orientation forms a 3' splice site, a polypyrimidine tract, and a branch point sequence that corrects the mutation, wherein the splicing signal directs the inclusion of exon 2 into the mRNA, thereby correcting the mutation.

[0334] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 63 (GAA_50-0).

[0335] In some embodiments, the present disclosure provides a system for correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the system comprising the following components:

[0336] (a) any one of the aforementioned donor polynucleotides;

[0337] (b) one or more gRNA molecules; and

[0338] (c) site-directed nucleases,

[0339] Wherein when the system is introduced into the cell, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA at a position close to the mutation by the site-directed nuclease, thereby correcting the mutation. In some embodiments, the site-directed nuclease is encoded by mRNA. In some embodiments, the site-directed nuclease is a polypeptide. In some embodiments, one or more gRNA molecules and the site-directed nuclease comprise a ribonucleoprotein. In some embodiments, the site-directed nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Streptococcus pyogenes ( S. pyogenes)Cas9 (SpCas9), or a homologue, derivative or modified form thereof. In some embodiments, the Cas nuclease is Staphylococcus aureus ( S. aureus )Cas9 (SaCas9), or a homolog, derivative or modified form thereof.

[0340] In some embodiments, one or more gRNA molecules comprise a modification selected from the group consisting of backbone modifications, sugar modifications, modified internucleoside linkages, or modified or non-natural nucleobases. In some embodiments, one or more gRNA molecules comprise a backbone modification. In some embodiments, the backbone modification is a phosphorothioate modification.

[0341] In some embodiments of the systems provided by the present disclosure, the donor polynucleotide, gRNA molecule, and site-directed nuclease are individually formulated or co-formulated in liposomes or lipid nanoparticles. In some embodiments, the donor polynucleotide, gRNA molecule, and site-directed nuclease are individually formulated in liposomes or lipid nanoparticles. In some embodiments, the donor polynucleotide, gRNA molecule, and site-directed nuclease are co-formulated in liposomes or lipid nanoparticles. In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 107 (CH32 mutant-CTX1 sgRNA spacer). In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 108 (CH34 mutant-CTX1 sgRNA spacer). In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 127 (mutant GAA sgRNA spacer).

[0342] In some embodiments, the disclosure provides cells comprising any of the aforementioned donor polynucleotides or systems. In some embodiments, the cells are dividing cells or non-dividing cells. In some embodiments, the cells are dividing cells. In some embodiments, the cells are non-dividing cells. In some embodiments, the cells are patient-specific induced pluripotent stem cells (iPSCs). In some embodiments, the cells are primary hepatocytes.

[0343] In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned donor polynucleotides and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned systems and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned cells and a pharmaceutically acceptable carrier.

[0344] In some embodiments, the present disclosure provides any of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions for use in treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: isolating cells from the patient, contacting the cells with the donor polynucleotide, system, or pharmaceutical composition, wherein when the donor polynucleotide, system, or pharmaceutical composition contacts the cells, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

[0345] In some embodiments, the present disclosure provides any of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions for use in treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: administering to the patient an effective amount of the donor polynucleotide, system, or pharmaceutical composition, wherein upon administration of the donor polynucleotide, system, or composition, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

[0346] In some embodiments, the present disclosure provides a method of correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: contacting the cell with any of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein when the donor polynucleotide, system, or composition contacts the cell, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

[0347] In some embodiments, the present disclosure provides a method of treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in the cell, the method comprising: isolating cells from the patient, contacting the cells with any of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein when the donor polynucleotide, system, or composition contacts the cells, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

[0348] In some embodiments, the present disclosure provides methods of treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: administering to the patient an effective amount of any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein upon administration of the donor polynucleotide, system, or composition, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation (in vivo).

[0349] In some embodiments of the methods provided by the present disclosure, the cells are patient-specific induced pluripotent stem cells (iPSCs). In some embodiments, the cells are hepatocytes. In some embodiments of the methods provided by the present disclosure, the method further comprises differentiating the iPSCs comprising the corrected mutation into differentiated cells; and implanting the differentiated cells into the patient.

[0350] In some embodiments of the methods provided by the present disclosure, treatment results in translation of mRNA transcribed from a genomic DNA molecule (gDNA) comprising an inserted donor polynucleotide, wherein the translation results in the formation of a translation product (protein) that alleviates the disease or does not cause or promote the disease.

[0351] In some embodiments, the present disclosure provides a kit comprising a container comprising any of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions for correcting a mutation in a genomic DNA molecule (gDNA) in a cell and a package insert comprising instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0352] Figure 1 A schematic diagram is provided showing the endogenous locus in the target nucleic acid and the locus after insertion of the donor polynucleotide. The relative positions of splice signals, splice sites, introns, and exons are indicated.

[0353] Figures 2A-2C Provided are dot plots depicting the percentage of total sequencing reads corresponding to specific nucleotide changes (e.g., correction edits) encoded by the donor polynucleotide, indicating NHEJ-mediated insertion of the donor polynucleotide into the target gene (e.g., G6PC exon 2) as determined by next generation sequencing (NGS). Figures 2A-2C Depicts the effects of three different gRNAs on G6PC Percentage of corrected editing of DSBs in the exon 2 genomic DNA (gDNA) locus.

[0354] Figure 3Provided is a line graph showing the percentage of corrective edits resulting from NHEJ-mediated insertion of a donor polynucleotide at the CH34 sgRNA target site G6PC Insertion of a donor polynucleotide containing additional splicing signals and nucleotide changes in the ORF results in exon 2 within the gDNA locus (DNA). G6PC Proofreading edits in DNA (black lines) also lead to G6PC The gene is transcribed, which produces mRNA containing the desired predicted edit (grey line). The level of corrected edits is also shown as a function of the amount of donor polynucleotide used (ng of donor).

[0355] Figure 4A Provided are dot plots showing the percentage of total sequencing reads corresponding to corrective edits resulting from NHEJ-mediated insertion of a donor polynucleotide into mouse hepatocytes. G6PC The hepatocytes are transfected with a transgenic mouse model comprising a donor polynucleotide, mRNA encoding Cas9, and a targeting G6PC gRNA was processed in lipid nanoparticles (LNP). Figure 4B Dot plots are provided showing the expression of Cas9 mRNA, donor polynucleotide, and target G6PC Following in vivo administration of gRNA-LNPs, the percentage of corrected editing resulting from complete NHEJ-mediated insertion of the donor polynucleotide into the gDNA and transcribed mRNA.

[0356] Figure 5 A schematic diagram is provided describing the design of a bidirectional donor polynucleotide. The donor is designed to incorporate a splicing sequence when inserted into a DNA DSB in either the forward or reverse orientation.

[0357] Figures 6A-6B Provides a description for inserting GAA Schematic diagram of the design of bidirectional intragenic donor polynucleotides. Figure 6A Splicing signals in the sense and antisense strands of the dsODN donor polynucleotide are depicted. Figure 6B Insertion of the desired splicing signal is depicted regardless of whether the dsODN donor is inserted in the forward or reverse orientation GAA within the cleavage site.

[0358] Figure 7A and Figure 7C A bar graph is provided showing the percentage of corrected editing in cells treated with a unidirectional donor polynucleotide when inserted in the forward direction. G6PC ( Figure 7A )or GAA Gene( Figure 7C), the unidirectional donor polynucleotide encodes the correction edit. Figure 7B and Figure 7D A bar graph is provided showing the percentage of corrected editing in cells treated with a bidirectional donor polynucleotide when inserted in the forward or reverse direction. G6PC ( Figure 7B )or GAA Gene( Figure 7D ), the bidirectional donor polynucleotide encodes the correction edit.

[0359] Figure 8 Provided is a bar graph showing the percentage of corrective edits resulting from a 50 nt bidirectional donor insertion in primary human hepatocytes. GAA Depicted are the percentages of total sequence reads that resulted from perfect forward insertions, perfect reverse insertions, and total perfect insertions combined from the donor.

[0360] Figure 9 Provided are line graphs showing the percentage of corrective editing resulting from insertion of bidirectional donors of varying lengths into Huh-7 cells. GAA Shown are the percentages of corrective edits resulting from perfect insertions, and insertions in the forward (eg, perfect insertion) or reverse (eg, perfect reverse insertion) orientations within DSBs induced in genes.

[0361] Figures 10A-10C A bar graph showing the percentage of corrective edits resulting from insertion of a bidirectional donor into fibroblasts is provided. GAA The fibroblasts were derived from a patient with Pompe disease. Figure 10A Shown is the percentage of sequence reads with corrected edited gDNA encoded by the donor polynucleotide, as determined by NGS. Figure 10B Shown in is the quantification of mRNA transcripts encoding exon 2 normalized to the amount of mRNA transcripts lacking exon 2 as determined by quantitative PCR (qPCR). Figure 10C Shown in FIG is the percentage increase in the number of sequence reads encoding exon 2 in cells transfected with Cas9 / gRNA with or without a donor polynucleotide compared to mock-transfected cells, as determined by NGS. DETAILED DESCRIPTION

[0362] The leading pathways for repairing DNA double-strand breaks (DSBs) are non-homologous end joining (NHEJ) pathways and homology-directed repair (HDR) pathways (e.g., also referred to as homologous recombination or HR). By inducing site-specific DSBs in a target gene using a site-directed nuclease (e.g., CRISPR / Cas9 endonuclease), homologous donor polynucleotides can be used as templates to repair the introduction of gene editing by HDR. However, HDR repair is inefficient, particularly in non-dividing cells. Therefore, using NHEJ to repair a method for inducing the desired gene editing provided by a donor polynucleotide is advantageous for editing both non-dividing and dividing cells. The present disclosure is based, at least in part, on the design of a donor polynucleotide that uses an NHEJ repair pathway to insert into a target gene at a DSB induced by a site-directed nuclease (e.g., CRISPR / Cas9 endonuclease). Donor polynucleotides are designed for insertion into NHEJ-mediated target gene DSBs to correct pathogenic mutations located in exons (e.g., protein coding mutations) and introns (e.g., splicing signal mutations) that result in abnormal protein production or function. Therefore, provided herein are methods and compositions comprising donor polynucleotides that provide desired changes and / or regulatory exon definitions in the nucleotide sequence of genomic DNA (gDNA) such that the desired changes are included in RNA transcripts (e.g., pre-mRNA) transcribed from the edited gDNA.

