Compositions and methods for improved gene editing

Abstract

The disclosure provides novel methods and compositions for gene editing. In particular, the disclosure relates to compositions and methods of making nucleic acid donor templates for highly efficient and precise gene editing.

1 . A method of enhancing homology directed repair efficiency of an isolated double-stranded nucleic acid donor sequence, the method comprising the steps of:
 1) denaturing the double-stranded nucleic acid donor sequence to form two single-stranded nucleic acid sequences; and   2) renaturing the two single-stranded nucleic acid sequences to form the double-stranded nucleic acid donor sequence, thereby enhancing the homology directed repair efficiency of the double-stranded nucleic acid donor sequence,   wherein the double-stranded nucleic acid donor sequence has enhanced homology directed repair efficiency relative to a double-stranded nucleic acid donor sequence that has not been denatured and renatured.   
     
     
         2 . The method of  claim 1 , wherein the renatured double-stranded nucleic acid donor sequence comprises one or more single-stranded DNA nucleic acid regions or a nick in at least one of the strands. 
     
     
         3 . The method of  claim 2 , wherein the one or more single-stranded regions is selected from the group consisting of a single-stranded overhang, a DNA mismatch, a DNA bubble, and a non-B DNA structure. 
     
     
         4 . The method of  claim 3 , wherein the non-B DNA structure is selected from the group consisting of a cruciform, a triplex, a hairpin, a tetraplex, and a left-handed Z DNA. 
     
     
         5 . The method of  claim 3 , wherein the DNA mismatch is selected from the group consisting of a G:T, G:A, G:G, C:T, C:A, C:C, A:G, A:C, A:A, T:G, T:C, and T:T mismatch. 
     
     
         6 . The method of any one of  claims 3 - 5 , wherein the DNA bubble is between about 2 and about 40 bases in length. 
     
     
         7 . The method of any one of  claims 3 - 5 , wherein the DNA bubble is between about 10 and about 20 bases in length. 
     
     
         8 . The method of any one of  claims 3 - 7 , wherein the single-stranded overhang is present at one or both of the 5′ end and 3′ end of one or both of each strand of the double-stranded nucleic acid donor sequence. 
     
     
         9 . The method of any one of  claims 3 - 7 , wherein the single-stranded overhang is present at the 5′ end of one or both of each strand of the double-stranded nucleic acid donor sequence. 
     
     
         10 . The method of any one of  claims 3 - 9 , wherein the single-stranded overhang is between about 10 nucleotides to about 120 nucleotides in length. 
     
     
         11 . The method of any one of  claims 1 - 10 , wherein the isolated double-stranded nucleic acid donor sequence comprises about 100 nucleotides to about 10000 nucleotides in length. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein the double-stranded nucleic acid donor sequence is heat denatured and/or chemical denatured. 
     
     
         13 . The method of  claim 12 , wherein the denaturation chemical is sodium hydroxide. 
     
     
         14 . The method of  claim 12 , wherein the chemical denatured donor sequence is renatured with the addition of phosphate buffer. 
     
     
         15 . The method of  claim 12 , wherein the donor sequence is heat denatured by incubating the double-stranded nucleic acid donor sequence at 80° C. to 100° C. 
     
     
         16 . The method of  claim 15 , wherein the heat denatured donor sequence is renatured by gradually decreasing the temperature from the initial heat denaturing temperature of 80° C. to 100° C. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the donor sequence is heat denatured and renatured following the steps comprising:
 1) incubating at 95° C. for about 10 seconds to about 10 minutes,   2) incubating at 85° C. for about 5 seconds to about 2 minutes,   3) incubating at 75° C. for about 5 seconds to about 2 minutes,   4) incubating at 65° C. for about 5 seconds to about 2 minutes,   5) incubating at 55° C. for about 5 seconds to about 2 minutes,   6) incubating at 45° C. for about 5 seconds to about 2 minutes,   7) incubating at 35° C. for about 5 seconds to about 2 minutes, and   8) incubating at 25° C. for about 5 seconds to about 2 minutes.   
     
     
         18 . The method of any one of  claims 1 - 16 , wherein the donor sequence is heat denatured and renatured following the steps comprising:
 1) incubating at 95° C. for about 2 minutes,   2) incubating at 85° C. for about 10 seconds,   3) incubating at 75° C. for about 10 seconds,   4) incubating at 65° C. for about 10 seconds,   5) incubating at 55° C. for about 1 minute,   6) incubating at 45° C. for about 30 seconds,   7) incubating at 35° C. for about 10 seconds, and   8) incubating at 25° C. for about 10 seconds.   
     
