COMPOSITIONS AND METHODS FOR SITE-DIRECTED MUTAGENESIS
The present disclosure provides improved genome editing compositions and methods for editing a double-strand DNA target site. The disclosure further provides genome edited cells produced by the compositions and methods described.
1 . A fusion polypeptide comprising, a DNA-binding domain and an I-OnuI homing endonuclease (HE) variant that binds and cleaves a selected double strand DNA (dsDNA) target site in a cell; a linker domain; and an exonuclease or biologically active fragment thereof.
2 . The fusion polypeptide of claim 1 , wherein the exonuclease is Trex2, ExoI, or ExoX, or biologically active fragment thereof.
3 .- 6 . (canceled)
7 . The fusion polypeptide of claim 1 , wherein the DNA-binding domain binds a dsDNA target site upstream of the I-OnuI HE variant dsDNA target site.
8 . The fusion polypeptide of claim 1 , wherein the DNA-binding domain comprises a TALE DNA-binding domain.
9 . The fusion polypeptide of claim 8 , wherein the TALE DNA domain comprises about 9.5 TALE repeat units to about 15.5 TALE repeat units.
10 . (canceled)
11 . The fusion polypeptide of claim 1 , wherein the DNA-binding domain comprises a zinc finger DNA-binding domain.
12 . (canceled)
13 . The fusion polypeptide of claim 1 , wherein the linker domain is a peptide linker.
14 .- 30 . (canceled)
31 . The fusion polypeptide of claim 1 , wherein the I-OnuI HE variant dsDNA target site is within a gene selected from the group consisting of: programmed cell death protein 1 (PD-1; PDCD1), lymphocyte activation gene 3 protein (LAG-3), T cell immunoglobulin domain and mucin domain protein 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), band T lymphocyte attenuator (BTLA), T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), V-domain Ig suppressor of T cell activation (VISTA), and killer cell immunoglobulin-like receptor (KIR), CCR5, TRAC (TCRa), TCRp, ILIORa, ILIORp, TGFBRI, TGFBR2, CBL-B, PCSK9, AHR, BTK, a-globin, p-globin, y-globin, BCLI IA, KLFI, SOX6, GATA1, LSDI, alpha folate receptor (FRa), avp6 integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CDI71, carcinoembryonic antigen (CEA), C-type lectin-like molecule-I (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T cell lymphoma-associated antigen 1 (CTAGEI), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fe Receptor Like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), Glypican-3 (GPC3), EGFR family including ErbB2 (HER2), IL-11Ra, IL-I3Ra2, Kappa, cancer/testis antigen 2 (LAGE-IA), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (LI-CAM), melanoma antigen gene (MAGE)-AI, MAGE-A3, MAGE-A4, MAGE-A6, MAGEAIO, melanoma antigen recognized by T cells 1 (MelanA or MART1), Mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related proteins B (MICB), neural cell adhesion molecule (NCAM), cancer/testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (RORI), synovial sarcoma, X breakpoint 2 (SSX2), Survivin, tumor associated glycoprotein 72 (TAG72), tumor endothelial marker 1 (TEM1/CD248), tumor endothelial marker 7-related (TEM7R), TEM5, TEM8, trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1) gene.
32 .- 33 . (canceled)
34 . The fusion polypeptide of claim 31 , wherein the TCRa gene target site comprises the amino acid sequence set forth in SEQ ID NO: 1, the CBL-B gene target site comprises the amino acid sequence set forth in SEQ ID NO: 2, or the PD-1 gene target site comprises the amino acid sequence set forth in SEQ ID NO: 3.
35 . (canceled)
36 . The fusion polypeptide of claim 2 , wherein the ExoX, or biologically active fragment thereof, comprises an amino acid an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in SEQ ID NO: 109.
37 . (canceled)
38 . The fusion polypeptide of claim 36 , comprising an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 46, 64, 73, and 82.
39 . (canceled)
40 . The fusion polypeptide of claim 2 , wherein the ExoI, or biologically active fragment thereof, comprises an amino acid an amino acid sequence having at least 85% identity to an amino acid sequence as set forth in SEQ ID NO: 112.
41 . (canceled)
42 . The fusion polypeptide of claim 40 , comprising an amino acid sequence having at least 85% identity to an amino acid sequence as set forth SEQ ID NOs: 43.
43 . (canceled)
44 . A polynucleotide, a mRNA, or a vector encoding the fusion polypeptide of claim 1 .
45 .- 53 . (canceled)
54 . A cell comprising the polypeptide of claim 1 .
55 .- 69 . (canceled)
70 . A method of site-directed mutagenesis comprising:
a) selecting a double-stranded DNA (dsDNA) target site, and
b) introducing into the cell a fusion polypeptide of claim 1 ;
wherein the fusion peptide generates directionally biased deletions having a deletion center near the selected dsDNA target cut site in the cell.
71 . (canceled)
72 . The method of claim 70 , wherein greater than 50% of the directionally biased deletions have a deletion center location on one side of the I-OnuI HE variant dsDNA target site center location.
73 .- 78 . (canceled)
79 . The method of claim 70 , wherein the deletion center location is on the same side as the DNA-binding domain target site relative to the I-OnuI HE variant dsDNA target site center location.
80 . The method of claim 70 , wherein the deletion center location is 5′ to the I-OnuI HE variant dsDNA target site center location.
81 . (canceled)
82 . The method of claim 70 , wherein at least 50% of deletions have a deletion center greater than 4 nucleotides away from the I-OnuI HE variant dsDNA target site center location, at least 10% of deletions have a deletion center greater than 8 nucleotides away from the I-OnuI HE variant dsDNA target site center location, or at least 35% of deletions are 12 bps in length or greater.
83 .- 110 . (canceled)
111 . The method of claim 70 , wherein the deletion extends into the DNA-binding domain target site or the deletion center location is within the DNA-binding domain target site.
112 .- 119 . (canceled)
120 . A method of treating, preventing, or ameliorating at least one symptom of a disease, or condition associated therewith, comprising harvesting a population of cells from a subject; editing the population of cells according to the method of claim 70 , and administering the edited population of cells to the subject.
121 .- 124 . (canceled)