[0363] Genome editing

[0364] Genome editing generally refers to the process of editing or changing the nucleotide sequence of a genome, preferably in an accurate, desired and / or predetermined manner. Examples of the compositions, systems and methods of genome editing described herein use site-directed nucleases to cut or cut DNA at a precise target position in the genome, thereby producing double-strand breaks (DSBs) in the DNA. Such breaks can be repaired by endogenous DNA repair pathways, such as homology-guided repair (HDR) and / or non-homologous end joining (NHEJ) repair (see, for example, Cox et al. (2015) Nature Medicine 21 (2): 121-31). One of the main obstacles to effective genome editing in non-dividing cells is the lack of homology-guided repair (HDR). If there is no HDR, non-dividing cells rely on non-homologous end joining (NHEJ) to repair the double-strand breaks (DSBs) that occur in the genome. The result of the DNA repair of the DSB mediated by NHEJ can include the correction repair of DSB or the deletion or insertion of one or more nucleotides or polynucleotides.

[0365] Donor polynucleotide

[0366] In humans, exon definition is determined by splice sites that span exons rather than intron pairing. Other splicing signals, including but not limited to branch point sequences, polypyrimidine tracts, and exon and intron splicing enhancers and silencers, also facilitate the proper splicing of exons together to form mature mRNA. During pre-mRNA splicing, the splicing mechanism searches for a pair of closely spaced splice sites (i.e., upstream 3' splice site and downstream 5' splice site) with exon polarity (Berget (1995) J Biol Chem 270: 2411-2414).

[0367] Accordingly, the present disclosure provides donor polynucleotides that, after being inserted into a DSB, correct or induce a mutation in a target nucleic acid (e.g., genomic DNA), and comprise splicing signals (e.g., one or more splicing signals) that, through exon-limited regulation, allow the splicing mechanism to partially incorporate the corrected mutation (or induced mutation) into a transcription product (e.g., pre-mRNA). In some embodiments, the donor polynucleotides provided by the present disclosure are recognized by the NHEJ mechanism of the cell and are used to repair a double-strand break (DSB) introduced into a target nucleic acid by a site-directed nuclease, wherein the repair of the DSB results in the insertion of the donor polynucleotide into the target nucleic acid. In some embodiments, the donor polynucleotides are configured for unidirectional insertion into a DSB. In some embodiments, the donor polynucleotides are configured for bidirectional insertion into a DSB.

[0368] Unidirectional donor polynucleotide

[0369] In some embodiments, the donor polynucleotide provided by the present disclosure is a linear, non-replicating double-stranded DNA molecule (dsDNA) comprising a nucleotide sequence that corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and a nucleotide sequence that comprises one or more splicing signals to control the processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), which is introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide comprises a first DNA strand and a second DNA strand, wherein the second DNA strand comprises a nucleotide sequence complementary to the first DNA strand.

[0370] In some embodiments, the donor polynucleotide comprises a nucleotide sequence that corrects or induces mutation, wherein the nucleotide sequence that corrects or induces mutation comprises a single nucleotide. In some embodiments, the nucleotide sequence that corrects or induces mutation comprises two or more nucleotides. In some embodiments, the nucleotide sequence that corrects or induces mutation comprises a codon. In some embodiments, the nucleotide sequence that corrects or induces mutation comprises one or more codons. In some embodiments, the nucleotide sequence that corrects or induces mutation comprises an exon sequence.

[0371] In some embodiments, the donor polynucleotide comprises a nucleotide sequence that corrects or induces a mutation, wherein the nucleotide sequence that corrects or induces a mutation comprises an intron sequence. In some embodiments, the nucleotide sequence that corrects or induces a mutation comprises a splicing signal.

[0372] In some embodiments, the donor polynucleotide sequence is identical or substantially identical to the endogenous sequence of the target nucleic acid (having at least one nucleotide difference). In some embodiments, the endogenous sequence comprises the genomic sequence of the cell. In some embodiments, the endogenous sequence comprises a chromosome or extrachromosomal sequence. In some embodiments, the sequence included in the donor polynucleotide sequence is substantially identical to a portion of the endogenous sequence at or near the DSB in the cell (comprising at least one nucleotide difference / change). In some embodiments, repairing the target nucleic acid molecule with a donor polynucleotide results in the insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides results in one or more amino acid changes in the protein expressed by the gene comprising the target sequence. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides results in one or more nucleotide changes in the RNA expressed by the target gene. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides changes the expression level of the target gene. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides results in an increase or decrease in the expression of the target gene. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides results in a gene knockdown. In some embodiments, the insertion, deletion, or substitution of one or more nucleotides results in a gene knockout. In some embodiments, repair of the target nucleic acid molecule with a donor polynucleotide results in replacement of an exonic sequence, an intronic sequence, a transcriptional control sequence, a translational control sequence, a sequence containing a splicing signal, or a non-coding sequence of the target gene.

[0373] In some embodiments, the donor polynucleotide has a suitable length to correct or induce the mutation in gDNA. In some embodiments, the donor polynucleotide comprises a length of 10, 15, 20, 25, 50, 75, 100 or more nucleotides. In some embodiments (such as those described herein, wherein the donor polynucleotide is incorporated into the nucleic acid of cutting as an insertion mediated by non-homologous end connection), the donor polynucleotide does not have a homology arm. In some embodiments, the length of the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides. In some embodiments, the length of the donor polynucleotide is about 10-500 nucleotides. In some embodiments, the length of the donor polynucleotide is about 10-400 nucleotides. In some embodiments, the length of the donor polynucleotide is about 10-300 nucleotides. In some embodiments, the length of the donor polynucleotide is about 10-200 nucleotides. In some embodiments, the length of the donor polynucleotide is about 10-100 nucleotides. In some embodiments, the length of the donor polynucleotide is about 20-80 nucleotides. In some embodiments, the length of the donor polynucleotide is about 30-70 nucleotides. In some embodiments, the length of the donor polynucleotide is about 40-60 nucleotides. In some embodiments, the length of the donor polynucleotide is 40, 41, 42, 43, 44, 45, 46, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides. In some embodiments, the length of the donor polynucleotide is 40 nucleotides. In some embodiments, the length of the donor polynucleotide is 41 nucleotides. In some embodiments, the length of the donor polynucleotide is 42 nucleotides. In some embodiments, the length of the donor polynucleotide is 43 nucleotides. In some embodiments, the donor polynucleotide is 44 nucleotides in length. In some embodiments, the donor polynucleotide is 45 nucleotides in length. In some embodiments, the donor polynucleotide is 46 nucleotides in length. In some embodiments, the donor polynucleotide is 47 nucleotides in length. In some embodiments, the donor polynucleotide is 48 nucleotides in length. In some embodiments, the donor polynucleotide is 49 nucleotides in length. In some embodiments, the donor polynucleotide is 50 nucleotides in length. In some embodiments, the donor polynucleotide is 51 nucleotides in length. In some embodiments, the donor polynucleotide is 52 nucleotides in length. In some embodiments, the donor polynucleotide is 53 nucleotides in length. In some embodiments, the donor polynucleotide is 54 nucleotides in length. In some embodiments, the donor polynucleotide is 55 nucleotides in length.In some embodiments, the donor polynucleotide is 56 nucleotides in length. In some embodiments, the donor polynucleotide is 57 nucleotides in length. In some embodiments, the donor polynucleotide is 58 nucleotides in length. In some embodiments, the donor polynucleotide is 59 nucleotides in length. In some embodiments, the donor polynucleotide is 60 nucleotides in length.

[0374] In some embodiments, the donor polynucleotides provided by the present disclosure comprise intron sequences. In some embodiments, the donor polynucleotides comprise intron sequences that correct or induce mutations in gDNA. In some embodiments, the donor polynucleotides comprise exon sequences. In some embodiments, the donor polynucleotides comprise exon sequences that correct or induce mutations in gDNA.

[0375] In some embodiments, donor polynucleotides provided by the present disclosure comprise one or more splicing signals to control the processing (e.g., splicing) of a precursor mRNA (pre-mRNA) transcribed from the gDNA into which the donor polynucleotide is inserted.

[0376] In some embodiments, the donor polynucleotides provided by the present disclosure comprise one or more splicing signals to control the processing (e.g., splicing) of precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are selected from:

[0377] (a) natural or enhanced 3' splice site;

[0378] (b) native or enhanced 5' splice site;

[0379] (c) Polypyrimidine tablets;

[0380] (d) branching points;

[0381] (e) exonic splicing enhancer (ESE);

[0382] (f) intronic splicing enhancer (ISE);

[0383] (g) exonic splicing silencer (ESS);

[0384] (h) intronic splicing silencers (ISSs); and

[0385] (i) A combination of any one of (a) to (h).

[0386] In some embodiments, the donor polynucleotide comprises a natural or enhanced 3' splice site. In some embodiments, the donor polynucleotide comprises a naturally occurring 3' splice site. In some embodiments, the donor polynucleotide comprises an enhanced 3' splice site. A natural 3' splice site is a naturally occurring 3' splice site. In some embodiments, the nucleotide sequence of a natural 3' splice site is identical or substantially identical (having at least one nucleotide difference) to the nucleotide sequence of a 3' splice site at or near the donor polynucleotide insertion site. The enhanced 3' splice site is a splice site comprising a nucleotide sequence identical or substantially identical (having at least one nucleotide difference) to the consensus sequence of the 3' splice site. The consensus sequence of the 3' splice site is the nucleotide sequence YAG, and wherein Y is a nucleotide comprising a core base selected from thymine (T) and cytosine (C). See, for example, Reed (1989) Genes Dev 3 (12B): 2113-2123, which further describes the organization of the 3' splice site sequence in mammals.