     
         19 . The method of any one of  claims 1 - 16 , wherein the donor sequence is heat denatured and renatured following the steps comprising:
 1) incubating at 95° C. for about 2 minutes,   2) incubating at 85° C. for about 1 minute,   3) incubating at 75° C. for about 1 minute,   4) incubating at 65° C. for about 1 minute,   5) incubating at 55° C. for about 1 minute,   6) incubating at 45° C. for about 1 minute,   7) incubating at 35° C. for about 1 minute, and   8) incubating at 25° C. for about 1 minute.   
     
     
         20 . The method of any of  claims 15 - 19 , wherein the temperature is decreased at a rate of about 0.1° C./second to about 2° C./second. 
     
     
         21 . The method of  claim 20 , wherein the temperature is decreased at a rate of about 0.1° C./second. 
     
     
         22 . The method of  claim 20 , wherein the temperature is decreased at a rate of about 1° C./second. 
     
     
         23 . The method of  claims 15  and  16 , wherein the temperature is decreased immediately by placing the denatured donor sequence at about 4° C. to about 10° C. or by placing the denatured donor sequence on ice. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein the donor sequence comprises one or both of: one or more terminal adaptors; and one or more terminal adaptor ligand moieties. 
     
     
         25 . The method of  claim 24 , wherein the one or more terminal adaptors or the one or more terminal adaptor ligand moieties are attached to the 5′ end and/or the 3′ end of the nucleic acid donor sequence. 
     
     
         26 . The method of  claim 24 , wherein the one or more terminal adaptors comprise one or more of ethylene glycol, polyethylene glycol (PEG), a polyamine having at least two amino groups, an alkanediol, and a single-stranded RNA (ssRNA). 
     
     
         27 . The method of  claim 26 , wherein the ethylene glycol, PEG, polyamine having at least two amino groups, or alkanediol is attached to the ssRNA. 
     
     
         28 . The method of  claim 26 , wherein the PEG is tetraethylene glycol or triethylene glycol linker. 
     
     
         29 . The method of  claim 26 , wherein the ssRNA further comprises one or more modified nucleotides. 
     
     
         30 . The method of  claim 29 , wherein the one or more modified nucleotides are selected from the group consisting of a 2′-O-alkyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a 2′-deoxy-modified nucleotide, a locked nucleic acid (LNA), a bridged nucleotide, a constrained nucleotide, a bicyclic nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a peptide nucleic acid, and a non-natural base comprising nucleotide. 
     
     
         31 . The method of  claim 29 , wherein the one or more modified nucleotides are 2′-O-methyl (2′-OMe) modified nucleotides. 
     
     
         32 . The method of any one of  claims 26 - 31 , wherein the ssRNA is about 1 base in length to about 50 bases in length. 
     
     
         33 . The method of  claim 32 , wherein the ssRNA is about 8 bases in length to about 30 bases in length. 
     
     
         34 . The method of any one of  claims 24 - 33 , wherein the terminal adaptors can bind a terminal adaptor ligand. 
     
     
         35 . The method of  claim 34 , wherein the terminal adaptors can bind the terminal adaptor ligand through nucleic acid base-pairing interactions. 
     
     
         36 . The method of  claim 34 , wherein the terminal adaptor ligand comprises a peptide nucleic acid (PNA), a ssRNA, or a ssDNA. 
     
     
         37 . The method of  claim 34 , wherein the terminal adaptor ligand is attached to a terminal adaptor ligand moiety that confers one or more functionalities to the nucleic acid donor sequence. 
     
     
         38 . The method of  claim 37 , wherein the one or more functionalities is selected from the group consisting of tissue targeting, PK-modification, and nuclear localization. 
     
     
         39 . The method of  claim 37 , wherein the terminal adaptor ligand moiety is a peptide, a carbohydrate, a lipid, a steroid, or a small molecule. 
     
     
         40 . The method of  claim 37 , wherein the terminal adaptor ligand moiety is a nuclear localization signal (NLS). 
     
     
         41 . The method of  claim 40 , wherein the NLS comprises PKKKRK. 
     
     
         42 . The method of any one of  claims 37 - 41 , wherein the terminal adaptor ligand is attached to the terminal adaptor ligand moiety through a linker. 
     
     
         43 . The method of  claim 42 , wherein the linker is selected from the group consisting of aminoethoxyethoxyacetate (AEEA), aminohexanoic acid, oligoglycine, PEG, amino C6, and amino C12. 
     
     
         44 . The method of any of  claims 1 - 43 , wherein homology-directed repair (HDR) or homology-independent targeted integration (HITI) efficiency of the donor sequence is enhanced by about 2-fold to about 20-fold relative to a donor sequence that has not been denatured and renatured or nicked. 
     
     
         45 . The method of any of  claims 1 - 44 , wherein homology-directed repair (HDR) or homology-independent targeted integration (HITI) efficiency of the donor sequence is enhanced by about 10-fold relative to a donor sequence that has not been denatured and renatured or nicked. 
     
     
         46 - 95 . (canceled)