SERINE RECOMBINASES FOR GENE EDITING
The disclosure relates to gene editing systems comprising serine recombinases and methods of using such serine recombinases for integration of nucleic acid sequences. More specifically, the disclosure relates to sequence-defined serine recombinases having attachment sites such as a bacterial genomic recombination sequence (attB). Methods are also provided for recombinant pro-duction of said recombinases.
1 . A gene editing system comprising:
a) a serine recombinase comprising at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214 or a nucleic acid encoding the serine recombinase; and
b) a nucleic acid comprising a donor polynucleotide and a first attachment site sequence.
2 . The gene editing system of claim 1 , wherein the first attachment site sequence is 5′ of the donor polynucleotide.
3 . The gene editing system of any one of claims 1-2 , wherein the nucleic acid encoding the serine recombinase further comprises a second attachment site sequence.
4 . The gene editing system of claim 3 , wherein the second attachment site sequence is 5′ of the serine recombinase.
5 . The gene editing system of any one of claims 3-4 , wherein the first attachment site sequence and the second attachment site sequence are capable of recombination.
6 . The gene editing system of any one of claims 1-5 , wherein the first attachment site sequence is a bacterial genomic recombination sequence (attB).
7 . The gene editing system of any one of claims 1-5 , wherein the first attachment site sequence is a phage genomic recombination sequence (attP).
8 . The gene editing system of any one of claims 3-7 , wherein the second attachment site sequence is a bacterial genomic recombination sequence (attB).
9 . The gene editing system of any one of claims 3-7 , wherein the second attachment site sequence is a phage genomic recombination sequence (attP).
10 . The gene editing system of any one of claims 6-9 , wherein the attB sequence comprises about 20 to about 500 nucleotides.
11 . The gene editing system of any one of claims 7-10 , wherein the attP sequence comprises about 20 to about 500 nucleotides.
12 . The gene editing system of any one of claims 6-11 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7151, 7155, 7159, 7163, 7167, 7171, 7175, 7179, 7188-7200, 7206-7210, 7215, 7220, 7225, 7226, 7233, 7238, 7243, 7248, 7253, 7258, 7263, 7264, 7271, 7277, 7282, 7287, 7292, 7297, 7302, 7307, 7312, 7317, 7322, 7327, 7332, 7337, 7342, 7347, 7352, 7357, 7362, 7367, 7372, 7377, 7382, 7387, 7392, 7397, and 7402.
13 . The gene editing system of any one of claims 6-12 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 5, 9, and 13.
14 . The gene editing system of any one of claims 7-13 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7152, 7156, 7160, 7164, 7168, 7172, 7176, 7180, 7183-7187, 7201-7205, 7217, 7222, 7228, 7229, 7235, 7240, 7245, 7250, 7255, 7260, 7266, 7267, 7273, 7279, 7284, 7289, 7294, 7299, 7304, 7309, 7314, 7319, 7324, 7329, 7334, 7339, 7344, 7349, 7354, 7359, 7364, 7369, 7374, 7379, 7384, 7389, 7394, 7399, and 7404.
15 . The gene editing system of any one of claims 7-14 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 2, 6, 10, and 14.
16 . The gene editing system of any one of claims 1-15 , wherein the nucleic acid comprising the donor polynucleotide and the first attachment sequence is delivered using plasmid, a nanoplasmid, a phagemid, a phage derivative, a virus, a bacmid, a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a yeast artificial chromosome (YAC), or a cosmid.
17 . The gene editing system of any one of claims 1-16 , wherein the nucleic acid encoding the serine recombinase is delivered using a plasmid, a nanoplasmid, a phagemid, a phage derivative, a virus, a bacmid, a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a yeast artificial chromosome (YAC), or a cosmid.
18 . The gene editing system of any one of claims 16-17 , wherein the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus.
19 . The gene editing system of claim 18 , wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.
20 . The gene editing system of claim 18 , wherein the herpesvirus is HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.