[0387] In some embodiments, the donor polynucleotide comprises a natural or enhanced 5' splice site. In some embodiments, the donor polynucleotide comprises a natural 5' splice site. In some embodiments, the donor polynucleotide comprises an enhanced 5' splice site. In some embodiments, the natural 5' splice site is a naturally occurring 5' splice site. In some embodiments, the nucleotide sequence of the natural 5' splice site is equal to or substantially equal to (having at least one nucleotide difference) the nucleotide sequence of the donor polynucleotide insertion site or near the 5' splice site. In some embodiments, the enhanced 5' splice site is a splice site comprising a nucleotide sequence that is equal to or substantially equal to (having at least one nucleotide difference) the consensus sequence of the 5' splice site. The consensus sequence of the 5' splice site is the nucleotide sequence GTRAG, and wherein R is a nucleotide comprising a core base selected from adenine (A) and guanine (G).

[0388] In some embodiments, the donor polynucleotide comprises a polypyrimidine tract. A polypyrimidine tract is a cis-acting sequence element that directs intron removal in pre-mRNA splicing. Gradual deletion of a polypyrimidine tract has been found to abolish correct lariate formation, spliceosome assembly, and splicing. Studies have shown that pyrimidine tracts containing consecutive uridines are strong pyrimidine tracts (Coolidge et al. (1997) Nucleic Acids Res 25(4):888-896). In some embodiments, the polypyrimidine tract is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides in length. In some embodiments, the polypyrimidine tract is 16 nucleotides in length. In some embodiments, the polypyrimidine tract is 13 nucleotides in length. In some embodiments, the polypyrimidine tract comprises the nucleotide sequence TTTTTTTCTTTTTT (SEQ ID NO: 54). In some embodiments, the polypyrimidine tract is 9 nucleotides in length.In some embodiments, the polypyrimidine tract comprises the nucleotide sequence TTTTTTTCT (SEQ ID NO: 53).

[0389] In some embodiments, the donor polynucleotide comprises a branch point. In some embodiments, the donor polynucleotide comprises a nucleotide sequence comprising a branch point. In higher eukaryotes, pre-mRNA splicing is mediated by degenerate splicing cis elements, which comprise a branch point sequence (BPS), a polypyrimidine tract (PPT), 5' and 3' splice sites, and exon / intron splicing enhancers / silencers, and pre-mRNA is spliced ​​in two sequential transesterification reactions mediated by the spliceosome. In some embodiments, the donor polynucleotide comprises a branch point comprising a nucleotide sequence that is identical or substantially identical (having at least one nucleotide difference) to a branch point consensus sequence. In some embodiments, the branch point consensus sequence is YTNAY, wherein Y = C or T, and wherein N = A, G, C, or T (SEQ ID NO: 49. In some embodiments, the branch point sequence is TATTAAC (SEQ ID NO: 50). In some embodiments, the branch point sequence is GTTAATA (SEQ ID NO: 51). In some embodiments, the branch point sequence is TACTGAC (SEQ ID NO: 52).

[0390] In some embodiments, the donor polynucleotide comprises an exonic splicing enhancer (ESE) (see, e.g., Blencowe (2000) Trends Biochem Sci 25(3): 106-110). In some embodiments, the donor polynucleotide comprises an exonic splicing silencer (ESS) (see, e.g., Wang et al. (2006) Mol Cell 23(1): 61-70). In some embodiments, the donor polynucleotide comprises an intronic splicing enhancer (ISE) (see, e.g., Wang et al. (2012) Nat Struct Mol Biol 19(10): 1044-1052). In some embodiments, the donor polynucleotide comprises an intronic splicing silencer (ISS) (see, e.g., Carstens et al. (2000) Mol Cell Biol 20(19): 7388-7400).

[0391] In some embodiments, the donor polynucleotide comprises a natural or enhanced 3' splice site and a polypyrimidine tract. In some embodiments, the donor polynucleotide comprises a natural or enhanced 3' splice site, a polypyrimidine tract, and a branch point.

[0392] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0393] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0394] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0395] (a) natural or enhanced 3' splice site;

[0396] (b) Polypyrimidine tablets;

[0397] (c) branching points;

[0398] (d) exonic splicing enhancer (ESE);

[0399] (e) intronic splicing enhancer (ISE);

[0400] (f) exonic splicing silencer (ESS);

[0401] (g) intronic splicing silencers (ISSs); and

[0402] (h) A combination of any one of (a) to (g),

[0403] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0404] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0405] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA (gDNA) molecule in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 5' splice site, wherein the first strand comprises intron sequences and exon sequences, wherein the exon sequences correct the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0406] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0407] wherein the one or more splicing signals are selected from:

[0408] (a) natural or enhanced 5' splice site;

[0409] (b) exonic splicing enhancer (ESE);

[0410] (c) intronic splicing enhancer (ISE);

[0411] (d) exonic splicing silencer (ESS);

[0412] (e) intronic splicing silencers (ISSs); and

[0413] (f) A combination of any one of (a) to (e),

[0414] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0415] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0416] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 3' splice site; and

[0417] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0418] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0419] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0420] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site and a polypyrimidine tract; and

[0421] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0422] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0423] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0424] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals are a combination of a native or enhanced 3' splice site, a polypyrimidine tract, and a branch point; and

[0425] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0426] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0427] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0428] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 3′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0429] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0430] wherein the one or more splicing signals are selected from:

[0431] (a) Polypyrimidine tablets;

[0432] (b) branching points;

[0433] (c) exonic splicing enhancer (ESE);

[0434] (d) intronic splicing enhancer (ISE);

[0435] (e) exonic splicing silencer (ESS);

[0436] (f) intronic splicing silencers (ISSs); and

[0437] (g) A combination of any one of (a) to (f),

[0438] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0439] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0440] (i) a first strand comprising, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the donor polynucleotide comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a native or enhanced 5' splice site; and

[0441] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0442] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0443] In some embodiments, insertion of the donor polynucleotide into the DSB results in exon formation in the gDNA comprising the exonic sequence. In some embodiments, one or more splicing signals direct the inclusion of the exon comprising the exonic sequence into the mRNA.

[0444] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0445] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is in the 5′ splice site, wherein the first strand comprises an intron sequence, optionally an exon sequence, wherein the intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0446] (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals are selected from the group consisting of:

[0447] (a) Exonic splicing enhancer (ESE);

[0448] (b) intronic splicing enhancer (ISE);

[0449] (c) exonic splicing silencer (ESS);

[0450] (d) intronic splicing silencers (ISSs); and

[0451] (e) A combination of any one of (a) to (d),

[0452] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) that is introduced into the gDNA proximal to the location of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0453] In some embodiments, insertion of the donor polynucleotide results in the formation of an intron comprising an intron sequence, wherein the intron sequence corrects the mutation.

[0454] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the first strand comprises the formula:

[0455] 5'-[B]a-[S1]b-[P]c-[S2]dXE-3', where

[0456] (i) B, if present, is a branchpoint sequence comprising a nucleotide sequence that conforms to the branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a is an integer whose value indicates the number of nucleotides comprised by B, wherein a=0 or 5-7;

[0457] (ii) P is a polypyrimidine tract comprising a nucleotide sequence comprising nucleobases selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by P, wherein c = 9-20, wherein the nucleotide sequence comprising P is about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases;

[0458] (iii) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0459] (iv) X is a nucleotide sequence comprising a 3' splice site; and

[0460] (v) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0461] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, B (if present), P, X (if present), and E (if present) comprise the sense strand, wherein B (if present), P, and X (if present) comprise one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0462] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0463] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprises, from 5' to 3', a nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in a cell, wherein the pathogenic mutation is a protein-coding mutation proximal to a 5' splice site, wherein the first strand comprises an intron sequence and an exon sequence, wherein the exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA,

[0464] wherein the first chain comprises the formula:

[0465] 5'-EYI-3', where

[0466] (i) E is an exon sequence comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C);

[0467] (ii) Y is a nucleotide sequence comprising a 5' splice site; and

[0468] (iii) I, if present, comprises an intron sequence comprising nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C),

[0469] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directed insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, E (if present), Y, and I (if present) comprise the sense strand, wherein Y comprises one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0470] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0471] Bidirectional donor polynucleotide

[0472] In some embodiments, the donor polynucleotide provided by the present disclosure is a linear dsDNA molecule comprising two free DNA ends. Thus, insertion of the donor polynucleotide into the DSB by the cell's NHEJ machinery can occur in one of two orientations: forward and reverse. Accordingly, in some aspects, the present disclosure provides donor polynucleotides configured for bidirectional insertion into a DSB introduced into gDNA by a site-directed nuclease, wherein when inserted into the DSB in either orientation, the donor polynucleotide corrects or induces mutations in a genomic DNA (gDNA) molecule in the cell and provides one or more splicing signals to control the processing of precursor mRNA (pre-mRNA) transcribed from the gDNA.

[0473] In some embodiments, the donor polynucleotide comprises a first splice signal comprising a branch point sequence, wherein the donor polynucleotide comprises:

[0474] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first branch point sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0475] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second branch point sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0476] Wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70 or about 40-60 nucleotides in length. In some embodiments, the nucleotide sequences of the first branch point sequence and the second branch point sequence conform to the branch point consensus sequence, and the nucleotide sequences of the first branch point sequence and the second branch point sequence are complementary. In some embodiments, the branch point consensus sequence is YTNAY (SEQ ID NO: 49), wherein Y is a nucleotide comprising a cytosine (C) or thymine (T) nucleobase, and wherein N is a nucleotide comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C). In some embodiments, the first branch point sequence is TATTAAC (SEQ ID NO: 50). In some embodiments, the second branch point sequence is GTTAATA (SEQ ID NO: 51). In some embodiments, the second branch point sequence is TACTGAC (SEQ ID NO: 52).

[0477] In some embodiments, the donor polynucleotide comprises a second splicing signal comprising a polypyrimidine tract, wherein the first strand comprises a first polypyrimidine tract downstream of the first branch point sequence; and the second strand comprises a second polypyrimidine tract downstream of the second branch point sequence.

[0478] In some embodiments, the nucleotide sequence comprising the first polypyrimidine tract and the second polypyrimidine tract each comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), and wherein the nucleotide sequence is about 100%, about 90%-100%, or about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is TTTTTTTCT (SEQ ID NO: 53). In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is TTTTTTTCTTTTTT (SEQ ID NO: 54). In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is CTTCTTCTCTTCTTCC (SEQ ID NO: 55).

[0479] In some embodiments, the first branch point sequence and the first polypyrimidine tract are adjacent to each other. In some embodiments, the second branch point sequence and the second polypyrimidine tract are adjacent to each other.

[0480] In some embodiments, the donor polynucleotide comprises a third splice signal comprising a 3' splice site, wherein the first strand comprises a nucleotide sequence comprising a first 3' splice site downstream of the first polypyrimidine tract, and the second strand comprises a nucleotide sequence comprising a second 3' splice site downstream of the second polypyrimidine tract. In some embodiments, the first 3' splice site and the second 3' splice site comprise the nucleotide sequence YAG, and wherein Y is a nucleotide comprising a nucleobase selected from thymine (T) and cytosine (C).

[0481] In some embodiments, the donor polynucleotide comprises a coding sequence, wherein the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first nucleotide sequence that corrects the mutation in the gDNA comprises the first coding sequence, wherein the second nucleotide sequence that corrects the mutation in the gDNA comprises the second coding sequence, wherein the first coding sequence is located downstream of a first 3' splice site, and wherein the second coding sequence is located downstream of a second 3' splice site. In some embodiments, the nucleotide sequence comprising the first coding sequence and the second coding sequence comprises a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C). In some embodiments, the coding sequences and / or splicing signals comprising (i) and (ii) are non-identical or non-complementary to reduce self-annealing.

[0482] In some embodiments, the donor polynucleotides provided by the present disclosure comprise one or more delimiter sequences comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or 1 nucleotide in length. In some embodiments, the one or more delimiter sequences are located between a first branch point sequence and a second branch point sequence. In some embodiments, the one or more delimiter sequences are located between a first branch point sequence and a first polypyrimidine stretch. In some embodiments, the one or more delimiter sequences are located between a second branch point sequence and a second polypyrimidine stretch.

[0483] In some embodiments, the donor polynucleotide is configured for bidirectional insertion into a DSB introduced into the gDNA by a site-directed nuclease, wherein the first splice signal and the second splice signal, optionally the third splice signal, and the coding sequence constitute the sense strand when the donor polynucleotide is inserted into the DSB in either orientation, thereby correcting the mutation and providing one or more splice signals to control processing of pre-mRNA transcribed from the gDNA.

[0484] In some embodiments, the present disclosure provides a donor polynucleotide comprising one or more splice signals comprising a 5' splice site, wherein the donor polynucleotide comprises:

[0485] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising a first 5′ splice site sequence and a first nucleotide sequence that corrects the mutation in the gDNA; and

[0486] (ii) a second strand comprising, from 5′ to 3′, a nucleotide sequence comprising a second 5′ splice site sequence and a second nucleotide sequence that corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

[0487] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the first strand comprises a first coding sequence and the second strand comprises a second coding sequence, wherein the first coding sequence is located upstream of a first 5' splice site, and wherein the second coding sequence is located upstream of a second 5' splice site, and wherein the coding sequences in the first strand and the second strand are non-complementary (or comprise one, two, three, four, or more mismatches) to reduce self-annealing. In some embodiments, the donor polynucleotide comprises a delimiter sequence between a first 5' splice site and a second 5' splice site, wherein the delimiter sequence comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of: adenine (A), guanine (G), thymine (T), and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or 1 nucleotides in length.

[0488] In some embodiments, the donor polynucleotide is configured for bidirectional insertion into a DSB, wherein when the donor polynucleotide is inserted into the DSB in a first orientation, the first 5' splice site and the first coding sequence constitute the sense strand, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, and wherein when the donor polynucleotide is inserted into the DSB in a second orientation, the second 5' splice site and the second coding sequence constitute the sense strand, thereby correcting the mutation and providing one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA.

[0489] In some embodiments, the donor polynucleotide comprises a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0490] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0491] (ii) a second strand comprising, from 5' to 3', a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0492] 5'-[P1]a-[S1]b-[B]c-[S2]d-[P2]e-3', where

[0493] (a) B comprises a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprised by B, wherein c = 5-7;

[0494] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T), and cytosine (C), wherein a and e are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a=9-20 and e=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, and wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide; and

[0495] (c) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and d = 0-20,

[0496] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B and P2 constitute the sense strand, and B and P2 comprise the first splicing signal and the second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B and P1 constitute the sense strand, and B and P2 comprise the first splicing signal and the second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0497] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0498] In some embodiments, the donor polynucleotide comprises a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0499] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is proximal to a 3' splice site, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0500] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0501] 5'-[P1]a-[S1]b-[B1]c-[S2]d-[B2]e-[S3]f-[P2]g-3', where

[0502] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein c and e are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein c = 0 or 5-7, wherein e = 5-7;

[0503] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein a=9-20 and g=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide; and

[0504] (c) S1, S2, and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b, d, and f are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein b and f = 1-20, and wherein d = 1-40,

[0505] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2 and P2 constitute the sense strand, and B2 and P2 comprise a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1 and P1 constitute the sense strand, and B1 and P1 provide a first splicing signal and a second splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0506] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0507] In some embodiments, the donor polynucleotide comprises a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0508] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0509] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:

[0510] 5'-[E1]a-X1-[P1]b-[S1]c-[B]d-[S2]e-[P2]f-X2-[E2]g-3', where

[0511] (a) B is a branchpoint sequence comprising a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein d is an integer whose value indicates the number of nucleotides comprised by B, wherein d = 5-7;

[0512] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b and f are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and f=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and is on opposite strands of the donor polynucleotide;

[0513] (c) E1 and E2 are each exonic sequences, each comprising a nucleotide sequence that corrects the mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and g are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exonic sequence comprising E1 is in opposite orientation relative to the exonic sequence comprising E2 and is on opposite strands of the donor polynucleotide, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0514] (d) X1 and X2 are each a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence constituting X1 is in the opposite orientation relative to the nucleotide sequence constituting X2 and is on opposite strands; and

[0515] (e) S1 and S2 (if either is present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein c and e are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c and e = 1-20,

[0516] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B, P2, E2, and X2 constitute the sense strand, wherein B, P2, and X2 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B, P1, E1, and X1 constitute the sense strand, wherein B, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA,

[0517] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0518] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0519] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0520] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0521] wherein the first and second chains each comprise the formula:

[0522] 5'-[E1]a-X1-[P1]b-[S1]c-[B1]d-[S2]e-[B2]f-[S3]g-[P2]h-X2-[E2]i-3', where

[0523] (a) B1 (if present) and B2 are each a branchpoint sequence, wherein B2 comprises a nucleotide sequence that conforms to a branchpoint consensus sequence on each strand of the donor polynucleotide, wherein the branchpoint sequence comprising B1 (if present) is in an opposite orientation relative to the branchpoint sequence comprising B2 and is on opposite strands of the donor polynucleotide, wherein B1 and B2 each comprise a nucleotide sequence comprising a nucleobase selected from the group consisting of: adenine, guanine, thymine, and cytosine, wherein d and f are integers whose values ​​indicate the number of nucleotides comprised by B1 and B2, respectively, wherein d and f = 5-7;

[0524] (b) P1 and P2 are polypyrimidine tracts, each comprising a nucleotide sequence comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b and h are integers whose values ​​indicate the number of nucleotides comprised by P1 and P2, respectively, wherein b=9-20 and h=9-20, wherein the nucleotide sequences comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90%, pyrimidine nucleobases, wherein P1 is in opposite orientation relative to P2 and on opposite strands of the donor polynucleotide;

[0525] (c) E1 and E2 are each exon sequences, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and i are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence comprising E1 is in opposite orientation relative to the exon sequence comprising E2 and on opposite strands, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0526] (d) X1 and X2 each comprise a nucleotide sequence comprising a 3' splice site, wherein the nucleotide sequence of X1 is in an opposite orientation relative to the nucleotide sequence of X2 and is on opposite strands of the donor polynucleotide; and

[0527] (e) S1, S2, and S3 (if any are present) are each a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein c, e, and g are each integers whose values ​​indicate the number of nucleotides comprised by the delimiter sequence, wherein c and g = 1-20, and wherein e = 1-40,

[0528] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2, P2, E2, and X2 constitute the sense strand, wherein B2, P2, and X2 each comprise a first splicing signal, a second splicing signal, and a third splicing signal, thereby correcting the mutation and providing splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in the second orientation, B1, P1, E1, and X1 constitute the sense strand, wherein B1, P1, and X1 comprise the first splicing signal, the second splicing signal, and the third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of the pre-mRNA transcribed from the gDNA,

[0529] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0530] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA) comprising

[0531] (i) a first strand comprising, from 5' to 3', a first nucleotide sequence that corrects a pathogenic mutation in a genomic DNA molecule (gDNA) in the cell, wherein the pathogenic mutation is a protein-coding mutation near a 3' splice site, wherein the first strand comprises a first intron sequence and a first exon sequence, wherein the first exon sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