21 . The gene editing system of any one of claims 1-20 , wherein the donor polynucleotide comprises a size of at least about 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, or more than 120 kb.
22 . The gene editing system of any one of claims 1-21 , wherein the donor polynucleotide encodes a therapeutic, a reporter, or a marker.
23 . The gene editing system of claim 22 , wherein the reporter comprises a fluorescent protein.
24 . The gene editing system of claim 23 , wherein the fluorescent protein is GFP, EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet, RFP, CFP, or a derivative thereof.
25 . The gene editing system of claim 22 , wherein the reporter is acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, or a derivative thereof.
26 . The gene editing system of any one of claims 22-25 , wherein the marker is an antibiotic resistance marker.
27 . The gene editing system of claim 26 , wherein the antibiotic resistance marker is kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or a derivative thereof.
28 . The gene editing system of any one of claims 22-27 , wherein the marker is a cell surface marker.
29 . A eukaryotic genome comprising a donor polynucleotide sequence; and an attL sequence 5′ to the donor polynucleotide sequence, wherein the attL sequence comprises a sequence selected from the group consisting of: SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7153, 7157, 7161, 7165, 7169, 7173, 7177, 7181, 7216, 7221, 7227, 7234, 7239, 7244, 7249, 7254, 7259, 7265, 7272, 7278, 7283, 7288, 7293, 7298, 7303, 7308, 7313, 7318, 7323, 7328, 7333, 7338, 7343, 7348, 7353, 7358, 7363, 7368, 7373, 7378, 7383, 7388, 7393, 7398, and 7403.
30 . The eukaryotic genome of claim 29 , further comprising an attR sequence 3′ to the donor polynucleotide sequence.
31 . A eukaryotic genome comprising a donor polynucleotide sequence; and an attL sequence 3′ to the donor polynucleotide sequence, wherein the attL sequence comprises a sequence selected from the group consisting of: SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7153, 7157, 7161, 7165, 7169, 7173, 7177, 7181, 7216, 7221, 7227, 7234, 7239, 7244, 7249, 7254, 7259, 7265, 7272, 7278, 7283, 7288, 7293, 7298, 7303, 7308, 7313, 7318, 7323, 7328, 7333, 7338, 7343, 7348, 7353, 7358, 7363, 7368, 7373, 7378, 7383, 7388, 7393, 7398, and 7403.
32 . The eukaryotic genome of claim 31 , further comprising an attR sequence 3′ to the donor polynucleotide sequence.
33 . A eukaryotic genome comprising:
a donor polynucleotide sequence;
an attL sequence 5′ or 3′ to the donor polynucleotide sequence, wherein the attL sequence comprises a sequence selected from the group consisting of: SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7153, 7157, 7161, 7165, 7169, 7173, 7177, 7181, 7216, 7221, 7227, 7234, 7239, 7244, 7249, 7254, 7259, 7265, 7272, 7278, 7283, 7288, 7293, 7298, 7303, 7308, 7313, 7318, 7323, 7328, 7333, 7338, 7343, 7348, 7353, 7358, 7363, 7368, 7373, 7378, 7383, 7388, 7393, 7398, and 7403; and
an attR sequence 5′ or 3′ to the donor polynucleotide sequence, wherein the attR sequence comprises a sequence selected from the group consisting of: SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7154, 7158, 7162, 7166, 7170, 7174, 7178, 7182, 7218, 7223, 7230, 7236, 7241, 7246, 7251, 7256, 7261, 7268, 7274, 7280, 7285, 7290, 7295, 7300, 7305, 7310, 7315, 7320, 7325, 7330, 7335, 7340, 7345, 7350, 7355, 7360, 7365, 7370, 7375, 7380, 7385, 7390, 7395, 7400, and 7405.
34 . The eukaryotic genome of any one of claim 30, 32, or 33 , wherein the attL sequence and the attR sequence are the same.