[0532] (ii) a second strand comprising, from 5′ to 3′, a second nucleotide sequence that corrects a pathogenic mutation in a gDNA in the cell, wherein the pathogenic mutation is a protein-coding mutation near the 3′ splice site, wherein the second strand comprises a second intron sequence and a second exon sequence, wherein the second exon sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

[0533] wherein the first and second chains each comprise the formula:

[0534] 5'-[E1]a-Y1-[S1]b-Y2-[E2]c-3', where

[0535] (a) E1 and E2 are each exon sequences, each comprising a nucleotide sequence that corrects a mutation, wherein the nucleotide sequence comprises a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein a and c are integers whose values ​​indicate the number of nucleotides comprised by E1 and E2, respectively, wherein the exon sequence comprising E1 is in opposite orientation relative to the exon sequence comprising E2 and on opposite strands, and wherein the nucleotide sequences comprising E1 and E2 are not complementary;

[0536] (b) Y1 and Y2 each comprise a nucleotide sequence comprising a 5' splice site, wherein the nucleotide sequence of Y1 is in an opposite orientation relative to the nucleotide sequence of Y2 and is on opposite strands of the donor polynucleotide; and

[0537] (c) S1, if present, is a delimiter sequence comprising one or more nucleotides comprising a nucleobase selected from the group consisting of adenine, guanine, thymine, and cytosine, wherein b is an integer whose value indicates the number of nucleotides comprised by the delimiter sequence, wherein b = 1-50,

[0538] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bidirectional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, E2 and Y2 constitute the sense strand, and E2 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, E1 and Y1 constitute the sense strand, and E1 comprises a splicing signal, thereby correcting the mutation and providing a splicing signal to control processing of a pre-mRNA transcribed from the gDNA,

[0539] And wherein when the donor polynucleotide is introduced into the cell in combination with the site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB, which is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation.

[0540] In some embodiments, the exon sequence comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the 3' splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the 5' splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the polypyrimidine tract comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising the branch point sequence comprising the sense strand corrects the mutation.

[0541] In some embodiments, the 5'-most nucleotide of each strand of any donor polynucleotide provided by the present disclosure comprises a 5' phosphate group. In some embodiments, the donor polynucleotide comprises two blunt ends. In some embodiments, the donor polynucleotide comprises one blunt end and includes one end comprising an overhang (e.g., a 5' or 3' overhang).

[0542] In some embodiments, the donor polynucleotide comprises a nucleotide sequence comprising one or more nucleotides that prevent the site-directed nuclease from recognizing and cleaving the donor polynucleotide.

[0543] In some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a nucleotide sequence that corrects a mutation in a genomic DNA molecule (gDNA) in a cell, the mutation causing glycogen storage disease 1a, wherein the mutation is located in the human G6PC gene on human chromosome 17q21 and results in the amino acid substitution R83C or R83H, the donor polynucleotide comprising:

[0544] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising an exon sequence that corrects the mutation, and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from a genomic DNA molecule, wherein the exon sequence comprises a codon encoding arginine (R) corresponding to the codon at position 83 in the G6PC gene, wherein the one or more splicing signals are a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

[0545] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0546] wherein the donor polynucleotide is about 40-70 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each chain of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, wherein the insertion of the donor polynucleotide forms an exon in the gDNA comprising an exon sequence that corrects the mutation, wherein the splicing signal directs the exon to be included in the mRNA, thereby correcting the mutation. In some embodiments, the branch point sequence comprises the nucleotide sequence TTCAT, wherein the polypyrimidine tract comprises the nucleotide sequence CTTGTTCTGTTTTTTT, wherein the 3' splice site comprises the nucleotide sequence TAG, and wherein the exon sequence comprises the nucleotide sequence GATTCTCTTTGGACAGCGCCCTTACT.

[0547] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 30 (CH34 54-0).

[0548] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 20 (CH32 50-0).

[0549] Methods for preparing and testing donor polynucleotides

[0550] The donor polynucleotides provided by the present disclosure are produced by suitable DNA synthesis methods or means known in the art. DNA synthesis is the natural or artificial production of deoxyribonucleic acid (DNA) molecules. The term DNA synthesis refers to DNA replication, DNA biosynthesis (e.g., in vivo DNA amplification), enzymatic DNA synthesis (e.g., polymerase chain reaction (PCR); in vitro DNA amplification), or chemical DNA synthesis.

[0551] In some embodiments, each strand of the donor polynucleotide is produced by oligonucleotide synthesis. Oligonucleotide synthesis is the chemical synthesis of relatively short single-stranded nucleic acid fragments or chains with a defined chemical structure (sequence). Methods for oligonucleotide synthesis are known in the art (see, for example, Reese (2005) Organic & Biomolecular Chemistry 3(21): 3851). The two strands can then be annealed together or duplexed to form the donor polynucleotide.

[0552] In some aspects, the insertion of the donor polynucleotide into the DSB is determined by suitable methods known in the art. For example, after the insertion event, the nucleotide sequence of the PCR amplicon generated using PCR primers flanking the DSB site is analyzed for the presence of the nucleotide sequence comprising the donor polynucleotide. Next generation sequencing (NGS) technology is used to determine the extent to which the donor polynucleotide inserts into the DSB by analyzing the PCR amplicon for the presence or absence of the donor polynucleotide sequence. Further, since each donor polynucleotide is a linear dsDNA molecule, it can be inserted in either of two orientations, so NGS analysis can be used to determine the extent of insertion of the donor polynucleotide in either direction.

[0553] In some aspects, the insertion of the donor polynucleotide and its ability to correct the mutation are determined by nucleotide sequence analysis of mRNA transcribed from the gDNA into which the donor polynucleotide is inserted. The mRNA transcribed from the gDNA containing the inserted donor polynucleotide is analyzed by suitable methods known in the art. For example, mRNA extracted from cells (which are treated or contacted with the donor polynucleotide or system provided by the present disclosure) is enzymatically converted into cDNA, which is further analyzed by NGS analysis to determine the extent of mRNA molecules containing the corrected mutation.

[0554] In other aspects, the insertion of the donor polynucleotide and its ability to correct mutations are determined by protein sequence analysis of polypeptides translated from mRNA transcribed from the gDNA into which the donor polynucleotide is inserted. In some embodiments, the donor polynucleotide corrects or induces mutations by incorporating codons into exons that make amino acid changes in the gene containing the gDNA molecule, wherein translation of the mRNA from the gene containing the inserted donor polynucleotide generates a polypeptide containing the amino acid change. The amino acid changes in the polypeptide are determined by protein sequence analysis using techniques including, but not limited to, Sanger sequencing, mass spectrometry, functional assays that measure enzymatic activity of the polypeptide, or immunoblotting using antibodies reactive to the amino acid changes.

[0555] Use of donor polynucleotides

[0556] DNA repair pathways

[0557] DNA breaks (such as DSBs) are repaired in cells primarily through two DNA repair pathways: the non-homologous end joining (NHEJ) repair pathway and the homology-directed repair (HDR) pathway.

[0558] During NHEJ, the Ku70 / 80 heterodimer binds to DNA ends and recruits DNA protein kinase (DNA-PK) (Cannan & Pederson (2015) J Cell Physiol 231:3–14). Once bound, DNA-PK activates its catalytic subunit (DNA-PKcs) and further recruits the endonuclease Artemis (also known as SNM1c). At a subset of DSBs, Artemis removes excess single-stranded DNA (ssDNA) and generates substrate for ligation by DNA ligase IV. DNA repair by NHEJ involves a sequence homology-independent blunt-end ligation mechanism via the canonical DNA-PKcs / Ku70 / 80 complex. During the cell cycle, NHEJ occurs predominantly in G0 / G1 and G2 (Chiruvella et al. (2013) Cold Spring Harb Perspect Biol 5:a012757). Current studies have shown that NHEJ is the only DSB repair pathway active in G0 and G1, while HDR functions primarily in S and G2 phases and plays a major role in the repair of replication-associated DSBs (Karanam et al. (2012) Mol Cell 47:320–329; Li and Xu (2016) Acta Biochim Biophys Sin 48(7):641-646). Unlike HDR, NHEJ is active in both dividing and non-dividing cells, not just dividing cells, which enables the development of genome editing-based therapies for non-dividing adult cells (e.g., cells of the eye, brain, pancreas, or heart).

[0559] During DNA repair by HDR, the DSB ends are resected to expose the 3' ssDNA tail, primarily by the MRE11-RAD50-NBS1 (MRN) complex (Heyer et al. (2010) Annu Rev Genet 44: 113–139). Under physiological conditions, the adjacent sister chromatid will serve as a repair template, providing homologous sequences, and the ssDNA will invade the template mediated by the recombinase Rad51, displacing the intact strand to form a D-loop. D-loop extension is followed by branch migration to produce a double Holliday junction, which resolubilizes to complete the repair cycle. HDR often requires error-prone polymerases but is generally considered error-free (Li and Xu (2016) Acta Biochim Biophys Sin 48(7):641-646).

[0560] The third repair mechanism is microhomology-mediated end joining (MMEJ), also known as "alternative NHEJ," which has similar genetic consequences to NHEJ in that small deletions and insertions can occur at the cut site. MMEJ utilizes homologous sequences of several nucleotides flanking the DNA break site to drive a more favorable DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanism of this process (Cho and Greenberg, (2015) Nature 518: 174-176; Mateos-Gomez et al. (2015) Nature 518, 254-257; Ceccaldi et al. (2015) Nature 528, 258-262).

[0561] Use of donor polynucleotides to correct or induce mutations

[0562] In some embodiments, by inserting a donor polynucleotide into a target nucleic acid (e.g., gDNA) at a cleavage site (e.g., DSB) induced by a site-directed nuclease (e.g., those described herein), the donor polynucleotide provided by the present disclosure is used to correct or induce a mutation in the gDNA in a cell. In some embodiments, the NHEJ DNA repair mechanism of the cell uses a donor polynucleotide to repair DSB, thereby inserting the donor polynucleotide into the DSB, and adding the nucleotide sequence of the donor polynucleotide to the gDNA. In some embodiments, the donor polynucleotide comprises a nucleotide sequence that corrects the pathogenic mutation in the gDNA in the cell, and includes a nucleotide sequence that comprises one or more splicing signals to control the processing of the precursor mRNA (pre-mRNA) transcribed by the gDNA. In some embodiments, the donor polynucleotide is inserted into a position close to the mutation, thereby correcting the mutation. In some embodiments, the mutation is a substitution, missense, nonsense, insertion, deletion, or frameshift mutation. In some embodiments, the mutation is in an exon. In some embodiments, the mutation is a substitution, insertion, or deletion, and is located in an intron. In some embodiments, the mutation is close to the splicing signal in the gDNA. In some embodiments, the mutation is near the 3' splice site in the gDNA. In some embodiments, the mutation is near the 5' splice site in the gDNA. In some embodiments, the mutation is in a splice signal or a sequence comprising a splice signal in the gDNA. In some embodiments, the mutation is in the 3' splice site in the gDNA. In some embodiments, the mutation is in the 5' splice site in the gDNA. In some embodiments, the mutation is in a polypyrimidine tract. In some embodiments, the mutation is in a branch point sequence. In some embodiments, the mutation is a protein-coding mutation. In some embodiments, the mutation is associated with or causes a disease.

[0563] In some embodiments, the nucleotide sequence that corrects the pathogenic mutation in the gDNA in the cell comprises a branch point sequence. In some embodiments, the nucleotide sequence that corrects the disease comprises a polypyrimidine tract. In some embodiments, the donor polynucleotide is inserted into the DSB by NHEJ DNA repair. In some embodiments, the donor polynucleotide, or a portion of the donor polynucleotide, is inserted into the target nucleic acid cleavage site by NHEJ DNA repair. In certain aspects, the insertion of the donor polynucleotide into the target nucleic acid via NHEJ repair can result in, for example, mutation, deletion, alteration, integration, gene correction, gene replacement, gene tagging, transgenic insertion, nucleotide deletion, gene disruption, translocation, and / or gene mutation of the endogenous gene sequence.

[0564] In some embodiments, the pathogenic mutation causes the disease glycogen storage disease 1a (GSD1a). In some embodiments, the pathogenic mutation is located in the human G6PC gene on human chromosome 17q21. In some embodiments, the pathogenic mutation is located in the G6PC gene and results in an R83C, R83H, or E110K amino acid substitution in the human G6PC protein. In some embodiments, the pathogenic mutation in the G6PC gene results in an R83C amino acid substitution in the human G6PC protein. In some embodiments, the pathogenic mutation in the G6PC gene results in an R83H amino acid substitution in the human G6PC protein.

[0565] In some embodiments, the pathogenic mutation in the G6PC gene leads to the E110K amino acid substitution in the human G6PC protein. In some embodiments, the present disclosure provides donor polynucleotides for use in repairing DSBs introduced into target nucleic acid molecules (e.g., gDNA) by site-directed nucleases (e.g., Cas9) in cells. In some embodiments, donor polynucleotides are used by the non-homologous end joining (NHEJ) repair pathway of cells to repair DSBs in target nucleic acid molecules. In some embodiments, the site-directed nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is Cas9. The site-directed nucleases described herein can introduce DSBs into target nucleic acids (e.g., genomic DNA) in cells. The DSBs induced by the site-directed nucleases are introduced into cell genomic DNA, which will stimulate endogenous DNA repair pathways, such as those described herein. In contrast, DSB repair by NHEJ occurs in non-dividing cells, but does not require the use of homologous polynucleotide sequences for repair. However, the NHEJ pathway can be used to insert polynucleotides (e.g., donor polynucleotides) into DSBs during repair.

[0566] Accordingly, in some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the dsDNA comprising a nucleotide sequence that corrects a mutation in a genomic DNA molecule (gDNA) in a cell, the mutation causing glycogen storage disease 1a, wherein the mutation is located in the human G6PC gene on human chromosome 17q21 and results in the amino acid substitution R83C or R83H, the donor polynucleotide comprising:

[0567] (i) a first strand comprising, from 5′ to 3′, a nucleotide sequence comprising an exon sequence that corrects the mutation, and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from a genomic DNA molecule, wherein the exon sequence comprises a codon encoding arginine (R) corresponding to the codon at position 83 in the G6PC gene, wherein the one or more splicing signals are a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

[0568] (ii) a second strand comprising a nucleotide sequence complementary to the first strand,

[0569] wherein the donor polynucleotide is about 40-70 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each chain of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA by the site-directed nuclease at a position close to the mutation, wherein the insertion of the donor polynucleotide forms an exon in the gDNA comprising an exon sequence that corrects the mutation, wherein the splicing signal directs the exon to be included in the mRNA, thereby correcting the mutation. In some embodiments, the branch point sequence comprises the nucleotide sequence TTCAT, wherein the polypyrimidine tract comprises the nucleotide sequence CTTGTTCTGTTTTTTT, wherein the 3' splice site comprises the nucleotide sequence TAG, and wherein the exon sequence comprises the nucleotide sequence GATTCTCTTTGGACAGCGCCCTTACT.

[0570] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 30 (CH34 54-0). In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 20 (CH32 50-0).

[0571] In some embodiments, the pathogenic mutation causes the disease Pompe disease. In some embodiments, the pathogenic mutation is located in the human glucosidase alpha (GAA) gene on human chromosome 17q25.3. In some embodiments, the mutation is in the splicing signal of GAA, which results in an mRNA transcript of the GAA gene lacking exon 2. In some embodiments, the mutation is in the splicing signal of GAA, which results in the activation of one or more cryptic splicing sites. Cryptic splicing sites are mRNA sequences that encode splicing signals and have the potential to interact with the spliceosome, but are not usually used for mRNA splicing to generate functional protein products. Mutations in genes can activate cryptic splicing signals, which result in the production of abnormal protein products.

[0572] Accordingly, in some embodiments, the present disclosure provides a donor polynucleotide comprising a non-replicating dsDNA molecule comprising a nucleotide sequence that corrects a mutation in a gDNA molecule in a cell, the mutation causing Pompe disease, wherein the mutation is in the splicing signal of GAA, which results in an mRNA transcript of an activated GAA gene lacking exon 2 and / or one or more cryptic splice sites, the donor polynucleotide comprising:

[0573] (i) a first strand comprising, from 5′ to 3′, a first nucleotide sequence that corrects the mutation, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

[0574] (ii) a second strand comprising, from 5′ to 3′, a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence, wherein the second strand is complementary to the first strand,

[0575] wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each strand of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into a cell in combination with a site-directed nuclease, the NHEJ DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease proximal to the location of the mutation, wherein the donor polynucleotide is configured for bidirectional insertion into the DSB break, wherein insertion in either orientation forms a 3' splice site, a polypyrimidine tract, and a branch point sequence that corrects the mutation, wherein the splicing signal directs the inclusion of exon 2 into the mRNA, thereby correcting the mutation.

[0576] In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 63 (GAA_50-0).

[0577] Accordingly, in some embodiments, multiple copies of a single donor polynucleotide or the same donor polynucleotide are provided. In other embodiments, two or more donor polynucleotides are provided so that repair can occur at two or more target sites. For example, different donor polynucleotides are provided to repair a single gene in a cell or two different genes in a cell. In some embodiments, different donor polynucleotides are provided with independent copy numbers.

[0578] In some embodiments, the donor polynucleotide is incorporated into the target nucleic acid as an insertion mediated by non-homologous end joining (NHEJ). In some embodiments, the donor polynucleotide sequence has no similarity to the nucleotide sequence near the cleavage site. In some embodiments, multiple copies of a single donor polynucleotide or identical donor polynucleotide are provided. In other embodiments, two or more donor polynucleotides with different sequences are inserted into two or more sites by non-homologous end joining. In some embodiments, different donor polynucleotides are provided with independent copy numbers.

[0579] CRISPR / Cas nuclease system

[0580] The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of acquired immunity in prokaryotes. CRISPR is an abbreviation for clustered regularly interspaced short palindromic repeats. It is a family of DNA sequences found in the genomes of bacteria and archaea that contains DNA (spacer DNA) fragments with similarity to foreign DNA previously exposed to cells, such as by viruses that have infected or attacked prokaryotes. These DNA fragments are used by prokaryotes to detect and destroy similar foreign DNA when reintroduced by similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of RNA molecules comprising spacer sequences that bind and target Cas (CRISPR-associated) proteins that can recognize and cut foreign exogenous DNA. Numerous types and species of CRISPR / Cas systems have been described (see, for example, Koonin et al. (2017) Curr Opin Microbiol 37: 67-78).

[0581] The modified version of the CRISPR / Cas system has been developed in many forms to mutate or edit the genomic DNA of cells from other species. The general method using the CRISPR / Cas system involves heterologous expression or introduction of a site-directed nuclease (such as Cas nuclease) in combination with a guide RNA (gRNA) in a cell, resulting in a DNA cleavage event (e.g., forming a single-strand or double-strand break (SSB or DSB)) at a precise targetable position, in the backbone of the genomic DNA of the cell. Wherein the DNA cleavage event provides the opportunity to edit the genome by adding, removing or modifying (replacing) DNA nucleotides or sequences (such as genes) by means of cell repair.