35 . The eukaryotic genome of any one of claims 29-34 , wherein the attL sequence is a recombined sequence of a first attachment site sequence and a second attachment site sequence.
36 . The eukaryotic genome of any one of claim 30 or 32-35 , wherein the attR sequence is a recombined sequence of a first attachment site sequence and a second attachment site sequence.
37 . The eukaryotic genome of any one of claims 35-36 , wherein the first attachment site sequence is a bacterial genomic recombination sequence (attB).
38 . The eukaryotic genome of any one of claims 35-36 , wherein the first attachment site sequence is a phage genomic recombination sequence (attP).
39 . The eukaryotic genome of any one of claims 35-38 , wherein the second attachment site sequence is a bacterial genomic recombination sequence (attB).
40 . The eukaryotic genome of any one of claims 35-38 , wherein the second attachment site sequence is a phage genomic recombination sequence (attP).
41 . The eukaryotic genome of any one of claims 37-40 , wherein the attB sequence comprises about 20 to about 500 nucleotides.
42 . The eukaryotic genome of any one of claims 38-41 , wherein the attP sequence comprises about 20 to about 500 nucleotides.
43 . The eukaryotic genome of any one of claims 37-42 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7151, 7155, 7159, 7163, 7167, 7171, 7175, 7179, 7188-7200, 7206-7210, 7215, 7220, 7225, 7226, 7233, 7238, 7243, 7248, 7253, 7258, 7263, 7264, 7271, 7277, 7282, 7287, 7292, 7297, 7302, 7307, 7312, 7317, 7322, 7327, 7332, 7337, 7342, 7347, 7352, 7357, 7362, 7367, 7372, 7377, 7382, 7387, 7392, 7397, and 7402.
44 . The eukaryotic genome of any one of claims 37-43 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 5, 9, and 13.
45 . The eukaryotic genome of any one of claims 38-44 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7152, 7156, 7160, 7164, 7168, 7172, 7176, 7180, 7183-7187, 7201-7205, 7217, 7222, 7228, 7229, 7235, 7240, 7245, 7250, 7255, 7260, 7266, 7267, 7273, 7279, 7284, 7289, 7294, 7299, 7304, 7309, 7314, 7319, 7324, 7329, 7334, 7339, 7344, 7349, 7354, 7359, 7364, 7369, 7374, 7379, 7384, 7389, 7394, 7399, and 7404.
46 . The eukaryotic genome of any one of claims 38-45 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 2, 6, 10, and 14.
47 . The eukaryotic genome of any one of claims 29-46 , wherein the attL sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7153, 7157, 7161, 7165, 7169, 7173, 7177, 7181, 7216, 7221, 7227, 7234, 7239, 7244, 7249, 7254, 7259, 7265, 7272, 7278, 7283, 7288, 7293, 7298, 7303, 7308, 7313, 7318, 7323, 7328, 7333, 7338, 7343, 7348, 7353, 7358, 7363, 7368, 7373, 7378, 7383, 7388, 7393, 7398, and 7403.
48 . The eukaryotic genome of any one of claims 29-47 , wherein the attR sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 3, 4, 7, 8, 11, 12, 15, 16, 7154, 7158, 7162, 7166, 7170, 7174, 7178, 7182, 7218, 7223, 7230, 7236, 7241, 7246, 7251, 7256, 7261, 7268, 7274, 7280, 7285, 7290, 7295, 7300, 7305, 7310, 7315, 7320, 7325, 7330, 7335, 7340, 7345, 7350, 7355, 7360, 7365, 7370, 7375, 7380, 7385, 7390, 7395, 7400, and 7405.
49 . A mammalian cell comprising the eukaryotic genome of any one of claims 29-48 .
50 . The mammalian cell of claim 49 , wherein the mammalian cell is a human cell.
51 . The mammalian cell of any one of claims 49-50 , further comprising a serine recombinase.
52 . The mammalian cell of claim 51 , wherein the serine recombinase comprises at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214.
53 . The mammalian cell of claim 51 , wherein the serine recombinase comprises at least about 80% sequence identity to SEQ ID NO: 21.
54 . The mammalian cell of claim 51 , wherein the serine recombinase comprises at least about 80% sequence identity to SEQ ID NO: 22.
55 . The mammalian cell of claim 51 , wherein the serine recombinase comprises at least about 80% sequence identity to SEQ ID NO: 23.
56 . The mammalian cell of claim 51 , wherein the serine recombinase comprises at least about 80% sequence identity to SEQ ID NO: 24.
57 . The mammalian cell of claim 51 , wherein the serine recombinase comprises an integration efficiency of at least about 5%.
58 . The mammalian cell of claim 51 , wherein the serine recombinase comprises an integration efficiency of at least about 25%.
59 . The mammalian cell of claim 51 , wherein the serine recombinase comprises an integration efficiency of at least about 50%.
60 . The mammalian cell of claim 51 , wherein the serine recombinase is capable of targeting genes comprising a catalase domain or synthase domain.
61 . The mammalian cell of claim 60 , wherein the catalase is manganese catalase.
62 . The mammalian cell of any one of claims 60-61 , wherein the synthase is Queuosine synthase.
63 . The mammalian cell of any one of claims 60-62 , wherein the serine recombinase is capable of targeting genes comprising a DUF4244 Pfam domain.
64 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214.
65 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 21.
66 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 22.
67 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 23.
68 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 24.
69 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 1848.
70 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7111.
71 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7115.
72 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7131.
73 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7136.
74 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7139.
75 . A eukaryotic cell comprising a serine recombinase comprising at least about 80% sequence identity to SEQ ID NO: 7140.
76 . The eukaryotic cell of any one of claims 64-75 , wherein the eukaryotic cell is a mammalian cell.
77 . The eukaryotic cell of any one of claims 64-75 , wherein the eukaryotic cell is a human cell.
78 . A vector comprising:
a) a nucleic acid encoding serine recombinase comprising at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214; and
b) one or more regulatory elements.
79 . The vector of claim 78 , wherein the one or more regulatory elements comprises a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, or a polyA signal.
80 . The vector of claim 79 , wherein the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or a derivative thereof.
81 . The vector of claim 79 , wherein the promoter is selected from the group consisting of:
CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and a derivative thereof.
82 . A vector comprising a nucleic acid encoding a serine recombinase comprising at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214, wherein the vector is selected from the group consisting of: a plasmid, a nanoplasmid, a phagemid, a phage derivative, a bacmid, a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a yeast artificial chromosome (YAC), and a cosmid.
83 . A method for gene editing, comprising:
a) providing or identifying a first attachment site sequence in a host genome;
b) providing a nucleic acid comprising a donor polynucleotide and a second attachment site sequence to a host cell; and
c) contacting the host cell with a serine recombinase comprising at least about 80% sequence identity to any one of SEQ ID NOs: 21-7060, 7105-7142, and 7211-7214 or a nucleic acid encoding the serine recombinase,
wherein the first attachment site sequence and the second attachment site sequence are capable of recombination.
84 . The method of claim 83 , wherein the first attachment site sequence is endogenous in the host genome.
85 . The method of claim 83 , wherein the first attachment site sequence is provided using viral delivery.
86 . The method of claim 83 , wherein the first attachment site sequence is provided using a transposase.
87 . The method of claim 83 , wherein the first attachment site sequence is provided using a nuclease.
88 . The method of claim 87 , wherein the nuclease is a double-strand nuclease.
89 . The method of claim 87 , wherein the nuclease is a Type II CRISPR endonuclease.
90 . The method of claim 87 , wherein the nuclease is a Type V CRISPR endonuclease.
91 . The method of claim 87 , wherein the nuclease is Cas9.
92 . The method of claim 76 , wherein the first attachment site sequence is provided using a reverse transcriptase.