[0582] Cas nucleases

[0583] In some embodiments, the present disclosure provides compositions and systems (e.g., CRISPR / Cas systems of transformation) comprising site-directed nucleases, wherein the site-directed nuclease is a Cas nuclease. The Cas nuclease may comprise at least one domain that interacts with a guide RNA (gRNA). In addition, the Cas nuclease is directed to a target sequence via a guide RNA. The guide RNA interacts with the Cas nuclease and the target sequence so that once directed to the target sequence, the Cas nuclease is able to cut the target sequence. In some embodiments, the guide RNA provides specificity for cutting the target sequence, and the Cas nuclease is universal and paired with different guide RNAs to cut different target sequences.

[0584] In some embodiments, the CRISPR / Cas system comprises components derived from type I, type II, or type III systems. An updated classification scheme for the CRISPR / Cas locus defines a type 1 and type 2 CRISPR / Cas system with types I to V or VI (Makarova et al. (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al. (2015) Mol Cell, 60:385-397). The type 2 CRISPR / Cas system has a single protein effector. The Cas proteins of types II, V, and VI are single-protein, RNA-guided endonucleases, referred to herein as “type 2 Cas nucleases.” Type 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. Cpf1 nuclease (Zetsche et al. (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0585] In some embodiments, the Cas nuclease is from a type II CRISPR / Cas system (e.g., a Cas9 protein from a CRISPR / Cas9 system). In some embodiments, the Cas nuclease is from a class 2 CRISPR / Cas system (a single-protein Cas nuclease, such as a Cas9 protein or a Cpf1 protein). The Cas9 and Cpf1 protein families are enzymes with DNA endonuclease activity, and they can be directed to cleave a desired nucleic acid target by designing appropriate guide RNAs, as further described herein.

[0586] Type II CRISPR / Cas system components are derived from Type IIA, Type IIB, or Type IIC systems. Cas9 and its orthologs are encompassed. Non-limiting exemplary species from which the Cas9 nuclease or other components are derived include Streptococcus pyogenes, Streptococcus thermophilus ( Streptococcus thermophilus ), Streptococcus species ( Streptococcus sp. ), Staphylococcus aureus, Listeria monocytogenes ( Listeria innocua ), Lactobacillus gasseri ( Lactobacillus gasseri ), the new killer Francisella ( Francisella novicida ), Wolinella succinicigenes ( Wolinella succinogenes ), Waldesartella ( Sutterella wadsworthensis ), γ-Proteobacteria ( Gamma proteobacterium ), Neisseria meningitidis ( Neisseria meningitidis jejuni ( Campylobacter jejuni ), Pasteurella multocida ( Pasteurella multocida ), Mycobacterium succinicum ( Fibrobacter succinogene ), Rhodospirillum rubrum ( Rhodospirillum rubrum ), Nocardiopsis dauricus ( Nocardiopsis dassonvillei ), Streptomyces elegans ( Streptomyces pristinaespiralis ), Streptomyces viridis ( Streptomyces viridochromogenes ), Streptomyces viridis, Streptomyces roseus ( Streptosporangium roseum ), Streptomyces roseus, Alicyclobacillus acidocaldarius ( Alicyclobacillus acidocaldarius ), Bacillus pseudomycoides ( Bacillus pseudomycoides ), Bacillus selenate-reducing ( Bacillus selenitireducens ), Exobacterium thaliana ( Exiguobacterium sibiricum ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), Lactobacillus salivarius ( Lactobacillus salivarius ), Lactobacillus buchneri ( Lactobacillus buchneri ), Treponema denticola ( Treponema denticola ), marine microtuberculosis ( Microscilla marina ), Burkholderiales bacteria ( Burkholderiales bacterium ), Acromonas naphthotrophica ( Polaromonas naphthalenivorans ), Polarmonas species ( Polaromonas sp. ), Crocodile Glomerella vashevii ( Crocosphaera watsonii ), Cyanobacteria species ( Cyanothece sp. ), Microcystis aeruginosa ( Microcystis aeruginosa ), Synechococcus species ( Synechococcus sp ), Arabinosaccharobacterium halogenum ( Acetohalobium arabaticum ), Bacillus dendrobatidis ( Ammonifex degensii ), Caldicelulosiruptor becscii 、 Candidatus Desulforudis , Botulinum toxin ( Clostridium botulinum ), Clostridium difficile ( Clostridium difficile ), Finegoldia macrophylla ( Finegoldia magna ), thermophilic alkali-requiring anaerobic bacteria ( Natranaerobius thermophilus ), thermophilic propionic acid degrading bacteria ( Pelotomaculum thermopropionicum ), Thermothiobacillus acidophilus ( Acidithiobacillus caldus ), Acidithiobacillus ferrooxidans ( Acidithiobacillus ferrooxidans ), Achromobacter vinifera ( Allochromatium vinosum ), Marinobacter species ( Marinobacter sp.), halophilic Nitrosococcus ( Nitrosococcus halophilus ), Nitrosococcus waaroni ( Nitrosococcus watsoni ), Pseudomonas aeruginosa ( Pseudoalteromonas haloplanktis ), clustered mycelial fungi ( Ktedonobacter racemifer ), Investigation of Methanogens ( Methanohalobium evestigatum ), Anabaena variabilis ( Anabaena variabilis )、Nyctolagus foamy Nodularia spumigena ), Nostoc species ( Nostoc sp. ), Arthrospira maxima ( Arthrospira maxima )、Arthrospira platensis ( Arthrospira platensis ), Arthrospira species ( Arthrospira sp. ), species of the genus Cladophora ( Lyngbya sp. ), Microcoleus philippinii ( Microcoleus chthonoplastes ), Oscillatoria species ( Oscillatoria sp. ), Sphaerotheca mobilis ( Petrotoga mobilis ), Thermus africanus ( Thermosipho africanus ), Streptococcus pasteurianus ( Streptococcus pasteurianus ), Neisseria griseus ( Neisseria cinerea ), Campylobacter spp. ( Campylobacter lari ), Leachate-feeding Bacillus microti ( Parvibaculum lavamentivorans ), Corynebacterium diphtheriae ( Corynebacterium diphtheria )or Acaryochloris marina . In some embodiments, the Cas9 protein is from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is from Neisseria meningitidis (NmCas9). In some embodiments, the Cas9 protein is from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is from Campylobacter jejuni (CjCas9).

[0587] In some embodiments, the Cas nuclease may include more than one nuclease domain. For example, the Cas9 nuclease may include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified to contain no functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified to contain no functional HNH-like nuclease domain. In some embodiments in which only one nuclease domain is functional, the Cas9 nuclease is a nicking enzyme capable of introducing a single-strand break ("nick") into the target sequence. In some embodiments, the conserved amino acids in the Cas9 nuclease domain are substituted to reduce or change the nuclease activity. In some embodiments, the Cas nuclease nickase is included in the amino acid replacement in the RuvC sample nuclease domain. The exemplary amino acid replacement in the RuvC sample nuclease domain includes D10A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nickase is included in the amino acid replacement in the HNH sample nuclease domain. The exemplary amino acid replacement in the HNH sample nuclease domain includes E762A, H840A, N863A, H983A and D986A (based on Streptococcus pyogenes Cas9 nuclease). In some embodiments, the nuclease system described herein includes a nickase and a pair of guide RNAs complementary to the sense strand and antisense strand of the target sequence, respectively. The guide RNA guides the nickase targeting and introduces DSB by generating a nick (i.e., a double nick) on the opposite strand of the target sequence. Using a chimeric Cas9 nuclease, a domain or region of the protein is replaced by a part of a differe...

Claims

1. A donor polynucleotide comprising a non-replicating double-stranded DNA molecule (dsDNA), the double-stranded DNA molecule comprising a nucleotide sequence that corrects a mutation in a genomic DNA (gDNA) molecule in a cell, the mutation causing glycogen storage disease 1a, wherein the mutation is a protein-coding mutation near the 3' splice site in the human G6PC gene, the donor polynucleotide comprising: (i) a first strand comprising, from 5′ to 3′, (a) a nucleotide sequence comprising an exon sequence that corrects a mutation in a cell and (b) a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and (ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the donor polynucleotide is 10-100 nucleotides in length, and wherein when the donor polynucleotide is introduced into a cell in combination with a site-directed nuclease, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB), and the DSB is introduced into the gDNA near the position of the mutation by the site-directed nuclease, thereby correcting the mutation, wherein the one or more splicing signals are a branch point, a polypyrimidine tract, or a native or enhanced 3' splice site, and wherein the branch point comprises the nucleotide sequence TTCAT, the polypyrimidine tract comprises the nucleotide sequence CTTGTTCTGTTTTTTTT (SEQ ID NO: 109), and the 3' splice site comprises the nucleotide sequence TAG.

2. The donor polynucleotide of claim 1, wherein insertion of the donor polynucleotide into a DSB results in exon formation in a gDNA comprising the exon sequence.

3. The donor polynucleotide of claim 2, wherein the one or more splicing signals direct the inclusion of an exon into the mRNA, the exon comprising an exonic sequence that corrects the mutation.

4. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide is 20-80 nucleotides in length.

5. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide is 30-70 nucleotides in length.

6. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide is 40-60 nucleotides in length.

7. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide is 40-70 nucleotides in length.

8. The donor polynucleotide of claim 7, wherein the donor polynucleotide is 50-60 nucleotides in length.

9. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.

10. The donor polynucleotide of claim 9, wherein the donor polynucleotide is 50 nucleotides in length.

11. The donor polynucleotide of any one of claims 1-3, wherein the site-directed nuclease constitutes a CRISPR / Cas system.

12. The donor polynucleotide of claim 11, wherein the CRISPR / Cas system comprises one or more guide RNAs (gRNAs).

13. The donor polynucleotide of claim 12, wherein the site-directed nuclease comprises a Cas9 polypeptide derived from Streptococcus pyogenes (SpCas9).

14. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide comprises natural nucleotides.

15. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide comprises one or more non-natural and / or modified nucleotides.

16. The donor polynucleotide of claim 15, wherein the one or more non-natural and / or modified nucleotides are 2'-O-methyl nucleotides.

17. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide comprises one or more backbone modifications.

18. The donor polynucleotide of claim 17, wherein the one or more backbone modifications are phosphorothioates.

19. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide comprises two blunt ends.

20. The donor polynucleotide of any one of claims 1-3, wherein the donor polynucleotide comprises one blunt end and includes one end comprising an overhang.

21. The donor polynucleotide of claim 20, wherein the overhang is a 5' or 3' overhang.

22. The donor polynucleotide of any one of claims 1-3, wherein the nucleotide sequence comprises one or more nucleotides that prevent a site-directed nuclease from recognizing and cleaving the donor polynucleotide.

23. The donor polynucleotide of claim 1, wherein the mutation is located in the human G6PC gene on human chromosome 17q21.

24. The donor polynucleotide of claim 1, wherein the mutation is an amino acid substitution of arginine (R) at position 83 in the G6PC gene, and wherein the exon sequence comprises a codon encoding arginine (R) corresponding to the codon at position 83 in the G6PC gene.

25. The donor polynucleotide of claim 1, wherein the mutation in the G6PC gene results in an R83C amino acid substitution in the human G6PC protein.

26. The donor polynucleotide of claim 1, wherein the mutation in the G6PC gene results in an R83H amino acid substitution in the human G6PC protein.

27. The donor polynucleotide of any one of claims 1-3, wherein the exon sequence comprises the nucleotide sequence GATTCTCTTTGGACAGCGCCCTTACT.

28. The donor polynucleotide of any one of claims 1-3, wherein the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 30 (CH3454-0).

29. The donor polynucleotide of any one of claims 1-3, wherein the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 20 (CH3250-0).

30. A donor polynucleotide comprising a non-replicating dsDNA molecule, the dsDNA molecule comprising a nucleotide sequence that corrects a mutation in a human glucosidase alpha (GAA) gene in a gDNA molecule in a cell, the mutation causing Pompe disease 1a, wherein the mutation in the splicing signal of GAA results in an mRNA transcript of an activated GAA gene lacking exon 2 and / or one or more cryptic splice sites, the donor polynucleotide comprising: (i) a first strand comprising, from 5′ to 3′, a first nucleotide sequence that corrects the mutation, wherein the first strand comprises a first intron sequence, wherein the first intron sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and (ii) a second strand comprising, from 5′ to 3′, a second intron sequence, wherein the second intron sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of pre-mRNA transcribed from the gDNA, wherein the one or more splicing signals comprise a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence, wherein the second strand is complementary to the first strand, wherein the donor polynucleotide is 10-100 nucleotides in length and comprises two blunt ends, wherein the 5'-most nucleotide of each strand of the donor polynucleotide comprises a 5' phosphate moiety, wherein when the donor polynucleotide is introduced into a cell in combination with a site-directed nuclease, the NHEJ DNA repair pathway inserts the donor polynucleotide into a DSB, wherein the DSB is introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, wherein the donor polynucleotide is configured for bidirectional insertion into the DSB break, wherein insertion in either direction forms a 3' splice site, a polypyrimidine tract, and a branch point sequence that corrects the mutation, wherein the splicing signal directs the inclusion of exon 2 into the mRNA, thereby correcting the mutation, wherein the branch point comprises the nucleotide sequence TATTAAC, GTTAATA, TACTGAC or TTAAC, the polypyrimidine tract comprises the nucleotide sequence CTTCTTCTCTTCTTCC or TTTTTTTTTTTTT, and the 3' splice site comprises the nucleotide sequence TAG or CAG.

31. The donor polynucleotide of claim 30, wherein the donor polynucleotide is 20-80 nucleotides in length.

32. The donor polynucleotide of claim 30, wherein the donor polynucleotide is 30-70 nucleotides in length.

33. The donor polynucleotide of claim 30, wherein the donor polynucleotide is 40-60 nucleotides in length.

34. The donor polynucleotide of claim 30, wherein the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 63 (GAA_50-0).

35. A system for correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the system comprising the following components: (a) a donor polynucleotide according to any one of claims 1 to 34; (b) one or more gRNA molecules; and (c) site-directed nucleases, When the system is introduced into cells, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break (DSB), and the DSB is introduced into the gDNA near the location of the mutation by a site-directed nuclease, thereby correcting the mutation.

36. The system of claim 35, wherein the site-directed nuclease is encoded by mRNA.

37. The system of claim 35, wherein the site-directed nuclease is a polypeptide.

38. The system of claim 37, wherein the one or more gRNA molecules and the site-directed nuclease comprise a ribonucleoprotein.

39. The system of any one of claims 35-38, wherein the site-directed nuclease is a Cas nuclease.

40. The system of claim 39, wherein the Cas nuclease is Streptococcus pyogenes Cas9 (SpCas9), or a homolog, derivative or modified form thereof.

41. The system of any one of claims 35-38, wherein the one or more gRNA molecules comprise a modification selected from the group consisting of a backbone modification, a sugar modification, a modified internucleoside linkage, or a modified or non-natural nucleobase.

42. The system of claim 41, wherein the one or more gRNA molecules comprise a backbone modification.

43. The system of claim 42, wherein the backbone modification is a phosphorothioate modification.

44. The system of any one of claims 35-38, wherein the donor polynucleotide, gRNA molecule, and site-directed nuclease are formulated individually or co-formulated in liposomes or lipid nanoparticles.

45. The system of claim 44, wherein the donor polynucleotide, gRNA molecule, and site-directed nuclease are individually formulated in liposomes or lipid nanoparticles.

46. ​​The system of claim 44, wherein the donor polynucleotide, gRNA molecule, and site-directed nuclease are co-formulated in liposomes or lipid nanoparticles.

47. The system of any one of claims 35-38, wherein the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 107 (CH32 mutant-CTX1 sgRNA).

48. The system of any one of claims 35-38, wherein the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 108 (CH34 mutant-CTX1 sgRNA).

49. The system of any one of claims 35-38, wherein the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 93 (mutant GAA sgRNA spacer).

50. A cell comprising the donor polynucleotide of any one of claims 1-34 or the system of any one of claims 35-38.

51. The cell of claim 50, wherein the cell is a dividing cell or a non-dividing cell.

52. The cell of claim 51, wherein the cell is a dividing cell.

53. The cell of claim 51, wherein the cell is a non-dividing cell.

54. The cell of claim 50, wherein the cell is a patient-specific induced pluripotent stem cell (iPSC).

55. The cell of claim 50, wherein the cell is a primary hepatocyte.

56. A pharmaceutical composition comprising a donor polynucleotide according to any one of claims 1-34 and a pharmaceutically acceptable carrier.

57. A pharmaceutical composition comprising the system according to claim 35 and a pharmaceutically acceptable carrier.

58. A pharmaceutical composition comprising the cell according to claim 50 and a pharmaceutically acceptable carrier.

59. The donor polynucleotide of any one of claims 1-34, the system of any one of claims 35-38, or the pharmaceutical composition of any one of claims 56-58, for use in treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: Cells are isolated from the patient and contacted with the donor polynucleotide, system or pharmaceutical composition, wherein when the donor polynucleotide, system or pharmaceutical composition contacts the cell, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

60. The donor polynucleotide of any one of claims 1-34, the system of any one of claims 35-38, or the pharmaceutical composition of any one of claims 56-58, for use in treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: An effective amount of the donor polynucleotide, system or pharmaceutical composition is administered to the patient, wherein when the donor polynucleotide, system or composition is administered, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

61. Use of the donor polynucleotide of any one of claims 1-34, the system of any one of claims 35-38, or the pharmaceutical composition of any one of claims 56-58 for the manufacture of a medicament for treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell.

62. Use of a donor polynucleotide according to any one of claims 1 to 34, a system according to any one of claims 35 to 38, or a pharmaceutical composition according to any one of claims 56 to 58 in the preparation of a medicament, wherein the medicament is prepared for use in a method for correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: The cell is contacted with the donor polynucleotide, the system, or the pharmaceutical composition, wherein when the donor polynucleotide, system or composition contacts the cell, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

63. Use of a donor polynucleotide according to any one of claims 1-34, a system according to any one of claims 35-38, or a pharmaceutical composition according to any one of claims 56-58 in the preparation of a medicament, wherein the medicament is prepared for use in a method of treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: Cells are isolated from the patient and contacted with the donor polynucleotide, the system, or the pharmaceutical composition, wherein when the donor polynucleotide, system, or composition contacts the cell, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

64. Use of a donor polynucleotide according to any one of claims 1-34, a system according to any one of claims 35-38, or a pharmaceutical composition according to any one of claims 56-58 in the preparation of a medicament, wherein the medicament is prepared for use in a method of treating a patient suffering from a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: An effective amount of the donor polynucleotide, the system, or the pharmaceutical composition is administered to the patient, wherein when the donor polynucleotide, system or composition is administered, the non-homologous end joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the double-stranded DNA break introduced into the gDNA proximal to the location of the mutation, thereby correcting the mutation.

65. The use of claim 62, wherein the cells are patient-specific induced pluripotent stem cells (iPSCs).

66. The use of claim 62, wherein the cell is a hepatocyte.

67. The use of claim 65, wherein the method further comprises differentiating the iPSC comprising the corrected mutation into differentiated cells; and implanting the differentiated cells into a patient.

68. The method of claim 62, wherein the treatment results in the translation of mRNA transcribed from a genomic DNA molecule (gDNA), the gDNA comprising an inserted donor polynucleotide, wherein the translation results in the formation of a translation product (protein) that alleviates the disease or does not cause or promote the disease.

69. A kit comprising a container and a package insert comprising instructions for use, wherein the container comprises a donor polynucleotide according to any one of claims 1-34, a system according to any one of claims 35-38, or a pharmaceutical composition according to any one of claims 56-58, which is used to correct a mutation in a genomic DNA molecule (gDNA) in a cell.

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