93 . The method of any one of claims 83-92 , wherein the second attachment site sequence is 5′ of the donor polynucleotide.
94 . The method of any one of claims 83-93 , wherein the first attachment site sequence is a bacterial genomic recombination sequence (attB).
95 . The method of any one of claims 83-94 , wherein the first attachment site sequence is a phage genomic recombination sequence (attP).
96 . The method of any one of claims 83-95 , wherein the second attachment site sequence is a bacterial genomic recombination sequence (attB).
97 . The method of any one of claims 83-96 , wherein the second attachment site sequence is a phage genomic recombination sequence (attP).
98 . The method of any one of claims 94-97 , wherein the attB sequence comprises about 20 to about 500 nucleotides.
99 . The method of any one of claims 95-98 , wherein the attP sequence comprises about 20 to about 500 nucleotides.
100 . The method of any one of claims 94-99 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7151, 7155, 7159, 7163, 7167, 7171, 7175, 7179, 7188-7200, 7206-7210, 7215, 7220, 7225, 7226, 7233, 7238, 7243, 7248, 7253, 7258, 7263, 7264, 7271, 7277, 7282, 7287, 7292, 7297, 7302, 7307, 7312, 7317, 7322, 7327, 7332, 7337, 7342, 7347, 7352, 7357, 7362, 7367, 7372, 7377, 7382, 7387, 7392, 7397, and 7402.
101 . The method of any one of claims 94-100 , wherein the attB sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 5, 9, and 13.
102 . The method of any one of claims 95-101 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 7152, 7156, 7160, 7164, 7168, 7172, 7176, 7180, 7183-7187, 7201-7205, 7217, 7222, 7228, 7229, 7235, 7240, 7245, 7250, 7255, 7260, 7266, 7267, 7273, 7279, 7284, 7289, 7294, 7299, 7304, 7309, 7314, 7319, 7324, 7329, 7334, 7339, 7344, 7349, 7354, 7359, 7364, 7369, 7374, 7379, 7384, 7389, 7394, 7399, and 7404.
103 . The method of any one of claims 95-102 , wherein the attP sequence comprises at least about 80% sequence identity to any one of SEQ ID NOs: 2, 6, 10, and 14.
104 . The method of any one of claims 83-103 , wherein the nucleic acid comprising the donor polynucleotide and the second attachment site sequence is delivered by a plasmid, a nanoplasmid, a phagemid, a phage derivative, a virus, a bacmid, a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a yeast artificial chromosome (YAC), or a cosmid.
105 . The method of any one of claims 83 - 106 , wherein the nucleic acid encoding the serine recombinase is delivered by a plasmid, a nanoplasmid, a phagemid, a phage derivative, a virus, a bacmid, a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a yeast artificial chromosome (YAC), or a cosmid.
106 . The method of any one of claims 104-105 , wherein the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus.
107 . The method of claim 106 , wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.
108 . The method of claim 106 , wherein the herpesvirus is HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.
109 . The method of any one of claims 83-108 , wherein the donor polynucleotide comprises a size of at least about 1 kilobase (kb), 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, or more than 120 kb.
110 . The method of any one of claims 83-109 , wherein the donor polynucleotide encodes a therapeutic, a reporter, or a marker.
111 . The method of claim 110 , wherein the reporter comprises a fluorescent protein.
112 . The method of claim 111 , wherein the fluorescent protein is GFP, EBFP, EBFP2, Azurite, mKalamal, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, YPet, RFP, CFP, or a derivative thereof.
113 . The method of claim 110 , wherein the reporter is acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), luciferase, or a derivative thereof.
114 . The method of any one of claims 110-113 , wherein the marker is an antibiotic resistance marker.
115 . The method of claim 114 , wherein the antibiotic resistance marker is kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or a derivative thereof.
116 . The method of any one of claims 110-113 , wherein the marker is a cell surface marker.